Mechanism of alternative splicing through competing 5’ splice sites

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Pillai, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7007499/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Dec, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Alternative splicing of precursor-messenger RNAs (pre-mRNA) bearing introns with competing and overlapping 5’ splice sites (5’SS) produces more mRNAs. We investigated the mechanism of alternative splicing via competing 5’SS by monitoring RNA and protein products of the yeast SRC1/HEH1 gene in different mutant strains. HEH1 alternative splicing requires a sixteen-nucleotide pre-mRNA segment spanning its two 5’SS. The nucleotides are decoded by U5 and U6 small nuclear RNAs (snRNA), supported by specific proteins of the spliceosomal B and Bact complexes, including Prp8. HEH1 alternative splicing became independent of the supporting proteins following recalibration of the pre-mRNA-snRNA base pairings through changes in the 5’SS or U5 and U6 snRNAs. Assisted by proteins that stabilize low-fidelity, high-efficiency spliceosome conformations, the competing HEH1 5’SS are marked for alternative splicing by U5 and U6 snRNAs during B to Bact transition of the spliceosome. Biological sciences/Molecular biology/RNA metabolism/Alternative splicing Biological sciences/Biochemistry/RNA Alternative 5’ splice site SRC1 Prp8 RES complex Ecm2 U5 snRNA U6 snRNA B complex Bact complex Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Splicing of precursor messenger RNAs (pre-mRNA) occurs through a multi-step process involving large and dynamic ribonucleoprotein (RNP) complexes called spliceosomes. Stage-specific spliceosomes are assembled on a pre-mRNA following intricate rearrangements of five distinct snRNPs containing cognate small nuclear RNAs (snRNA) and associated proteins. Specific snRNPs recognise splicing signals in pre-mRNAs, including the donor 5’ splice site (5’SS) ‘GU’, the acceptor 3’ splice site (3’SS) ‘AG’, and the branchpoint adenosine ‘A’. Two steps of transesterification reactions, catalysed by divalent metal ions and U6 snRNA in the spliceosomes, remove introns and join exons to form functional mRNA 1 , 2 . The spliceosome machinery is essential for constitutive and alternative splicing. More mRNAs from a gene through can be produced through alternative splicing. The process amplifies protein diversity from limited gene pools in an organism 3 , 4 . Different classes of alternative splicing exist in eukaryotes. Key among them occurs through alternative selection of competing 5’SS 5 and 3’SS 4 , and alternative splicing through competing 5’SS is a prevalent mode. A majority of pre-mRNAs in the budding yeast Saccharomyces cerevisiae have the highly conserved GUAUGU hexanucleotide as 5’SS donors 6 , but the sequences are highly variable in intron-rich eukaryotes, where alternative 5’SS selection is widespread 7 . Two major classes of 5’SS (NN/GURAG and AG/GUNNN; ‘/’ indicates exon-intron junction), distinguished by the presence of ‘G’ at -1 and + 5 positions, have been reported 8 . Alternative 5’SS are often found 4 nucleotides across the dominant 5’SS, and such occurrences in the human genome are estimated to be large 9 , 10 . The canonical ‘GU’ dinucleotide deviates to ‘GC’ in 0.87% of the 5’SS 11 . The HEH1 gene (also called SRC1 ) in S. cerevisiae is alternatively spliced 12 – 14 . Its pre-mRNA has alternative donors with ‘GC’ and ‘GU’ dinucleotides embedded into two non-canonical 5’SS, / GC AAGU and / GU GAGU, arranged as a partially overlapping sequence / GC AA/ GU GAGU. / GU GAGU is the dominant 5’SS and splicing through this site generates mRNA-encoding a longer full-length protein, Heh1L. Splicing through the upstream / GC AAGU is tightly regulated and produces mRNA-encoding a shorter protein, Heh1S (the removal of GCAA tetranucleotide gains an in-frame stop codon early in HEH1S mRNA). The two proteins share a common N-terminus but differ in their C-termini, thus attaining different topologies within the inner nuclear membrane and have distinct functions 12 , 13 , 15 , 16 . 5’SS selection relies on early pre-mRNA recognition by trans-acting U1 snRNA and associated proteins 17 – 19 . The role of specific RBPs (RNA-binding proteins), SR (serine/arginine-rich) proteins, and hnRNPs (heterogeneous nuclear ribonucleoproteins) has also been reported to be crucial for 5’SS selection 5 , 20 – 25 . These proteins act as splicing enhancers and silencers, and regulate spliceosome assembly on pre-mRNA targets in association with the U1 snRNP. The role of successive spliceosomal complexes in 5’SS selection has been recently reported 26 . The HEH1 gene is an interesting tool for exploring RNA and protein factors needed for alternative splicing through competing and overlapping 5’SS. Its alternative splicing requires non-covalent associations of the ubiquitin-like protein Hub1 (UBL5 in humans) with the U1 and U2 snRNP-bridging RNA helicase Prp5 27 , and the U4/U6.U5 tri-snRNP protein Snu66 14 . Thus, Hub1 acts early in spliceosome assembly by activating the RNA helicase, as well as later with the tri-snRNP component 28 . The latter activity suggests a possible role of the spliceosome core in HEH1 5’SS selection. We find that HEH1 alternative splicing required optimal pairing of U5 and U6 snRNAs across a sixteen-nucleotide segment of the pre-mRNA across the two 5’SS at the exon1-intron boundary. The selection of the competing 5’SS by U5 and U6 snRNAs and alternative splicing was supported by specific proteins of the spliceosome core, including Prp8. RESULTS Additional trans-acting proteins for HEH1 alternative splicing Between to two 5’SS of SRC1/HEH1 pre-mRNA / GC AA/ GU GAGU, the usage of the upstream 5’SS / GC AAGU requires non-covalent binding of the ubiquitin-like protein Hub1 to Snu66. The downstream 5’SS / GU GAGU was dominant and selected independently of this complex 14 . We searched for additional HEH1 alternative splicing factors in S. cerevisiae through the following approaches. (i) HEH1 –LACZ alternative splicing reporters HEH1S – LACZ and HEH1L – LACZ were prepared (Extended Data Fig. 1a), on the line of RP51 – LACZ splicing reporter 29 , by fusing a HEH1 gene segment containing parts of the exons across the intron upstream of the b galactosidase-encoding LACZ gene. In-frame translatable reporter mRNA would be made only after precise 5’SS selection and excision of the intron. Reporter activities were monitored in yeast strains grown on solid (X-Gal overlay assay) and liquid (ONPG assay) media. Splicing from the alternative 5’SS was diminished in the deletion strains of the RES (Retention and Splicing complex) complex subunits Snu17, Bud13, Pml1, and Urn1. The deletion of the NTC (Nineteen Complex) factor Ecm2 showed similar defects (Extended Data Fig.1b and c). By contrast, splicing from the dominant 5’SS remained unaltered in the mutants, suggesting a role of these proteins in the selection of the weaker alternative 5’SS. (ii) Heh1 protein isoforms were monitored in deletion and point mutants of splicing factors by western blot assays. The gene was expressed with N-terminal epitope tags from a plasmid or tagged chromosomally. Supporting the data with the reporter assays (Extended Data Fig.1a-c), Heh1S protein was strongly diminished in the knockout mutants of Snu17, Bud13, Pml1, and Urn1, in addition to Hub1 (Extended Data Fig. 1d). The previously reported D22A and H63L mutants of Hub1 were defective in HEH1 alternative splicing. Potential RNA-binding mutant of Hub1, as deduced from cryo-EM studies of spliceosomes 30,31 , showed a similar lack of Heh1S protein (Extended Data Fig. 1h). Alternative splicing defect was also seen in the Hub1-binding deficient Snu66-HIND mutant (Extended Data Fig. 1j). Prp8 alleles were also screened for HEH1 alternative splicing. Heh1S level was strongly reduced in the prp8-101 (E1960K) allele. No other Prp8 mutant tested showed similar defects. While Heh1S protein was diminished in the above mutants, they showed more Heh1L than wt (Figure 1a), possibly because the lack of competition from HEH1S 5’SS led to increased usage of the HEH1L site. (iii) HEH1 mRNA isoforms in the yeast mutants were monitored by reverse-transcription (RT) PCR assays with primers binding to HEH1 exons, followed by sequencing of the cDNAs. The two mRNAs show a single band. Since they differ in GCAA nucleotides at the exon junction ( HEHL gains GCAA due to the usage of downstream 5’SS), electropherograms show two mixed peaks after the common exon. Areas under the peaks were integrated to estimate the relative abundance of the two mRNAs. Consistent with the findings from reporter assays and protein analysis, the above mutants showed diminished levels of HEH1S mRNA, confirming the 5’SS selection defects at the RNA level (Figure 1c). The electropherograms also showed increased HEH1L mRNA level in the mutants, compared to the wt strain (Figure 1d), consistent with higher protein levels seen in Figure 1a. Thus, spliceosomes defective in using HEH1S 5’SS used the dominant HEH1L 5’SS more efficiently in the absence of competition. (iv) Splicing factors essential for cell viability could not be explored with deletion strains. Hub1 proximity to them was used to explore their potential role in HEH1 alternative splicing. Snu66-binding-deficient hub1(D22A) in the free form was defective in the usage of HEH1 alternative 5’SS, but its linear fusion to Snu66, Prp38 and Prp8 restored alternative splicing in cells lacking Hub1 14 . Interestingly, Heh1S protein was restored after anchoring Hub1 to Prp3, Prp6, and Prp31, but not to other proteins or the components of U1 and U2 snRNPs (Figure 1e). Thus, Hub1’s proximity to specific proteins of the spliceosome B and Bact complexes (Snu66, Prp38, Prp8, Prp3, Prp6, and Prp31) promoted HEH1 alternative splicing. The data discussed in i-iv indicated the need for specific proteins of the spliceosome core in HEH1 alternative splicing. However, their excess did not push the selection towards HEH1S 5’SS. Overexpression of none of these proteins increased the Heh1S isoform or suppressed the Heh1L (Extended Data Fig. 1 m-n). Hub1’s excess in the spliceosome through its chromosomal fusions to two HEH1S splicing-promoting factors, Snu66 and Prp38, could not alter the Heh1S/Heh1L ratio (Extended Data Fig. 1o). Thus, since the proteins played only regulatory roles, how are the competing 5’SS decoded? U5 and U6 snRNAs decode pre-mRNA cis elements for alternative 5’SS selection From the splicing reporters discussed above, and known binding sites of U1, U5, and U6 snRNAs at exon-intron boundaries, we narrowed a 16-nucleotide pre-mRNA segment (1913-1928 of the HEH1 gene) and studied its importance for alternative 5’SS selection by mutagenesis (Figure 2a). Proteins originating from the variants were detected by western blots (Figure 2b), and mRNAs by RT-PCR followed by cDNA sequencing (Figure 2c). Almost any change in this segment lost alternative splicing (barring the G(−1)A variant of HEH1S ) and produced either one of the Heh1 isoforms. Changes leading to stop codons were studied by cDNA sequencing. G C AA to G U AA change of the upstream 5’SS gains an in-frame stop codon of HEH1L mRNA, abolishing the Heh1L protein (this data explains the choice of the ‘GC’ donor for HEH1S splicing; a ‘GU’ would have abolished Heh1L). These results highlighted the invariability of nucleotides around the competing 5’SS. The alternative splicing defects in the pre-mRNA variants, however, did not correlate with predicted U1 snRNA-5’SS interactions. Strikingly, we noticed a perfect correlation in the 5’SS choice and their expected base-pairing strengths with U5 and U6 snRNAs. 5’SS that paired stronger to U5 or U6 snRNAs was the preferred donor, and the use of the second site was minimal (Figure 2 and Extended Data Fig. 2). U5 and U6 snRNAs can promote alternative 5’SS selection without the protein factors From Figure 2, U5 and U6 (but not U1) snRNA variants with modified base pairing to the competing sites were expected to bias the selection. Indeed, the respective 5’SS was used more efficiently upon overexpression of U5 and U6 snRNA variants with strengthened base pairing (Figure 3b). Similar overexpression of the U1 snRNA variant did not alter 5’SS choice. U5 snRNA U(98)G and U6 snRNA A(45)G variants with stronger binding to HEH1L 5’SS increased Heh1L protein (Extended Data Fig. 3). Conversely, U5 snRNA U(98)C and U6 snRNA U(46)C A(45)U variants with stronger binding to HEH1S 5’SS increased Heh1S mRNA and protein (Figure 3b-c and Extended Data Fig. 3a-b and 3e-f). Thus, 46 th and 45 th nucleotides of U6 snRNA, beyond the ACAGA box, were also crucial for selecting the alternative 5’SS by base pairing at +7 and +8 nucleotides in the intron. If the proteins discussed above acted by stabilizing U5 and U6 snRNAs on the pre-mRNA substrate, the snRNA variants pairing strongly to HEH1S 5’SS should bypass the need for the proteins. Indeed, overexpression of U(98)C variant of U5 snRNA and U(46)C,A(45)U variants of U6 snRNA restored alternative splicing defects in D hub1 , D snu17 , D ecm2, and D urn1 mutants (Figure 3 and Extended Data Fig. 3). Notably, the defect in prp8-101 strain was partially restored by overexpression of the U5 and U6 snRNA variants, suggesting an additional role of the core spliceosome protein Prp8 in 5’SS selection. The Prp8-101 surface is critical for processing the ‘GC’ donor Prp8 is reported to exist in two distinct conformations to maintain equilibrium between the two catalytic steps of splicing; consequently, two opposing alleles of Prp8 rescue splicing defects in one another 32 . Combining a second step Prp8 allele, prp8-161 , with prp8-101 did not restore the defects (Extended Data Fig. 4d). Thus, the alternative splicing defects in the first step Prp8 allele, prp8-101 , could not be explained by the above mechanism. In a pre-mRNA variant (#7), the upstream 5’SS /GCA U GU with ‘GC’ donor was preferred over the downstream /GUGAGU in wt, D hub1 , D snu17 , D ecm2, and D urn1 strains (Figure 4b and Extended Data Fig. 4b), likely due to the stronger pairing of +4U with the AC A GA box of U6 snRNA. However, the prp8-101 allele could not use this site. The defect in prp8-101 was explored using the ACT1–CUP1 splicing reporter with a single 5’SS 33 . The 5’SS /GCA U GU was used efficiently in all mutants, except for D hub1 and prp8-101 . C(+2)U-exchanged /G U A U GU was efficiently used in all strains, including D hub1 and prp8-101 (Figure 4c). Thus, the donor with +2C was poorly used in prp8-101 and D hub1 , leading to the loss of alternative splicing. Similarly, splicing from a +3C 5’SS variant /GU C UGU was defective in D hub1 , D snu66, and prp8-101 strains (Extended Data Fig. 4e). Thus, the Prp8-101 surface promotes selection (and catalysis?) from non-canonical 5’SS containing +2C and +3C. Specific competing 5’SS allow alternative splicing without the regulatory proteins Optimal decoding of competing 5’SS by U5 and U6 snRNAs, together with the use of ‘GC’ donor by Prp8, is the primary determinant of alternative splicing. The process is facilitated by specific proteins of the spliceosome. Thus, competing 5’SS with modified binding to the snRNAs should allow alternative selection in the absence of the regulatory proteins. More pre-mRNA variants were assayed to identify such 5’SS. Two 5’SS variants allowed alternative splicing not only in wt yeast (Extended Data Fig. 2) but also in strains mutated for the regulatory proteins (Figure 4b and Extended Data Fig. 4b). U5 and U6 snRNAs’ base pairing was recalibrated in #15 with two non-canonical 5’SS /GCAA/GU AUA U. In contrast, 5’SS pairing with U6 snRNAs was strongest in #18, containing two canonical 5’SS /G U A U /GU AU GU. The sites being canonical were readily used without the need for the protein factors, including the Prp8-101 surface. The efficient use of canonical 5’SS in the absence of the protein factors was further supported by HEH1–CUP1 reporters (Extended Data Fig. 4f-h). Ecm2, Urn1, and the RES proteins bring weaker 5’SS in competition HEH1S expression from /GCAA/GU AUA U 5’SS (#15) in cells lacking the protein factors was intriguing, since the changes in this variant were beyond the hexanucleotide /GCAAGU. The outcomes could be explained by two possibilities: (i) weakening of the HEH1L 5’SS /GUGAGU to /GU AUA U rebalanced the competition in favor of the HEH1S site, or (ii) improved binding of +7A and +8U in /GCAAGU AU to U6 snRNA nucleotides U46 and A45 favored the HEH1S 5’SS. To study these possibilities, alternative splicing reporters, HEH1S–CUP1 and HEH1L–CUP1 , were made (Figure 5b). HEH1L 5’SS was weakened into /GUGAG A (#10; it also lacks potential HEH1S -U6 pairing at +7 and +8 positions) or /GU AUAA (#20; with enhanced U6 pairing at +7 and +8 positions of HEH1S ) (Figure 5a and c). HEH1S 5’SS selection in D hub1 improved for reporters with enhanced U6 pairing at +7 and +8 positions of the 5’SS, and the defect was partially restored in the prp8-101 allele. The data confirmed the importance of U6 snRNA’s U46 and A45 nucleotides for alternative splicing discussed earlier in Figure 3. The 5’SS selection in D snu17 , D urn1, and D ecm2, on the other hand, was improved by both enhanced U6 snRNA pairing to HEH1S and weakening the competition from HEH1L (Figure 5d). Thus, Hub1 and Prp8 promote selection of 5’SS with +2C by stabilizing U5 and U6 snRNA on the target pre-mRNA. On the contrary, Snu17, Urn1, and Ecm2 promote competition for the weaker 5’SS by stabilizing snRNA-pre-mRNA interactions. DISCUSSION Pre-mRNA-snRNA interactions in alternative 5’SS selection The data presented support dominant roles of U5 and U6 snRNAs and regulatory roles of B and Bact proteins in alternative 5’SS selection. U1 snRNA and associated factors recognize 5′SS early in the splicing cycle and have been reported to promote alternative 5’SS selection 34 . However, U1 snRNA, its associated proteins, and spliceosomes before the pre-B stage were not critical for HEH1 alternative splicing. At the B stage of the spliceosome, U1 snRNA hands over the 5’SS to U6 snRNA. The ACAGA box of U6 base pairs with +3 to +6 nucleotides of the 5’SS, and U5 snRNA’s loop 1 base pairs with the upstream four nucleotides of the exon 35 . Moreover, the 45 th and 46 th nucleotides of U6 snRNA appear to play a critical role in decoding +7 and +8 positions of 5’SS. Optimal strength of U5 and U6 snRNAs base pairing was essential for the selection of HEH1 competing 5’SS. Any deviations in pairing led to the loss of alternative splicing and produced the isoform which had relatively better snRNAs-pre-mRNA interactions. The snRNA variants with reinforced pairing to the weaker site not only favored their selection but also rescued the defects in the protein mutants. In addition, certain pre-mRNA variants with recalibrated U5 and U6 snRNA pairing to the competing 5’SS were alternatively spliced in the absence of the protein factors. The marking of the pre-mRNA target by the snRNAs in the B complex induces U4/U6 snRNA unwinding and concomitant U2/U6 snRNA pairing in the Bact complex 36 . The switch may define the branching site for the first catalytic step for the +2C containing 5’SS in the B* complex, likely with the help of the Prp8-101 surface. Thus, RNA-RNA interactions at the B stage of the spliceosome are essential for alternative 5’SS selection, centered around pre-mRNA recognition by U5 and U6 snRNAs (Figure 6). Specific B and Bact proteins facilitate alternative 5’SS selection The trans-acting proteins critical for the alternative selection of HEH1 5’SS reported earlier 14 and identified in this study belong to the spliceosome B or Bact complexes. Importantly, alternative 5’SS of HEH1 is the first in vivo target of these factors in S. cerevisiae , including the prp8-101 allele. The proteins appear to facilitate alternative 5’SS selection by stabilizing the spliceosome in a low-fidelity, high-efficiency conformation. Supporting this idea, the Hub1-Snu66 complex enhances the selection of non-canonical 5’SS and error-prone splicing 14,27 . The RES complex proteins Snu17, Bud13, and Urn1 promote non-canonical splicing 37–39 , and Ecm2 facilitates selection of non-canonical and competing 5’SS 40 . The proteins seem to promote competition for the alternative 5’SS at the expense of the dominant site. Splicing from HEH1S 5’SS needed these proteins, yet splicing from the HEH1L 5’SS was even better in their absence. Prp8 and associated proteins in alternative 5’SS selection: Among the fourteen distinct Prp8 alleles 41 tested in this study, only prp8-101 (E1960K) mutant 42 was defective in splicing from the alternative 5'SS. The mutation lies in its RNaseH/RH domain. The Prp8 RH domain toggles between transitional and catalytic conformations 43 . It remains in a partially closed conformation till the B complex and promotes splicing fidelity over efficiency. Prp8’s first step alleles 44 and Prp38 45 have been shown to stabilize the high-fidelity conformation. The RH domain switches to a completely closed conformation in the Bact complex 43,46 and positions the RNA for first-step catalysis. The defective splicing in prp8-101 from +2C and +3C containing 5’SS, suggests high-fidelity, low-efficiency nature of this mutant and the role of Prp8 in selection (or catalysis) of +2C and +3C containing 5’SS. In contrast to the full rescue of HEH1 alternative splicing upon overexpression of U5 and U6 snRNA variants in the deletion of the splicing proteins, the partial rescue of prp8-101 defects points to a potential role of this Prp8 surface in catalysis from the ‘GC’ donor. The role of Prp3, Prp6 and Prp31 in alternative 5’SS selection was revealed through proximity probing with Hub1. These proteins surround the core Prp8 and undergo a major switch during the conversion of pre-B to B. The 180° shift in the RH domain from pre-B to B along with the tri-snRNP factors 47,48 appears to be important for the recognition of HEH1 alternative 5’SS. Thus, conformational changes in the spliceosome during B to Bact transition appears to be critical for HEH1 alternative splicing. Hub1 in alternative 5’SS selection: Hub1 joins spliceosome through non-covalent binding to Prp5 or Snu66. The Prp38 complex subunits, Prp38, Snu13 and Spp381/MFAP1, associate with the tri-snRNP and help stabilise the B complex. The D22A surface of Hub1 binds Snu66 in multiple eukaryotes 14,49–51 . Interestinlgy. the same Hub1 surface binds MFAP1 in the human B spliceosome 48,52 . The D21 residue of Hub1 also interacts with the same surface of MFAP1 48 . An additional Hub1 surface, N7 and K13, that binds the 5’ exon at -3 and -4 positions in human spliceosomes 52 , was critical for HEH1 alternative 5’SS selection. Thus, Hub1 is likely recruited to the early B complex through Snu66-HIND 14 and promote alternative 5’SS selection after switching to pre-mRNA and MFAP1 in the mature B complex 48,52 . The B to Bact transition : Brr2 helicase unwinds the U4/U6 duplex, triggering the release of U4 snRNP and associated proteins (Prp3, Prp6, Prp31, Snu66, Hub1). Prp38 complex is also leaves at this stage, a key step for full activation of the spliceosome. U6 snRNA forms new interactions with U2 snRNA, assembling the catalytic core. The NTC protein Ecm2 and the RES complex proteins are associated with the Bact complex, highlighting the role of the RES in the remodelling of Bact via Prp2. The RES complex leaves the spliceosome during Bact to B* conversion 53,54 . In conclusion, S. cerevisiae HEH1/SRC1 pre-mRNA is an important substrate for studying alternative splicing through competing and overlapping 5’SS. Data presented here, along with the literature, support a plausible mechanism of alternative 5’SS selection in the B and Bact spliceosomes orchestrated by U5 and U6 snRNAs as the primary determinants. RNA-RNA interaction is thus central to alternative splicing through competing 5’SS. Splicing factors promoting selection of weak 5’SS, reported previously (Hub1, Snu66, Prp38, Prp8), and identified in this study (the B complex proteins Prp3, Prp6, Prp31, and Snu66; the RES complex subunits Bud13, Snu17, Pml1 and Urn1, and the NTC complex Ecm2), function from or through the B or Bact spliceosomes. U5 and U6 snRNAs are essential components of both these assemblies. With the help of the trans-acting proteins, U5 and U6 snRNAs pair with target pre-mRNAs for alternative 5’SS selection and catalysis. STAR METHOD KEY RESOURCE TABLE Reagent Resource Identifier Rabbit polyclonal anti-c-myc antibody Sigma Aldrich C3956 Rabbit Peroxidase anti-peroxidase soluble antibody Sigma Aldrich A0545 Goat anti-rabbit IgG peroxidase antibody Sigma Aldrich P1291 High-Capacity cDNA Reverse Transcription Kit Applied Biosystem 4368813 NuPAGE™ Bis-Tris Mini Protein Gels, 4–12% Invitrogen™ NP0323BOX RQ1 RNase-Free DNase Promega M610A Zymo-Spin II Columns Zymo Research C1008-250 Vent DNA polymerase NEB M0254L Softwares Fiji-windows 64 NIH https://imagej.net/software/fiji AlphaFold3 DeepMind (Google) https://alphafoldserver.com/ METHOD DETAILS Plasmids, yeast strains, S. cerevisiae transformation, and growth assays Plasmids and strains used in this study are listed in Table 1 and Table 2, respectively. The yJU75 yeast strain, plasmids PRP8 and prp8-101, and ACT1–CUP1 reporter were kind gifts from C. Guthrie and M. Konarska's lab. Prp8 mutant strains were made by shuffling out the Ura+ PRP8 wildtype plasmid with the Trp+ prp8 mutants on 5-fluoroorotic acid (FOA) plates. The S. cerevisiae deletion strains of splicing factors were obtained from the Euroscarf haploid deletion library. Competent cell preparation and transformation were performed following previously published protocols. 55,56 . Chromosomal tagging for western blot, double knockout strains for genetic interactions, and splicing factors deletion in the yJU75 background were made by using the reported protocol. 55,56 . Protein overexpression was achieved by expressing clones under a strong gal promoter . For growth/spot assays, 5-fold serial dilutions of cells were spotted on the indicated agar plates, and plates were incubated at temperatures indicated in the figure. Chromosomal fusion of hub1 – D22A to the C-termini of splicing factors Splicing by Overlap Extension (SOE) PCR was done to make hub1(D22A) C-terminal fusion to essential splicing factors. This fusion was done based on the published protocol 55 . Three sets of primers were used to amplify three fragments. First set of primers amplified a 200-300 bp fragment of the C termini of the desired gene, excluding the stop codon. The reverse primer of this had an overhang of 20-25 bps that was complementary to the 20-25 bps of hub1(D22A) N-terminus. The second set of primers amplified the full-length hub1(D22A) with the Nat antibiotic selection marker cassette from the plasmid clones of pFA6a- hub1(D22A)-natNT2 . The third set of primers amplified a fragment of 200-300 bp from the stop codon of the gene that was going to be tagged. The forward primer for this had an overhang of 20-25 bp with the end of the antibiotic-resistance marker. All three fragments were amplified using Vent DNA polymerase. These fragments were confirmed based on their size on the agarose gel and the correct fragments were gel-purified. All three fragments were mixed and joined by SOE PCR using Vent DNA polymerase using the forward primer of the first fragment and the reverse primer of the third fragment. The joined fragment was confirmed by agarose gel electrophoresis and transformed into the S. cerevisiae strain. The transformants were selected on agar plates with antibiotic. The chromosomal fusion was confirmed by colony PCR using a forward primer specific to the gene and a reverse primer specific to the antibiotic cassette. Splicing reporters The splicing reporters used in this study are listed in Table 3. Splicing reporters are modified forms of the conventional ACT1 – CUP1 33 and RP51 – LACZ 29 . The HEH1S–CUP1 reporters have a 121 bp segment of the S. cerevisiae HEH1 gene (1874-2095 nucleotides), including the intron, using BamHI and KpnI restriction sites. The HEH1S mRNA is spliced using GCAAGU 5’SS, generating in-frame CUP1 mRNA for translation. The methionine initiation codon was introduced by inserting a point mutation at C1881G. Initiation codon and variants of the 5’SS were made by site-directed mutagenesis (SDM) using specific primers, and the change was confirmed using Sanger sequencing. For HEH1L–CUP1 reporter , a 120 bp segment of the HEH1 gene (1874-2094 nucleotides) was used to bring the HEH1L mRNA spliced using GUGAGU 5’SS in-frame with the CUP1 gene. For reporter assays, competent cells from the S. cerevisiae strain yJU75 were transformed with the reporters, and transformants were selected in media lacking leucine. 1 OD 600 cells were spotted on solid media with different concentrations of CuSO 4 . The plates were incubated for 2-3 days at 30°C. Similarly, for HEH1–LACZ reporters, the RP51 part of the RP51 – LACZ reporter (gift from M. Rosbash) was replaced with a HEH1 fragment of 399 bp (1792-2191 nucleotides) and 398 bp (1792-2190 nucleotides) to obtain HEH1S – LACZ and HEH1L – LACZ reporters , respectively. β-galactosidase assay β-galactosidase activity was observed on both solid and liquid media. The X-gal overlay and the ONPG assay were performed by following published protocols 57 . RNA isolation and cDNA synthesis RNA isolation and cDNA synthesis were done as described previously 58 . Briefly, five OD 600 cells in the log phase were harvested. Total RNA was isolated by the hot acid phenol method using 15 mL phase lock tubes for phase separation. Residual DNA was removed by treating RNA with DNase I for 15 min at room temperature, followed by RNA clean-up using a Zymo‐Spin TM II column. cDNA synthesis from 2μg total RNA was done using reverse transcriptase (RT) and random‐hexamer primers at 37°C for 2h. Splicing defects were monitored by detecting intron-containing transcripts or post-splicing mature transcripts using exonic primers across the intron and analysed by agarose gel electrophoresis. For cDNA sequencing, five identical PCR reactions were performed. The PCR products were pooled and kept overnight for precipitation after adding 2.5 times the volume of isopropanol and 1/10 th the volume of 3M sodium acetate at -20°C. DNA was pelleted by centrifugation for 15 min at 4°C at maximum speed. The pellet was washed twice with 70% ethanol. Dried pellet was dissolved in 30μl of nuclease-free water, and 10μl of the DNA was sequenced by Sanger sequencing using a primer nested inside the forward primer used in amplification. Quantification of HEH1 mRNA isoforms HEH1 alternative splicing was analysed using RT-PCR followed by cDNA sequencing. HEH1S and HEH1L mRNAs differ in GCAA nucleotides at their exon junctions ( HEHL gains them due to the usage of downstream 5’SS), the electropherogram shows mixed peaks corresponding to the two isoforms after the exon-exon junction. The area under the peaks was integrated to find the relative abundance of the two mRNAs. Both common and isoform-specific peaks were analysed within the region corresponding to the 5’exon/intron boundary. To understand alternative 5’SS usage, common nucleotides after the junction in the two isoforms was excluded from further analysis. For each site, we recorded the nucleotide proportions possible for both the cDNAs and calculated the ratio between the two nucleotides, normalising their combined total to 100%. The average value of both forms is directly related to their respective 5’SS usage. Relative expression of HEH1L in wild-type and yeast mutants was obtained from the cDNA electropherogram. The areas under 10 common and 10 mixed peaks (before and after the junction, respectively), were integrated and averaged. Similarly, an area under the peaks specific to HEH1L was integrated and averaged. The ratio of common and HEHL peaks for different strains was obtained. The fold difference in HEH1L expression in the mutants was calculated against the wildtype strain by dividing the average of the area under HEH1L peaks (after the junction) by the average of the area under common peaks (before the junction). Western blots For protein western blots (immunoblotting), logarithmically growing cells equivalent to 2 OD 600 were harvested. Whole protein was extracted using the trichloroacetic acid (TCA) method 55 . Total proteins used for western blots were denatured by heating cells in a high urea buffer at 65°C for 15 min and then centrifuged. The soluble protein was separated on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) 8% gels, or NuPAGE TM 4%-12% Bis-Tris gels (Invitrogen), transferred to a polyvinylidene difluoride (PVDF) membrane, and probed with specific primary and secondary antibodies. Protein bands were quantified using ImageJ following the published method 59 . Declarations RESOURCE AVAILABILITY A.K.B. or S.K.M. may be contacted for resources used in this study. ACKNOWLEDGEMENT We thank Stefan Jentsch (MPI Biochemistry, Martinsried, Germany) for his support. The work in S.K.M. laboratory has been supported by IISER Mohali and the Centre for Protein Science Design and Engineering (CPSDE) of the Ministry of Human Resource and Development (MHRD), Government of India; the Max Planck Society, Germany; and the Wellcome Trust/DBT India Alliance Fellowship/grant awarded to S.K.M. A.K.B. was supported by ICMR fellowship and IISER Mohali. We acknowledge Anupa T. Anil and B. Mohapatra for their inputs and technical guidance. AUTHOR CONTRIBUTION S.K.M. initiated the study in the Jentsch laboratory at the MPI Biochemistry, Martinsried, Germany. All authors designed and performed the experiments and analysed the data. A.K.B. and S.K.M. collated the data and prepared the manuscript with inputs from all authors. DECLARATION OF INTEREST The authors declare no conflict of interest. References Wilkinson, M. E., Charenton, C. & Nagai, K. Annual Review of Biochemistry RNA Splicing by the Spliceosome. 01 , 46 (2025). Fica, S. M. & Nagai, K. Cryo-electron microscopy snapshots of the spliceosome: Structural insights into a dynamic ribonucleoprotein machine. Nature Structural and Molecular Biology vol. 24 791–799 Preprint at https://doi.org/10.1038/nsmb.3463 (2017). WANG, Y. et al. Mechanism of alternative splicing and its regulation. Biomed Rep 3 , 152–158 (2015). Zhang, Y., Qian, J., Gu, C. & Yang, Y. Alternative splicing and cancer: a systematic review. 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ID Name Plasmids Description Ref. 3658 WT YC111 pGAL-3MYC– HEH1 -tADH S. cerevisiae HEH1 gene under GAL promoter with 3MYC tag at the N-terminus and ADH terminator Mishra et al. 2011 D1401 YC111 pGAL-3MYC–heh1 (A1913T)-tADH A1913T mutation pGAL 3MYC–heh1 tADH This study D1402 YC111 pGAL-3MYC–heh1 (A1915T)-tADH A1915T mutation pGAL 3MYC–heh1 tADH This study D1403 YC111 pGAL-3MYC–heh1 (G1916T)-tADH G1916T mutation pGAL 3MYC–heh1 tADH This study D1404 YC111 pGAL-3MYC–heh1 (G1916A)-tADH G1916A mutation pGAL 3MYC–heh1 tADH This study D1405 YC111 pGAL-3MYC–heh1 (C1918A)-tADH C1918A mutation pGAL 3MYC–heh1 tADH This study D1406 YC111 pGAL-3MYC–heh1 (C1918T)-tADH C1918T mutation pGAL 3MYC–heh1 tADH This study D1407 YC111 pGAL-3MYC–heh1 (A1920T)-tADH A1920T mutation pGAL 3MYC–heh1 tADH This study D1408 YC111 pGAL-3MYC–heh1 (G1923A)-tADH G1923A mutation pGAL 3MYC–heh1 tADH This study D1409 YC111 pGAL-3MYC–heh1 (A1924T)-tADH A1924T mutation pGAL 3MYC–heh1 tADH This study D1410 YC111 pGAL-3MYC–heh1 (T1926A)-tADH T1926A mutation pGAL 3MYC–heh1 tADH This study D1411 YC111 pGAL-3MYC–heh1 (A1920T, A1924T)-tADH A1920T, A1924T mutation pGAL 3MYC–heh1 tADH This study D1412 YC111 pGAL-3MYC–heh1 (G1923C, A1924T)-tADH G1923C, A1924T mutation pGAL 3MYC–heh1 tADH This study D1413 YC111 pGAL-3MYC–heh1 (C1918T, A1919C, A1920T)-tADH C1918T, A1919C, A1920T mutation pGAL 3MYC–heh1 tADH This study D1414 YC111 pGAL-3MYC–heh1 (A1920T, G1923A, A1924T)-tADH A1920T, G1923A, A1924T mutation pGAL 3MYC–heh1 tADH This study D1415 YC111 pGAL-3MYC–heh1 (G1923A, A1924T, G1925A)-tADH G19223A, A1924T, G1925A mutation pGAL 3MYC–heh1 tADH This study D1416 YC111 pGAL-3MYC–heh1 (G1923A, A1924T, G1925T, T1926A)-tADH G1923A, A1924T, G1925T, T1926A mutation pGAL 3MYC–heh1 tADH This study D1417 YC111 pGAL-3MYC–heh1 (G1923A, A1924T, G1925A, T1926A, A1927C)-tADH G1923A, A1924T, G1925A, T1926A, A1927C mutation pGAL 3MYC–heh1 tADH This study D1418 YC111 pGAL-3MYC–heh1 (C1918T, A1920T, G1923A, A1924T)-tADH C1918T, A1920T, G1923A, A1924T mutation pGAL 3MYC–heh1 tADH This study D1419 YC111 pGAL-3MYC–heh1 (G1921A)-tADH G1921A mutation pGAL 3MYC–heh1 tADH This study D1420 YC111 pGAL-3MYC–heh1 (G2046C)-tADH G2046C mutation pGAL 3MYC–heh1 tADH This study D897 YE112 pADH-3myc–Snu66-tADH S. cerevisiae SNU66 gene under GAL promoter with 3MYC tag at the N-terminus and ADH terminator This study D909 YE112 pADH-3myc–snu66-RRAA-tADH R16A, R47A mutation pGAL 3MYC–snu66 tADH This study D605 YC333 pHUB1-Hub1-tADH S. cerevisiae HUB1 gene under its own promoter and terminator This study D1382 YC333 pHUB1-Hub1(R9A)-tADH R9A mutation pHUB1-Hub1-tHUB1 This study D1383 YC333 pHUB1-Hub1(K13A)-tADH K13A mutation pHUB1-Hub1-tHUB1 This study D1384 YC333 pHUB1-Hub1(R9A, K13A)-tADH R9A, K13A mutation pHUB1-Hub1-tHUB1 This study D739 YC333 pHUB1-Hub1(D22A)-tADH D22A mutation pHUB1-Hub1-tHUB1 This study D740 YC333 pHUB1-Hub1(H63L)-tADH H63L mutation pHUB1-Hub1-tHUB1 This study 3743 PRP8+ WT TRP CEN PRP8 with its own promoter and terminator This study D1421 prp8-161 prp8 (P986T) TRP CEN prp8 (P986T) with its own promoter and terminator This study D1422 prp8* prp8 (P1384L) TRP CEN prp8 (P1384L) with its own promoter and terminator This study D1423 syf77 prp8 (L1577F) TRP CEN prp8 (L1577F) with its own promoter and terminator This study D1424 prp8-107 prp8 (F1834L) TRP CEN prp8 (F1834L) with its own promoter and terminator This study D1425 prp8-cat prp8 (F1851L) TRP CEN prp8 (F1851L) with its own promoter and terminator This study D1426 prp8-cat prp8 (V1860D) TRP CEN prp8 (V1860D) with its own promoter and terminator This study D1427 prp8-201 prp8 (T1861P) TRP CEN prp8 (T1861P) with its own promoter and terminator This study D1428 prp8-cat prp8 (V1862D) TRP CEN prp8 (V1862D) with its own promoter and terminator This study D1429 prp8-D143 prp8 (K1864E) TRP CEN prp8 (K1864E) with its own promoter and terminator This study D1430 prp8 (T1865K) TRP CEN prp8 (T1865K) with its own promoter and terminator This study D1431 prp8-162 prp8 (V1870N) TRP CEN prp8 (V1870N) with its own promoter and terminator This study 3745 prp8-101 prp8 (E1960K) TRP CEN prp8 (E1960K) with its own promoter and terminator D1432 prp8-153 prp8 (E1982A) TRP CEN prp8 (E1982A) with its own promoter and terminator This study D1433 prp8-161, prp8-101 prp8 (P986T) TRP CEN prp8 (P986T, E1960K) with its own promoter and terminator This study D1434 prp8-162 prp8 (V1870N) TRP CEN prp8 (F1834L, E1960K) with its own promoter and terminator This study D1435 YC111 pGAL- SNU17 -tADH S. cerevisiae SNU17 gene under GAL promoter and ADH terminator This study D1436 YC111 pGAL- ECM2 -tADH S. cerevisiae ECM2 gene under GAL promoter and ADH terminator This study D1437 YC111 pGAL- URN1 -tADH S. cerevisiae URN1 gene under GAL promoter and ADH terminator This study D1438 U1•{WT} pYE195 SNR19-WT + S. cerevisiae SNR19-WT+ gene with its own promoter(1000bp) and terminator(310bp) This study D1439 U1•{A7G} pYE195 snr19 (A7G) S. cerevisiae snr19 (A7G) gene with its own promoter and terminator This study D1440 U5•{WT} pYE195 SNR7-L-WT + S. cerevisiae SNR 7-L-WT+ gene with its own promoter(100bp) and terminator(294bp) This study D1441 U5•{U(98)C} pYE195 snr7-L(U98C) S. cerevisiae snr7-L (U98C) gene with its own promoter and terminator This study D1442 U5•{U(98)G} pYE195 snr7-L(U98G) S. cerevisiae snr7-L (U98G) gene with its own promoter and terminator This study D1443 U5•{U(98)A} pYE195 snr7-L(U98A) S. cerevisiae snr7-L (U98A) gene with its own promoter and terminator This study D1444 U6•{WT} pYE195 SNR6-WT + S. cerevisiae SNR6-WT+ gene with its own promoter(1000bp) and terminator(374bp) This study D1445 U6•{A51G} pYE195 snr6(A51G) S. cerevisiae snr6 (A51G) gene with its own promoter and terminator This study D1446 U6•{A51U} pYE195 snr6(A51U) S. cerevisiae snr6 (A51U) gene with its own promoter and terminator This study D1447 U6•{G50U} pYE195 snr6(G50U) S. cerevisiae snr6 (G50U) gene with its own promoter and terminator This study D1448 U6•{G50C} pYE195 snr6(G50C) S. cerevisiae snr6 (G50C) gene with its own promoter and terminator This study D1449 U6•{A49U} pYE195 snr6(A49U) S. cerevisiae snr6 (A49U) gene with its own promoter and terminator This study D1450 U6•{U46C} pYE195 snr6(U46C) S. cerevisiae snr6 (U46C) gene with its own promoter and terminator This study D1451 U6•{A45U} pYE195 snr6(A45U) S. cerevisiae snr6 (A45U) gene with its own promoter and terminator This study D1452 U6•{A45G} pYE195 snr6(A45G) S. cerevisiae snr6 (A45G) gene with its own promoter and terminator This study D1453 U6•{T46C, A45T} pYE195 snr6(T46C, A45T) S. cerevisiae snr6 (T46C, A45T) gene with its own promoter and terminator This study Table 2: Yeast strains used in this study. Strain Genotype Ref. YJU75 (SC11) MATa ade2 cup1 D ::ura3 his3 leu2 lys2 prp8 D ::LYS2 trp1; pJU169 (PRP8 URA3 CEN ARS) Gift from C. Guthrie SC87 YJU75 hub1:: KanMX6 This study SC196 YJU75 PRP8 TRP This study SC197 YJU75 prp8-101 TRP This study SC198 YJU75 PRP8 TRP hub1:: KanMX6 This study SC200 YJU75 P CUP1-1 TAP–SRC1::natNT2 This study SC214 YJU75 prp8-101P CUP1-1 TAP–SRC1::natNT2 This study SC224 YJU75 hub1::KanMX6 P CUP1-1 TAP–SRC1::natNT2 This study SC297 SC200 nam8:: KanMX6 This study SC321 SC11 . Dib1 – hub1(D22A) :: natNT2 This study SC322 SC11 . Prp31 – hub1(D22A) :: natNT2 This study SC323 SC11 . Sad1 – hub1(D22A) :: natNT2 This study SC324 SC87 . Dib1 – hub1(D22A) :: natNT2 This study SC325 SC87 . Prp31 – hub1(D22A) :: natNT2 This study SC326 SC87 . Sad1 – hub1(D22A) :: natNT2 This study BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 This study YSKM601 BY4741 hub1 ::KanMX6 P CUP1-1 TAP–SRC1::natNT2 This study YSKM602 BY4741 lea1::KanMX6 P CUP1-1 TAP–SRC1::natNT2 This study YSKM603 BY4741 ecm2::KanMX6 P CUP1-1 TAP–SRC1::natNT2 This study YSKM604 BY4741 bud13::KanMX6 P CUP1-1 TAP–SRC1::natNT2 This study YSKM605 BY4741 nup60::KanMX6 P CUP1-1 TAP–SRC1::natNT2 This study YSKM606 BY4741 lsm1::KanMX6 P CUP1-1 TAP–SRC1::natNT2 This study YSKM607 BY4741 sky1::KanMX6 P CUP1-1 TAP–SRC1::natNT2 This study YSKM608 BY4741 snu17::KanMX6 P CUP1-1 TAP–SRC1::natNT2 This study YSKM609 BY4741 pml1::KanMX6 P CUP1-1 TAP–SRC1::natNT2 This study YSKM610 BY4741 urn1::KanMX6 P CUP1-1 TAP–SRC1::natNT2 This study YSKM611 BY4741 mlp2::KanMX6 P CUP1-1 TAP–SRC1::natNT2 This study YSKM612 BY4741 mlp1::KanMX6 P CUP1-1 TAP–SRC1::natNT2 This study YSKM613 BY4741 pml39::KanMX6 P CUP1-1 TAP–SRC1::natNT2 This study YSKM614 BY4741 brr1::KanMX6 P CUP1-1 TAP–SRC1::natNT2 This study YSKM615 BY4741 mud2::KanMX6 P CUP1-1 TAP–SRC1::natNT2 This study YSKM616 BY4741 mud1::KanMX6 P CUP1-1 TAP–SRC1::natNT2 This study YSKM617 BY4741 prp17::KanMX6 P CUP1-1 TAP–SRC1::natNT2 This study YSKM618 BY4741 npl3::KanMX6 P CUP1-1 TAP–SRC1::natNT2 This study YSKM619 BY4741 cwc15::KanMX6 P CUP1-1 TAP–SRC1::natNT2 This study YSKM620 BY4741 cwc21::KanMX6 P CUP1-1 TAP–SRC1::natNT2 This study YSKM621 BY4741 cwc27::KanMX6 P CUP1-1 TAP–SRC1::natNT2 This study YSKM622 BY4741 ntc20::KanMX6 P CUP1-1 TAP–SRC1::natNT2 This study YSKM623 BY4741 ntc30::KanMX6 P CUP1-1 TAP–SRC1::natNT2 This study YSKM624 BY4741 ntc31::KanMX6 P CUP1-1 TAP–SRC1::natNT2 This study W303 ade2-1 his3-11, 15 leu2-3, 112 ura3 trp1-1 ssd1 can1-100 Gift from K. Nasmyth YSKM625 W303a P CUP1-1 TAP–SRC1::KanMX6 YSKM626 W303a P CUP1-1 TAP–SRC1::KAN Prp8–hub1(D22A) This study YSKM627 W303a P CUP1- 1 TAP– SRC1::KAN Snu66 – hub1(D22A) This study YSKM628 W303a P CUP1- 1 TAP– SRC1::KAN Prp38 – hub1(D22A) This study YSKM629 W303a P CUP1- 1 TAP– SRC1::KAN Snu66 – hub1(D22A) Prp38 – hub1(D22A) This study YSKM630 W303a hub1::HIS3MX6 P CUP1-1 TAP–SRC1::KanMX6 SNU17– hub1(D22A) :: natNT2 This study YSKM631 W303a hub1::HIS3MX6 P CUP1-1 TAP–SRC1::KanMX6 PRP4– hub1(D22A) :: natNT2 This study YSKM632 W303a hub1::HIS3MX6 P CUP1-1 TAP–SRC1::KanMX6 BRR2– hub1(D22A) :: natNT2 This study YSKM633 W303a hub1::HIS3MX6 P CUP1-1 TAP–SRC1::KanMX6 PRP6– hub1(D22A) :: natNT2 This study YSKM634 W303a hub1::HIS3MX6 P CUP1-1 TAP–SRC1::KanMX6 PRP3– hub1(D22A) :: natNT2 This study YSKM635 W303a hub1::HIS3MX6 P CUP1-1 TAP–SRC1::KanMX6 LEA1– hub1(D22A) :: natNT2 This study YSKM636 W303a hub1::HIS3MX6 P CUP1-1 TAP–SRC1::KanMX6 PRP9– hub1(D22A) :: natNT2 This study YSKM637 W303a hub1::HIS3MX6 P CUP1-1 TAP–SRC1::KanMX6 PRP11– hub1(D22A) :: natNT2 This study YSKM638 W303a hub1::HIS3MX6 P CUP1-1 TAP–SRC1::KanMX6 MUD1– hub1(D22A) :: natNT2 This study YSKM639 W303a hub1::HIS3MX6 P CUP1-1 TAP–SRC1::KanMX6 NAM8– hub1(D22A) :: natNT2 This study SC186 W303a snu66::HIS3MX6 This study Table 3: Splicing reporters used in this study. Splicing Reporters Description ACT1 – CUP1 reporter 5’SS GUAUGU (Gift from C. Guthrie) 5’SS GCAUGU (D1454) 5’SS GCAAGU (D1455) HEH1L – CUP1 reporter ACT1 exon1-intron-exon2 in ACT1 – CUP1 reporter is replaced with HEH1 exon1-intron-exon2 (nucleotides 1874-2094) to keep HEH1L 5’SS GUGAGU in frame with CUP1. Its variants were made using SDM. 5’SS GUGAGU AC (D1456) 5’SS GUGAGA AC (D1457) 5’SS GUAUAU AC (D1458) 5’SS GUAUAA AC (D1459) 5’SS GUAUGU AC (D1460) HEH1S – CUP1 reporter ACT1 exon1-intron-exon2 in ACT1 – CUP1 reporter is replaced with HEH1 exon1-intron-exon2 (nucleotide 1874-2095) to keep HEH1S 5’SS GCAAGU in frame with CUP1. Its variants were made using SDM. 5’SS GCAAGU GUGAGUAC (D1461) 5’SS GCAAGU GUGAGAAC (D1462) 5’SS GCAACU GUAUAUAC (D1463) 5’SS GCAAGU GUAUAAAC (D1464) 5’SS GCAAGU GUAUGUAC (D1465) HEH1L – LACZ reporter RP51 exon1-intron-exon2 is replaced with HEH1 exon1-intron-exon2 (nucleotide 1792-2190) is fused with the LACZ gene to keep HEH1L 5’SS GUGAGU in frame with LACZ. HEH1S – LACZ reporter RP51 exon1-intron-exon2 is replaced with HEH1 exon1-intron-exon2 (nucleotide 1792-2191) is fused with the LACZ gene to keep HEH1S 5’SS GCAAGU in frame with LACZ. Additional Declarations There is NO Competing Interest. Supplementary Files ExtendedData.docx Extended Data Figures Cite Share Download PDF Status: Published Journal Publication published 16 Dec, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7007499","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":481908363,"identity":"4f1361a3-ca19-4341-b279-c06fc195e148","order_by":0,"name":"Ankita Katoch Banyal","email":"","orcid":"","institution":"Indian Institute of Science Education and Research Mohali","correspondingAuthor":false,"prefix":"","firstName":"Ankita","middleName":"Katoch","lastName":"Banyal","suffix":""},{"id":481908364,"identity":"5b982321-af49-43c2-89e6-3367af23b402","order_by":1,"name":"Poulami Choudhuri","email":"","orcid":"","institution":"Indian Institute of Science Education and Research Mohali","correspondingAuthor":false,"prefix":"","firstName":"Poulami","middleName":"","lastName":"Choudhuri","suffix":""},{"id":481908365,"identity":"3595bdaf-15b2-4602-b327-58d63ff9ce18","order_by":2,"name":"Balashankar R. Pillai","email":"","orcid":"","institution":"Indian Institute of Science Education and Research Mohali","correspondingAuthor":false,"prefix":"","firstName":"Balashankar","middleName":"R.","lastName":"Pillai","suffix":""},{"id":481908366,"identity":"00b77513-bdd8-4d5a-87b4-796490107bae","order_by":3,"name":"Amjadudheen Varikkapulakkal","email":"","orcid":"","institution":"Indian Institute of Science Education and Research Mohali","correspondingAuthor":false,"prefix":"","firstName":"Amjadudheen","middleName":"","lastName":"Varikkapulakkal","suffix":""},{"id":481908362,"identity":"6cdb9fe3-284a-4378-8ee1-3c28ac7e965b","order_by":4,"name":"Shravan Kumar Mishra","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABD0lEQVRIiWNgGAWjYDACHgaGA4wNDAz8zCASCAwYmBsOgBhshLRINsO1MBLWwgBSbHAAKmDAANWLC8j3nE48+HPHPXnj48zNHz7u2SZnzn6w8QBDjR0DnzR2rQZnezcc5j1TbLjtMGOb5Ixnt40texKBDjuWzMAmcwC7Fn7eDUDFCYwgLcw8B24nbjgA0sIGRBIJ2B3Wz7vh4M+2BPvNzYzNn/+AtJx/CNTyD7cWBqDDDvC2JSRuAAayNANIyw2gLYxtuLUYnDkL9EtbQvIMkF96Dtw2NrgBtCWxL5kHp8N6cjd/BDrMtr//+OMPPw7cljM4n3z4w4dvdnLyM3A4DDtIgETYKBgFo2AUjAIyAQA63GwDnzQHxQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-3899-0495","institution":"Indian Institute of Science Education and Research Mohali","correspondingAuthor":true,"prefix":"","firstName":"Shravan","middleName":"Kumar","lastName":"Mishra","suffix":""}],"badges":[],"createdAt":"2025-06-30 07:30:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7007499/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7007499/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-67659-8","type":"published","date":"2025-12-16T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87002072,"identity":"ada8e0c3-a380-4917-9087-87537fddff7d","added_by":"auto","created_at":"2025-07-18 07:30:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4584557,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eHEH1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e alternative splicing needs B and Bact proteins.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e The abundance of Heh1 L and S protein isoforms in the mutants of the indicated spliceosomal proteins was monitored by western blots. Data shown as mean ± s.d. (n=3 independent replicates). Heh1 isoforms in wt yeast (labelled as ‘Positive’) are used as the reference for alternative splicing. \u003cstrong\u003e(b) \u003c/strong\u003eRT-PCR assays to monitor of \u003cem\u003eHEH1\u003c/em\u003e pre-mRNA splicing in deletions and mutant strains of indicated splicing factors. \u003cem\u003ehub1\u003c/em\u003e, \u003cem\u003esnu17\u003c/em\u003e, \u003cem\u003eecm2\u003c/em\u003e, and \u003cem\u003eurn1\u003c/em\u003e mutant strains are respective gene knockouts. \u003cem\u003esnu66-AA \u003c/em\u003emutant refers to R16A and R47A variant of Snu66, its Hub1-binding-deficient HIND mutant. \u003cem\u003eprp8-101 \u003c/em\u003eis the E1960K allele of Prp8. The mutants were identified from different targeted screens shown in Figure S1a-f, h-j, and k-l. \u003cstrong\u003e(c) \u003c/strong\u003eSpliced cDNA bands were purified from mutants in (b) and sequenced to estimate the extent of \u003cem\u003eHEH1\u003c/em\u003e alternative splicing from the area under peaks of isoform-specific nucleotides. The values for UU\u003cu\u003eA\u003c/u\u003eUCACC indicate the relative abundance of \u003cem\u003eHEH1S\u003c/em\u003e, and for GC\u003cu\u003eA\u003c/u\u003eAUUAU shows the relative abundance of \u003cem\u003eHEH1L\u003c/em\u003e. Peak for the underlined ‘A’, being common to both isoforms, were omitted from the quantitation. The values on the right of the peaks indicate relative % abundance of respective mRNA isoforms. \u003cstrong\u003e(d) \u003c/strong\u003eRelative abundance of \u003cem\u003eHEH1L\u003c/em\u003e mRNA in different strains was estimated from cDNA electropherogram in (c). \u003cstrong\u003e(e)\u003c/strong\u003e Splicing factors from distinct spliceosomal complexes were probed by linear chromosomal fusions of a Snu66-binding-deficient \u003cem\u003ehub1(D22A)\u003c/em\u003e mutant, indicated with ‘\u003cstrong\u003e○\u003c/strong\u003e’, at the C-termini of respective splicing factors. Rescue of alternative splicing defects (gain of Heh1S protein) following \u003cem\u003ehub1(D22A)\u003c/em\u003e fusion in the \u003cem\u003e∆hub1\u003c/em\u003e background was monitored in western blots. Upward triangle (▲) marks \u003cem\u003eDhub1\u003c/em\u003e strain harboring splicing factor–\u003cem\u003ehub1(D22A) \u003c/em\u003efusions, where Heh1S protein were restored. Note that the otherwise free \u003cem\u003ehub1(D22A)\u003c/em\u003e is deficient in \u003cem\u003eHEH1\u003c/em\u003e alternative splicing\u003csup\u003e14\u003c/sup\u003e (also see Extended Data Fig.1h).\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7007499/v1/040e0c3a301200dc318e98fe.png"},{"id":87002075,"identity":"5c44d620-df10-4257-a20c-04cdea461985","added_by":"auto","created_at":"2025-07-18 07:30:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4223060,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSequence at the exon-intron boundary determines alternative 5’SS selection.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eThe 16 nucleotides segment at the exon/intron (1913-1926) boundary of \u003cem\u003eHEH1\u003c/em\u003e was investigated for alternative splicing by mutagenesis. The sequence was selected based on reporter assays described earlier, pairing of U5 snRNA loop1 and U6 snRNA ACAGA box. \u003cem\u003eHEH1S\u003c/em\u003e (in orange color) and \u003cem\u003eHEH1L \u003c/em\u003e(in red color) sequences represent isoforms of \u003cem\u003eHEH1 \u003c/em\u003epre-mRNA. \u003cstrong\u003e(b) \u003c/strong\u003e\u003cem\u003eHEH1\u003c/em\u003e gene was mutated by site-directed mutagenesis sequentially from left to right (except for the essential ‘G’ of the splicing donors) and alternative selection was monitored by western blot. Data shown as mean ± s.d. (n=3 independent repeats). \u003cstrong\u003e(c)\u003c/strong\u003e Analysis of pre-mRNA variants paring with U5 snRNA (in blue color) and U6 snRNA (in green color) for both \u003cem\u003eHEH1S\u003c/em\u003e and \u003cem\u003eHEH1L\u003c/em\u003e was tabulated. The mutants were analyzed for presumed U5 and U6 snRNA pairings adapted from\u003csup\u003e60,61\u003c/sup\u003e. Western blot lanes are taken from (b). The number in the left column shows variants ID. Resulting changes for \u003cem\u003eHEH1S\u003c/em\u003e and \u003cem\u003eHEH1L\u003c/em\u003e are shown in curly brackets {}. Filled dots (●) shows non-Watson-Crick pairing, standing lines (\u003cstrong\u003eı\u003c/strong\u003e) represent Watson-Crick pairing, upward arrows (↑) mean increase in U5 / U6 pairing compared to WT \u003cem\u003eHEH1S \u003c/em\u003eor \u003cem\u003eHEH1L\u003c/em\u003e, downward arrows (↓) show decrease in U5 / U6 pairing compared to WT, and double upward arrows (↑↑) represent the yeast canonical 5’SS (GUAUGU or GUAAGU. The variants leading to premature stop codons, indicated by asterisk (*), could be analysed only by cDNA sequencing. The values on the right of the peaks indicate relative % abundance of respective mRNA isoforms.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7007499/v1/bf2286f118a3ba4ad3e81b94.png"},{"id":87002079,"identity":"fbcc203f-8c8b-4055-9ead-fe8622732750","added_by":"auto","created_at":"2025-07-18 07:30:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4202931,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRole of U5/U6 pairing in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eHEH1 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ealternative splicing.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eThe pairing of U5 snRNA and its variant U5•{U(98)C} and U6 snRNA and its variant U6•{A(45)U,U(46)C} to \u003cem\u003eHEH1\u003c/em\u003e pre-mRNA. The snRNA variants were identified (Extended Data Fig. 3a-b and 3e-f) by mutagenesis of U5 Loop1 and U6 ACAGA box. Up/down arrows (↑/↓) refer to an increase/decrease in U5/U6 pairing strengths to\u003cem\u003e HEH1\u003c/em\u003eisoforms. \u003cem\u003eHEH1\u003c/em\u003e mRNA and proteins were assayed after overexpressing variants of U5 and U6 snRNAs from multicopy plasmids. \u003cstrong\u003e(b) \u003c/strong\u003e\u003cem\u003eHEH1\u003c/em\u003e cDNAs from yeast strains overexpressing U5 and U6 snRNAs and their variants were sequenced to monitor alternative 5’SS selection. Area under the peak was integrated (similar to Figure 1) the find the relative abundance of the two mRNA isoforms. The values on the right of the peaks indicate relative % abundance of respective mRNA isoforms. \u003cstrong\u003e(c) \u003c/strong\u003eThe expression of Heh1 protein isoforms upon overexpression of U5 and U6 snRNA variants monitored in different mutants by western blots. Heh1 isoforms in wt yeast (labelled as ‘Positive’) are used as the reference for alternative splicing. Data shown as mean ± s.d. (n=3 independent replicates). An orange upward triangle (▲) indicates snRNA overexpression, resulting in a relatively higher amount of Heh1S protein.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7007499/v1/aef3aa685b2e2049ccc37e6c.png"},{"id":87002601,"identity":"f8c13ae8-226c-4b45-bd7b-d67dd7649f83","added_by":"auto","created_at":"2025-07-18 07:38:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4110728,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe protein factors are dispensable for alternative splicing from specific 5’SS variants.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003ePairing of U5 and U6 snRNAs to \u003cem\u003eHEH1\u003c/em\u003e \u003cem\u003eWT\u003c/em\u003e pre-mRNA and its variants #7 \u003cem\u003eHEH1S\u003c/em\u003e•{A(+4)U} or \u003cem\u003eHEH1L\u003c/em\u003e•{A(−1)U}; #15 {\u003cem\u003eHEH1S\u003c/em\u003e•{G(+7)A,A(+8)U,G(+9)A} or \u003cem\u003eHEH1L\u003c/em\u003e•{G(+3)A,A(+4)U,G(+5)A}; and #18 \u003cem\u003eHEH1S\u003c/em\u003e•{C(+2)U,A(+4)U,G(+7)A,A(+8)U} or \u003cem\u003eHEH1L\u003c/em\u003e•{C(−3)U,A(−1)U,G(+3)A,A(+4)U}. \u003cstrong\u003e(b) \u003c/strong\u003eAlternative splicing of \u003cem\u003eHEH1\u003c/em\u003e variants #7, 15, and 18 was analyzed in different mutant strains by western blot. Partial rescue Heh1S in prp8-101 strain is shown in dotted box. Data shown as mean ± s.d. (n=3 independent repeats). \u003cstrong\u003e(c) \u003c/strong\u003e\u003cem\u003eACT1\u003c/em\u003e–\u003cem\u003eCUP1\u003c/em\u003e reporter with a single 5’SS was used to understand the exclusive production of Heh1S from #7 {A(+4)U}. U5 and U6 snRNAs pairing to \u003cem\u003eHEH1S\u003c/em\u003e 5’SS GCAAGU, GCA\u003cu\u003eU\u003c/u\u003eGU and G\u003cu\u003eU\u003c/u\u003eA\u003cu\u003eU\u003c/u\u003eGU were compared by the reporter assay. The 5’SS mutants were analyzed by growth at indicated CuSO\u003csub\u003e4\u003c/sub\u003e concentrations (in mM), which correlates with their splicing efficiency.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7007499/v1/c64ae7fd7136ac466ef6530d.png"},{"id":87002074,"identity":"04c4e0f6-bc2b-4cb2-9750-e69565667995","added_by":"auto","created_at":"2025-07-18 07:30:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2594167,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e5’SS competition versus U6 snRNA pairing.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003ePairing of U6 snRNA with \u003cem\u003eHEH1\u003c/em\u003e \u003cem\u003eWT\u003c/em\u003e pre-mRNA\u003cem\u003e, \u003c/em\u003evariant #10 \u003cem\u003eHEH1S\u003c/em\u003e•{U(+10)A} or \u003cem\u003eHEH1L\u003c/em\u003e•{U(+6)A}; #15 {\u003cem\u003eHEH1S\u003c/em\u003e•{G(+7)A,A(+8)U,G(+9)A} or \u003cem\u003eHEH1L\u003c/em\u003e•{G(+3)A,A(+4)U,G(+5)A}; and #20 {\u003cem\u003eHEH1S\u003c/em\u003e•{G(+7)A,A(+8)U,G(+9)A,U(+10)A} or \u003cem\u003eHEH1L\u003c/em\u003e•{G(+3)A,A(+4)U,G(+5)A,U(+6)A}. Up/down arrows (↑/↓) indicate increase/decrease in U6 pairing and competition among the two sites. \u003cstrong\u003e(b) \u003c/strong\u003e\u003cem\u003eHEH1\u003c/em\u003e–\u003cem\u003eCUP1\u003c/em\u003e alternative splicing reporters made by replacing \u003cem\u003eACT1\u003c/em\u003e part with\u003cem\u003e HEH1\u003c/em\u003e segment in \u003cem\u003eACT1\u003c/em\u003e–\u003cem\u003eCUP1\u003c/em\u003e reporter. In \u003cem\u003eHEH1S\u003c/em\u003e–\u003cem\u003eCUP1, HEH1S \u003c/em\u003emRNA\u003cem\u003e \u003c/em\u003eis in frame with\u003cem\u003e CUP1\u003c/em\u003e, and in\u003cem\u003e HEH1L\u003c/em\u003e–\u003cem\u003eCUP1, HEH1L \u003c/em\u003emRNA\u003cem\u003e \u003c/em\u003eis in frame with\u003cem\u003e CUP1.\u003c/em\u003e The sequence used was from 1874-2095 for \u003cem\u003eHEH1S\u003c/em\u003e–\u003cem\u003eCUP1\u003c/em\u003e and 1874-2094 for \u003cem\u003eHEH1L\u003c/em\u003e–\u003cem\u003eCUP1\u003c/em\u003e. \u003cstrong\u003e(c)\u003c/strong\u003e Reporter activities were tested at different\u003cstrong\u003e \u003c/strong\u003eCuSO\u003csub\u003e4\u003c/sub\u003e concentrations. (◄) The left pointed triangle denotes the abolished \u003cem\u003eHEH1L \u003c/em\u003e5’SS in #10 and weakened site in #20. Mutated \u003cem\u003eHEH1L\u003c/em\u003e 5’SS do not compete with the \u003cem\u003eHEH1S\u003c/em\u003e site. \u003cstrong\u003e(d) \u003c/strong\u003eThe \u003cem\u003eHEH1S\u003c/em\u003e–\u003cem\u003eCUP1 \u003c/em\u003ereporter variants were tested for their splicing efficiency by yeast growth at different CuSO\u003csub\u003e4 \u003c/sub\u003econcentrations. Loss of competition restored splicing from \u003cem\u003eHEH1S\u003c/em\u003e 5’SS in \u003cem\u003esnu17\u003c/em\u003e, \u003cem\u003eecm2\u003c/em\u003e and \u003cem\u003eurn1\u003c/em\u003e mutants, but not in \u003cem\u003ehub1\u003c/em\u003e and \u003cem\u003eprp8-101\u003c/em\u003e mutants. Gain of U6 snRNA pairing at +7 and +8 positions of \u003cem\u003eHEH1S\u003c/em\u003e 5’SS restored splicing defects fully in most mutant strains and partially in \u003cem\u003eprp8-101\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-7007499/v1/04324e984b83705d47d618e0.png"},{"id":87002602,"identity":"7555ce35-48e5-4b4e-a35f-b8b0aa8e40e0","added_by":"auto","created_at":"2025-07-18 07:38:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2248586,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlausible mechanism of alternative 5’SS selection.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e The schematics show changes from B to Bact complex during selection of the alternative\u003cem\u003e HEH1\u003c/em\u003e 5’SS. Snu66 escorts Hub1 to the spliceosome\u003csup\u003e14\u003c/sup\u003e towards the 5’ exon in the early B complex, and hands over to the Spp381/MFAP1 (a subunit of Prp38 complex)\u003csup\u003e48,52 \u003c/sup\u003ein the late B complex. Thus, Hub1 binding to Snu66 and Spp381/MFAP1 may be mutually exclusive. Hub1 also interacts with the 5’ exon at the exon-intron boundary. The tri-snRNP factors Prp6, Prp3 and Prp31 are present throughout the B complex. They likely stabilise the U4/U6 snRNA duplex and prevent its premature unbinding\u003csup\u003e62\u003c/sup\u003e. Prp8 converts from a partially open conformation to a closed state during transition from B to Bact state\u003csup\u003e45,60\u003c/sup\u003e. The changes in Prp8 RH domain β-hairpin to loop conformation orchestrate the release of tri-snRNP factors and incorporation of NTC into the spliceosome\u003csup\u003e43,60\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-7007499/v1/a0434a32209e5ad7d088e63d.png"},{"id":101032194,"identity":"0da2ec4a-7925-4f64-971a-d76ded1b022a","added_by":"auto","created_at":"2026-01-24 08:07:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":28279306,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7007499/v1/9c9f5a2e-ad94-4255-bf70-fb7b4d2ab06a.pdf"},{"id":87002080,"identity":"919c320a-f438-4186-a583-78732c8b2663","added_by":"auto","created_at":"2025-07-18 07:30:26","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6071731,"visible":true,"origin":"","legend":"Extended Data Figures","description":"","filename":"ExtendedData.docx","url":"https://assets-eu.researchsquare.com/files/rs-7007499/v1/8b5f59a5ca9c57d3efb29bc9.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eMechanism of alternative splicing through competing 5’ splice sites\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eSplicing of precursor messenger RNAs (pre-mRNA) occurs through a multi-step process involving large and dynamic ribonucleoprotein (RNP) complexes called spliceosomes. Stage-specific spliceosomes are assembled on a pre-mRNA following intricate rearrangements of five distinct snRNPs containing cognate small nuclear RNAs (snRNA) and associated proteins. Specific snRNPs recognise splicing signals in pre-mRNAs, including the donor 5\u0026rsquo; splice site (5\u0026rsquo;SS) \u0026lsquo;GU\u0026rsquo;, the acceptor 3\u0026rsquo; splice site (3\u0026rsquo;SS) \u0026lsquo;AG\u0026rsquo;, and the branchpoint adenosine \u0026lsquo;A\u0026rsquo;. Two steps of transesterification reactions, catalysed by divalent metal ions and U6 snRNA in the spliceosomes, remove introns and join exons to form functional mRNA\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The spliceosome machinery is essential for constitutive and alternative splicing.\u003c/p\u003e\u003cp\u003eMore mRNAs from a gene through can be produced through alternative splicing. The process amplifies protein diversity from limited gene pools in an organism\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Different classes of alternative splicing exist in eukaryotes. Key among them occurs through alternative selection of competing 5\u0026rsquo;SS\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e and 3\u0026rsquo;SS\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, and alternative splicing through competing 5\u0026rsquo;SS is a prevalent mode. A majority of pre-mRNAs in the budding yeast \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e have the highly conserved GUAUGU hexanucleotide as 5\u0026rsquo;SS donors\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, but the sequences are highly variable in intron-rich eukaryotes, where alternative 5\u0026rsquo;SS selection is widespread\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Two major classes of 5\u0026rsquo;SS (NN/GURAG and AG/GUNNN; \u0026lsquo;/\u0026rsquo; indicates exon-intron junction), distinguished by the presence of \u0026lsquo;G\u0026rsquo; at -1 and +\u0026thinsp;5 positions, have been reported\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Alternative 5\u0026rsquo;SS are often found 4 nucleotides across the dominant 5\u0026rsquo;SS, and such occurrences in the human genome are estimated to be large\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The canonical \u0026lsquo;GU\u0026rsquo; dinucleotide deviates to \u0026lsquo;GC\u0026rsquo; in 0.87% of the 5\u0026rsquo;SS\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eHEH1\u003c/em\u003e gene (also called \u003cem\u003eSRC1\u003c/em\u003e) in \u003cem\u003eS. cerevisiae\u003c/em\u003e is alternatively spliced\u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Its pre-mRNA has alternative donors with \u0026lsquo;GC\u0026rsquo; and \u0026lsquo;GU\u0026rsquo; dinucleotides embedded into two non-canonical 5\u0026rsquo;SS, /\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGC\u003c/span\u003eAAGU and /\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGU\u003c/span\u003eGAGU, arranged as a partially overlapping sequence /\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGC\u003c/span\u003eAA/\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGU\u003c/span\u003eGAGU. /\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGU\u003c/span\u003eGAGU is the dominant 5\u0026rsquo;SS and splicing through this site generates mRNA-encoding a longer full-length protein, Heh1L. Splicing through the upstream /\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGC\u003c/span\u003eAAGU is tightly regulated and produces mRNA-encoding a shorter protein, Heh1S (the removal of GCAA tetranucleotide gains an in-frame stop codon early in \u003cem\u003eHEH1S\u003c/em\u003e mRNA). The two proteins share a common N-terminus but differ in their C-termini, thus attaining different topologies within the inner nuclear membrane and have distinct functions\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e5\u0026rsquo;SS selection relies on early pre-mRNA recognition by trans-acting U1 snRNA and associated proteins\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The role of specific RBPs (RNA-binding proteins), SR (serine/arginine-rich) proteins, and hnRNPs (heterogeneous nuclear ribonucleoproteins) has also been reported to be crucial for 5\u0026rsquo;SS selection\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. These proteins act as splicing enhancers and silencers, and regulate spliceosome assembly on pre-mRNA targets in association with the U1 snRNP. The role of successive spliceosomal complexes in 5\u0026rsquo;SS selection has been recently reported\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eHEH1\u003c/em\u003e gene is an interesting tool for exploring RNA and protein factors needed for alternative splicing through competing and overlapping 5\u0026rsquo;SS. Its alternative splicing requires non-covalent associations of the ubiquitin-like protein Hub1 (UBL5 in humans) with the U1 and U2 snRNP-bridging RNA helicase Prp5\u003csup\u003e27\u003c/sup\u003e, and the U4/U6.U5 tri-snRNP protein Snu66\u003csup\u003e14\u003c/sup\u003e. Thus, Hub1 acts early in spliceosome assembly by activating the RNA helicase, as well as later with the tri-snRNP component\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The latter activity suggests a possible role of the spliceosome core in \u003cem\u003eHEH1\u003c/em\u003e 5\u0026rsquo;SS selection. We find that \u003cem\u003eHEH1\u003c/em\u003e alternative splicing required optimal pairing of U5 and U6 snRNAs across a sixteen-nucleotide segment of the pre-mRNA across the two 5\u0026rsquo;SS at the exon1-intron boundary. The selection of the competing 5\u0026rsquo;SS by U5 and U6 snRNAs and alternative splicing was supported by specific proteins of the spliceosome core, including Prp8.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eAdditional trans-acting proteins for \u003cem\u003eHEH1\u0026nbsp;\u003c/em\u003ealternative splicing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBetween to two 5\u0026rsquo;SS of\u0026nbsp;\u003cem\u003eSRC1/HEH1\u003c/em\u003e pre-mRNA /\u003cu\u003eGC\u003c/u\u003eAA/\u003cu\u003eGU\u003c/u\u003eGAGU, the usage of the upstream 5\u0026rsquo;SS /\u003cu\u003eGC\u003c/u\u003eAAGU requires non-covalent binding of the ubiquitin-like protein Hub1 to Snu66. The downstream 5\u0026rsquo;SS /\u003cu\u003eGU\u003c/u\u003eGAGU was dominant and selected independently of this complex\u003csup\u003e14\u003c/sup\u003e. We searched for additional\u0026nbsp;\u003cem\u003eHEH1\u003c/em\u003e alternative splicing factors in \u003cem\u003eS. cerevisiae\u003c/em\u003e through the following approaches.\u003c/p\u003e\n\u003cp\u003e(i) \u003cem\u003eHEH1\u003c/em\u003e\u003cem\u003e\u0026ndash;LACZ\u003c/em\u003e alternative splicing reporters\u0026nbsp;\u003cem\u003eHEH1S\u003c/em\u003e\u0026ndash;\u003cem\u003eLACZ\u003c/em\u003e and \u003cem\u003eHEH1L\u003c/em\u003e\u0026ndash;\u003cem\u003eLACZ\u003c/em\u003e were prepared (Extended Data Fig. 1a),\u003cem\u003e\u0026nbsp;\u003c/em\u003eon\u0026nbsp;the line of \u003cem\u003eRP51\u003c/em\u003e\u003cem\u003e\u0026ndash;\u003c/em\u003e\u003cem\u003eLACZ\u003c/em\u003e splicing reporter\u003csup\u003e29\u003c/sup\u003e, by fusing a \u003cem\u003eHEH1\u003c/em\u003e gene segment containing parts of the exons across the intron upstream of the\u0026nbsp;b\u0026nbsp;galactosidase-encoding \u003cem\u003eLACZ\u003c/em\u003e gene. In-frame translatable reporter mRNA would be made only after precise 5\u0026rsquo;SS selection and excision of the intron. Reporter activities were monitored in yeast strains grown on solid (X-Gal overlay assay) and liquid (ONPG assay) media. Splicing from the alternative 5\u0026rsquo;SS was diminished in the deletion strains of the RES (Retention and Splicing complex) complex subunits Snu17, Bud13, Pml1, and Urn1. The deletion of the NTC (Nineteen Complex) factor Ecm2 showed similar defects (Extended Data Fig.1b and c). By contrast, splicing from the dominant 5\u0026rsquo;SS remained unaltered in the mutants, suggesting a role of these proteins in the selection of the weaker alternative 5\u0026rsquo;SS.\u003c/p\u003e\n\u003cp\u003e(ii) Heh1 protein isoforms were monitored in deletion and point mutants of splicing factors by western blot assays. The gene was expressed with N-terminal epitope tags from a plasmid or tagged chromosomally. Supporting the data with the reporter assays (Extended Data Fig.1a-c), Heh1S protein was strongly diminished in the knockout mutants of Snu17, Bud13, Pml1, and Urn1, in addition to Hub1 (Extended Data Fig. 1d). The previously reported D22A and H63L mutants of Hub1 were defective in \u003cem\u003eHEH1\u003c/em\u003e alternative splicing. Potential RNA-binding mutant of Hub1, as deduced from cryo-EM studies of spliceosomes\u003csup\u003e30,31\u003c/sup\u003e, showed a similar lack of Heh1S protein (Extended Data Fig. 1h). Alternative splicing defect was also seen in the Hub1-binding deficient Snu66-HIND mutant (Extended Data Fig. 1j). Prp8 alleles were also screened for \u003cem\u003eHEH1\u003c/em\u003e alternative splicing. Heh1S level was strongly reduced in the \u003cem\u003eprp8-101\u003c/em\u003e (E1960K) allele. No other Prp8 mutant tested showed similar defects. While Heh1S protein was diminished in the above mutants, they showed more Heh1L than wt (Figure 1a), possibly because the lack of competition from \u003cem\u003eHEH1S\u003c/em\u003e 5\u0026rsquo;SS led to increased usage of the \u003cem\u003eHEH1L\u003c/em\u003e site.\u003c/p\u003e\n\u003cp\u003e(iii) \u003cem\u003eHEH1\u003c/em\u003e mRNA isoforms in the yeast mutants were monitored by reverse-transcription (RT) PCR assays with primers binding to \u003cem\u003eHEH1\u003c/em\u003e exons, followed by sequencing of the cDNAs. The two mRNAs show a single band. Since they differ in GCAA nucleotides at the exon junction (\u003cem\u003eHEHL\u003c/em\u003e gains GCAA due to the usage of downstream 5\u0026rsquo;SS), electropherograms show two mixed peaks after the common exon. Areas under the peaks were integrated to estimate the relative abundance of the two mRNAs. Consistent with the findings from reporter assays and protein analysis, the above mutants showed diminished levels of \u003cem\u003eHEH1S\u003c/em\u003e mRNA, confirming the 5\u0026rsquo;SS selection defects at the RNA level (Figure 1c). The electropherograms also showed increased \u003cem\u003eHEH1L\u003c/em\u003e mRNA level in the mutants, compared to the wt strain (Figure 1d), consistent with higher protein levels seen in Figure 1a. Thus, spliceosomes defective in using \u003cem\u003eHEH1S\u003c/em\u003e 5\u0026rsquo;SS used the dominant \u003cem\u003eHEH1L\u003c/em\u003e 5\u0026rsquo;SS more efficiently in the absence of competition.\u003c/p\u003e\n\u003cp\u003e(iv) Splicing factors essential for cell viability could not be explored with deletion strains. Hub1 proximity to them was used to explore their potential role in \u003cem\u003eHEH1\u003c/em\u003e alternative splicing. Snu66-binding-deficient \u003cem\u003ehub1(D22A)\u003c/em\u003e in the free form was defective in the usage of \u003cem\u003eHEH1\u003c/em\u003e alternative 5\u0026rsquo;SS, but its\u003cem\u003e\u0026nbsp;\u003c/em\u003elinear fusion to Snu66, Prp38 and Prp8 restored alternative splicing in cells lacking Hub1\u003csup\u003e14\u003c/sup\u003e. Interestingly, Heh1S protein was restored after anchoring Hub1 to Prp3, Prp6, and Prp31, but not to other proteins or the components of U1 and U2 snRNPs (Figure 1e). Thus, Hub1\u0026rsquo;s proximity to specific proteins of the spliceosome B\u0026nbsp;and Bact\u003csup\u003e\u0026nbsp;\u003c/sup\u003ecomplexes (Snu66, Prp38, Prp8, Prp3, Prp6, and Prp31) promoted \u003cem\u003eHEH1\u003c/em\u003e alternative splicing.\u003c/p\u003e\n\u003cp\u003eThe data discussed in i-iv indicated the need for specific proteins of the spliceosome core in \u003cem\u003eHEH1\u003c/em\u003e alternative splicing. However, their excess did not push the selection towards \u003cem\u003eHEH1S\u003c/em\u003e 5\u0026rsquo;SS. Overexpression of none of these proteins increased the Heh1S isoform or suppressed the Heh1L (Extended Data Fig. 1 m-n). Hub1\u0026rsquo;s excess in the spliceosome through its chromosomal fusions to two \u003cem\u003eHEH1S\u003c/em\u003e splicing-promoting factors, Snu66 and Prp38, could not alter the Heh1S/Heh1L ratio (Extended Data Fig. 1o). Thus, since the proteins played only regulatory roles, how are the competing 5\u0026rsquo;SS decoded?\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eU5 and U6 snRNAs decode pre-mRNA cis elements for alternative 5\u0026rsquo;SS selection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrom the splicing reporters discussed above, and known binding sites of U1, U5, and U6 snRNAs at exon-intron boundaries, we narrowed a 16-nucleotide pre-mRNA segment (1913-1928 of the \u003cem\u003eHEH1\u003c/em\u003e gene) and studied its importance for alternative 5\u0026rsquo;SS selection by mutagenesis (Figure 2a). Proteins originating from the variants were detected by western blots (Figure 2b), and mRNAs by RT-PCR followed by cDNA sequencing (Figure 2c). Almost any change in this segment lost alternative splicing (barring the G(\u0026minus;1)A variant of \u003cem\u003eHEH1S\u003c/em\u003e) and produced either one of the Heh1 isoforms. Changes leading to stop codons were studied by cDNA sequencing. G\u003cu\u003eC\u003c/u\u003eAA to G\u003cu\u003eU\u003c/u\u003eAA change of the upstream 5\u0026rsquo;SS gains an in-frame stop codon of \u003cem\u003eHEH1L\u003c/em\u003e mRNA, abolishing the Heh1L protein (this data explains the choice of the \u0026lsquo;GC\u0026rsquo; donor for \u003cem\u003eHEH1S\u003c/em\u003e splicing; a \u0026lsquo;GU\u0026rsquo; would have abolished Heh1L). These results highlighted the invariability of nucleotides around the competing 5\u0026rsquo;SS.\u003c/p\u003e\n\u003cp\u003eThe alternative splicing defects in the pre-mRNA variants, however, did not correlate with predicted U1 snRNA-5\u0026rsquo;SS interactions. Strikingly, we noticed a perfect correlation in the 5\u0026rsquo;SS choice and their expected base-pairing strengths with U5 and U6 snRNAs. 5\u0026rsquo;SS that paired stronger to U5 or U6 snRNAs was the preferred donor, and the use of the second site was minimal (Figure 2 and Extended Data Fig. 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eU5 and U6 snRNAs can promote alternative 5\u0026rsquo;SS selection without the protein factors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrom Figure 2, U5 and U6 (but not U1) snRNA variants with modified base pairing to the competing sites were expected to bias the selection. Indeed, the respective 5\u0026rsquo;SS was used more efficiently upon overexpression of U5 and U6 snRNA variants with strengthened base pairing (Figure 3b). Similar overexpression of the U1 snRNA variant did not alter 5\u0026rsquo;SS choice. U5 snRNA U(98)G and U6 snRNA A(45)G variants with stronger binding to \u003cem\u003eHEH1L\u003c/em\u003e 5\u0026rsquo;SS increased Heh1L protein (Extended Data Fig. 3). Conversely, U5 snRNA U(98)C and U6 snRNA U(46)C A(45)U variants with stronger binding to \u003cem\u003eHEH1S\u003c/em\u003e 5\u0026rsquo;SS increased Heh1S mRNA and protein (Figure 3b-c and Extended Data Fig. 3a-b and 3e-f). Thus, 46\u003csup\u003eth\u003c/sup\u003e and 45\u003csup\u003eth\u003c/sup\u003e nucleotides of U6 snRNA, beyond the ACAGA box, were also crucial for selecting the alternative 5\u0026rsquo;SS by base pairing at +7 and +8 nucleotides in the intron.\u003c/p\u003e\n\u003cp\u003eIf the proteins discussed above acted by stabilizing U5 and U6 snRNAs on the pre-mRNA substrate, the snRNA variants pairing strongly to \u003cem\u003eHEH1S\u003c/em\u003e 5\u0026rsquo;SS should bypass the need for the proteins. Indeed, overexpression of U(98)C variant of U5 snRNA and U(46)C,A(45)U variants of U6 snRNA restored alternative splicing defects in \u003cem\u003eD\u003c/em\u003e\u003cem\u003ehub1\u003c/em\u003e, \u003cem\u003eD\u003c/em\u003e\u003cem\u003esnu17\u003c/em\u003e, \u003cem\u003eD\u003c/em\u003e\u003cem\u003eecm2,\u003c/em\u003e and \u003cem\u003eD\u003c/em\u003e\u003cem\u003eurn1\u003c/em\u003e mutants (Figure 3 and Extended Data Fig. 3). Notably, the defect in \u003cem\u003eprp8-101\u003c/em\u003e strain was partially restored by overexpression of the U5 and U6 snRNA variants, suggesting an additional role of the core spliceosome protein Prp8 in 5\u0026rsquo;SS selection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Prp8-101 surface is critical for processing the \u0026lsquo;GC\u0026rsquo; donor\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrp8 is reported to exist in two distinct conformations to maintain equilibrium between the two catalytic steps of splicing; consequently, two opposing alleles of Prp8 rescue splicing defects in one another\u003csup\u003e32\u003c/sup\u003e. Combining a second step Prp8 allele,\u003cem\u003e\u0026nbsp;prp8-161\u003c/em\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003ewith\u003cem\u003e\u0026nbsp;prp8-101\u0026nbsp;\u003c/em\u003edid not restore the defects (Extended Data Fig. 4d). Thus, the alternative splicing defects in the first step Prp8 allele, \u003cem\u003eprp8-101\u003c/em\u003e, could not be explained by the above mechanism.\u003c/p\u003e\n\u003cp\u003eIn a pre-mRNA variant (#7), the upstream 5\u0026rsquo;SS /GCA\u003cu\u003eU\u003c/u\u003eGU with \u0026lsquo;GC\u0026rsquo; donor was preferred over the downstream /GUGAGU in wt, \u003cem\u003eD\u003c/em\u003e\u003cem\u003ehub1\u003c/em\u003e, \u003cem\u003eD\u003c/em\u003e\u003cem\u003esnu17\u003c/em\u003e, \u003cem\u003eD\u003c/em\u003e\u003cem\u003eecm2,\u003c/em\u003e and \u003cem\u003eD\u003c/em\u003e\u003cem\u003eurn1\u003c/em\u003e strains (Figure 4b and Extended Data Fig. 4b), likely due to the stronger pairing of +4U with the AC\u003cu\u003eA\u003c/u\u003eGA box of U6 snRNA. However, the \u003cem\u003eprp8-101\u003c/em\u003e allele could not use this site. The defect in \u003cem\u003eprp8-101\u003c/em\u003e was explored using the \u003cem\u003eACT1\u0026ndash;CUP1\u003c/em\u003e splicing reporter with a single 5\u0026rsquo;SS\u003csup\u003e33\u003c/sup\u003e. The 5\u0026rsquo;SS /GCA\u003cu\u003eU\u003c/u\u003eGU was used efficiently in all mutants, except for \u003cem\u003eD\u003c/em\u003e\u003cem\u003ehub1\u003c/em\u003e and \u003cem\u003eprp8-101\u003c/em\u003e. C(+2)U-exchanged /G\u003cu\u003eU\u003c/u\u003eA\u003cu\u003eU\u003c/u\u003eGU was efficiently used in all strains, including\u003cem\u003e\u0026nbsp;D\u003c/em\u003e\u003cem\u003ehub1\u003c/em\u003e and \u003cem\u003eprp8-101\u003c/em\u003e (Figure 4c). Thus, the donor with +2C was poorly used in\u003cem\u003e\u0026nbsp;prp8-101\u003c/em\u003e and\u003cem\u003e\u0026nbsp;D\u003c/em\u003e\u003cem\u003ehub1\u003c/em\u003e, leading to the loss of alternative splicing. Similarly, splicing from a +3C 5\u0026rsquo;SS variant /GU\u003cu\u003eC\u003c/u\u003eUGU was defective in\u003cem\u003e\u0026nbsp;D\u003c/em\u003e\u003cem\u003ehub1\u003c/em\u003e, \u003cem\u003eD\u003c/em\u003e\u003cem\u003esnu66,\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;prp8-101\u003c/em\u003e strains (Extended Data Fig. 4e). Thus, the Prp8-101 surface promotes selection (and catalysis?) from non-canonical 5\u0026rsquo;SS containing +2C and +3C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpecific competing 5\u0026rsquo;SS allow alternative splicing without the regulatory proteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOptimal decoding of competing 5\u0026rsquo;SS by U5 and U6 snRNAs, together with the use of \u0026lsquo;GC\u0026rsquo; donor by Prp8, is the primary determinant of alternative splicing. The process is facilitated by specific proteins of the spliceosome. Thus, competing 5\u0026rsquo;SS with modified binding to the snRNAs should allow alternative selection in the absence of the regulatory proteins. More pre-mRNA\u0026nbsp;variants were assayed to identify such 5\u0026rsquo;SS.\u003c/p\u003e\n\u003cp\u003eTwo 5\u0026rsquo;SS variants allowed alternative splicing not only in wt yeast (Extended Data Fig. 2) but also in strains mutated for the regulatory proteins (Figure 4b and Extended Data Fig. 4b).\u0026nbsp;U5 and U6 snRNAs\u0026rsquo; base pairing was recalibrated in #15 with two non-canonical 5\u0026rsquo;SS /GCAA/GU\u003cu\u003eAUA\u003c/u\u003eU. In contrast, 5\u0026rsquo;SS pairing with U6 snRNAs was strongest in #18, containing two canonical 5\u0026rsquo;SS /G\u003cu\u003eU\u003c/u\u003eA\u003cu\u003eU\u003c/u\u003e/GU\u003cu\u003eAU\u003c/u\u003eGU. The sites being canonical were readily used without the need for the protein factors, including the Prp8-101 surface. The efficient use of canonical 5\u0026rsquo;SS in the absence of the protein factors was further supported by \u003cem\u003eHEH1\u0026ndash;CUP1\u0026nbsp;\u003c/em\u003ereporters (Extended Data Fig. 4f-h).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEcm2, Urn1, and the RES proteins bring weaker 5\u0026rsquo;SS in competition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHEH1S\u003c/em\u003e expression from /GCAA/GU\u003cu\u003eAUA\u003c/u\u003eU 5\u0026rsquo;SS (#15) in cells lacking the protein factors was intriguing, since the changes in this variant were beyond the hexanucleotide /GCAAGU. The outcomes could be explained by two possibilities: (i) weakening of the \u003cem\u003eHEH1L\u003c/em\u003e 5\u0026rsquo;SS /GUGAGU to /GU\u003cu\u003eAUA\u003c/u\u003eU rebalanced the competition in favor of the \u003cem\u003eHEH1S\u003c/em\u003e site, or (ii) improved binding of +7A and +8U in /GCAAGU\u003cu\u003eAU\u003c/u\u003e to U6 snRNA nucleotides U46 and A45 favored the\u003cem\u003e\u0026nbsp;HEH1S\u003c/em\u003e 5\u0026rsquo;SS. To study these possibilities, alternative splicing reporters, \u003cem\u003eHEH1S\u0026ndash;CUP1\u0026nbsp;\u003c/em\u003eand \u003cem\u003eHEH1L\u0026ndash;CUP1\u003c/em\u003e, were made (Figure 5b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHEH1L\u003c/em\u003e 5\u0026rsquo;SS was weakened into /GUGAG\u003cu\u003eA\u003c/u\u003e (#10; it also lacks potential \u003cem\u003eHEH1S\u003c/em\u003e-U6 pairing at +7 and +8 positions) or /GU\u003cu\u003eAUAA\u003c/u\u003e (#20; with enhanced U6 pairing at +7 and +8 positions of \u003cem\u003eHEH1S\u003c/em\u003e) (Figure 5a and c). \u003cem\u003eHEH1S\u003c/em\u003e 5\u0026rsquo;SS selection in\u0026nbsp;\u003cem\u003eD\u003c/em\u003e\u003cem\u003ehub1\u003c/em\u003e improved for reporters with enhanced U6 pairing at +7 and +8 positions of the 5\u0026rsquo;SS, and the defect was partially restored in the \u003cem\u003eprp8-101\u003c/em\u003e allele. The data confirmed the importance of U6 snRNA\u0026rsquo;s U46 and A45 nucleotides for alternative splicing discussed earlier in Figure 3. The 5\u0026rsquo;SS selection in\u0026nbsp;\u003cem\u003eD\u003c/em\u003e\u003cem\u003esnu17\u003c/em\u003e,\u0026nbsp;\u003cem\u003eD\u003c/em\u003e\u003cem\u003eurn1,\u003c/em\u003e and\u0026nbsp;\u003cem\u003eD\u003c/em\u003e\u003cem\u003eecm2,\u003c/em\u003e on the other hand, was improved by both enhanced U6 snRNA pairing to \u003cem\u003eHEH1S\u003c/em\u003e and weakening the competition from \u003cem\u003eHEH1L\u003c/em\u003e (Figure 5d). Thus, Hub1 and Prp8 promote selection of 5\u0026rsquo;SS with +2C by stabilizing U5 and U6 snRNA on the target pre-mRNA. On the contrary, Snu17, Urn1, and Ecm2 promote competition for the weaker 5\u0026rsquo;SS by stabilizing snRNA-pre-mRNA interactions.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e\u003cstrong\u003ePre-mRNA-snRNA interactions in alternative 5\u0026rsquo;SS selection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data presented support dominant roles of U5 and U6 snRNAs and regulatory roles of B and Bact proteins in alternative 5\u0026rsquo;SS selection.\u0026nbsp;U1 snRNA and associated factors\u0026nbsp;recognize 5\u0026prime;SS early in the splicing cycle\u0026nbsp;and have been reported to promote alternative 5\u0026rsquo;SS selection\u003csup\u003e34\u003c/sup\u003e. However, U1 snRNA, its associated proteins, and spliceosomes before the pre-B stage\u0026nbsp;were not critical for \u003cem\u003eHEH1\u003c/em\u003e alternative splicing.\u0026nbsp;At the B stage of the spliceosome, U1 snRNA hands over the 5\u0026rsquo;SS to U6 snRNA. The\u0026nbsp;ACAGA\u0026nbsp;box of U6 base pairs with +3 to +6 nucleotides of the 5\u0026rsquo;SS, and U5 snRNA\u0026rsquo;s loop 1 base pairs with the upstream four nucleotides of the exon\u003csup\u003e35\u003c/sup\u003e. Moreover, the 45\u003csup\u003eth\u003c/sup\u003e and 46\u003csup\u003eth\u003c/sup\u003e nucleotides of U6 snRNA appear to play a critical role in decoding +7 and +8 positions of 5\u0026rsquo;SS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOptimal strength of U5 and U6 snRNAs base pairing was essential for the selection of \u003cem\u003eHEH1\u003c/em\u003e competing 5\u0026rsquo;SS. Any deviations in pairing led to the loss of alternative splicing and produced the isoform which had relatively better snRNAs-pre-mRNA interactions. The snRNA variants with reinforced pairing to the weaker site not only favored their selection but also rescued the defects in the protein mutants. In addition, certain pre-mRNA variants with recalibrated U5 and U6 snRNA pairing to the competing 5\u0026rsquo;SS were alternatively spliced in the absence of the protein factors. The marking of the pre-mRNA target by the snRNAs in the B complex induces U4/U6 snRNA unwinding and concomitant U2/U6 snRNA pairing in the Bact complex\u003csup\u003e36\u003c/sup\u003e. The switch may define the branching site for the first catalytic step for the +2C containing 5\u0026rsquo;SS in the B* complex, likely with the help of the Prp8-101 surface. Thus, RNA-RNA interactions at the B stage of the spliceosome are essential for alternative 5\u0026rsquo;SS selection, centered around pre-mRNA recognition by U5 and U6 snRNAs (Figure 6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpecific B and Bact proteins facilitate alternative 5\u0026rsquo;SS selection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe trans-acting proteins critical for the alternative selection of \u003cem\u003eHEH1\u003c/em\u003e 5\u0026rsquo;SS reported earlier\u003csup\u003e14\u003c/sup\u003e and identified in this study belong to the spliceosome B or Bact complexes. Importantly, alternative 5\u0026rsquo;SS\u003cem\u003e\u0026nbsp;\u003c/em\u003eof \u003cem\u003eHEH1\u003c/em\u003e is the first \u003cem\u003ein vivo\u003c/em\u003e target of these factors in \u003cem\u003eS. cerevisiae\u003c/em\u003e, including the \u003cem\u003eprp8-101\u003c/em\u003e allele. The proteins appear to facilitate alternative 5\u0026rsquo;SS selection by stabilizing the spliceosome in a low-fidelity, high-efficiency conformation. Supporting this idea, the Hub1-Snu66 complex enhances the selection of non-canonical 5\u0026rsquo;SS and error-prone splicing\u003csup\u003e14,27\u003c/sup\u003e. The RES complex proteins Snu17, Bud13, and Urn1 promote non-canonical splicing\u003csup\u003e37\u0026ndash;39\u003c/sup\u003e, and Ecm2 facilitates selection of non-canonical and competing 5\u0026rsquo;SS\u003csup\u003e40\u003c/sup\u003e. The proteins seem to promote competition for the alternative 5\u0026rsquo;SS at the expense of the dominant site. Splicing from \u003cem\u003eHEH1S\u003c/em\u003e 5\u0026rsquo;SS needed these proteins, yet splicing from the \u003cem\u003eHEH1L\u003c/em\u003e 5\u0026rsquo;SS was even better in their absence.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePrp8 and associated proteins in alternative 5\u0026rsquo;SS selection:\u003c/em\u003e Among the fourteen distinct Prp8 alleles\u003csup\u003e41\u003c/sup\u003e tested in this study, only \u003cem\u003eprp8-101\u0026nbsp;\u003c/em\u003e(E1960K) mutant\u003csup\u003e42\u003c/sup\u003e was defective in splicing from the alternative 5\u0026apos;SS. The mutation lies in its RNaseH/RH domain. The Prp8 RH domain toggles between transitional and catalytic conformations\u003csup\u003e43\u003c/sup\u003e. It remains in a partially closed conformation till the B complex and promotes splicing fidelity over efficiency. Prp8\u0026rsquo;s first step alleles\u003csup\u003e44\u003c/sup\u003e and Prp38\u003csup\u003e45\u003c/sup\u003e have been shown to stabilize the high-fidelity conformation. The RH domain switches to a completely closed conformation in the Bact complex\u003csup\u003e43,46\u003c/sup\u003e and positions the RNA for first-step catalysis. The\u0026nbsp;defective splicing in \u003cem\u003eprp8-101\u003c/em\u003e from +2C and +3C containing 5\u0026rsquo;SS, suggests high-fidelity, low-efficiency nature of this mutant and the role of Prp8 in selection (or catalysis) of +2C and +3C containing 5\u0026rsquo;SS. In contrast to the full rescue of \u003cem\u003eHEH1\u003c/em\u003e alternative splicing upon overexpression of U5 and U6 snRNA variants in the deletion of the splicing proteins, the partial rescue of \u003cem\u003eprp8-101\u003c/em\u003e defects points to a potential role of this Prp8 surface in catalysis from the \u0026lsquo;GC\u0026rsquo; donor.\u003c/p\u003e\n\u003cp\u003eThe role of Prp3, Prp6 and Prp31 in alternative 5\u0026rsquo;SS selection was revealed through proximity probing with Hub1. These proteins surround the core Prp8 and undergo a major switch during the conversion of pre-B to B. The 180\u0026deg; shift in the RH domain from pre-B to B along with the tri-snRNP factors\u003csup\u003e47,48\u003c/sup\u003e appears to be important for the recognition of \u003cem\u003eHEH1\u0026nbsp;\u003c/em\u003ealternative 5\u0026rsquo;SS.\u0026nbsp;Thus, conformational changes in the spliceosome during B to Bact transition appears to be critical for \u003cem\u003eHEH1\u003c/em\u003e alternative splicing.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHub1 in alternative 5\u0026rsquo;SS selection:\u0026nbsp;\u003c/em\u003eHub1 joins spliceosome through non-covalent binding to Prp5 or Snu66. The Prp38 complex subunits, Prp38, Snu13 and Spp381/MFAP1, associate with the tri-snRNP and help stabilise the B complex. The D22A surface of Hub1 binds Snu66 in multiple eukaryotes\u003csup\u003e14,49\u0026ndash;51\u003c/sup\u003e. Interestinlgy. the same Hub1 surface binds MFAP1 in\u0026nbsp;the human B spliceosome\u003csup\u003e48,52\u003c/sup\u003e. The D21 residue of Hub1 also interacts with the same surface of MFAP1\u003csup\u003e48\u003c/sup\u003e. An additional Hub1 surface, N7 and K13, that binds the 5\u0026rsquo; exon at -3 and -4 positions in human spliceosomes\u003csup\u003e52\u003c/sup\u003e, was critical for \u003cem\u003eHEH1\u003c/em\u003e alternative 5\u0026rsquo;SS selection. Thus, Hub1 is likely recruited to the early B complex through Snu66-HIND\u003csup\u003e14\u003c/sup\u003e and promote alternative 5\u0026rsquo;SS selection after switching to pre-mRNA and MFAP1 in the mature B complex\u003csup\u003e48,52\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe B to Bact transition\u003c/em\u003e: Brr2 helicase unwinds the U4/U6 duplex, triggering the release of U4 snRNP and associated proteins (Prp3, Prp6, Prp31, Snu66, Hub1). Prp38 complex is also leaves at this stage, a key step for full activation of the spliceosome. U6 snRNA forms new interactions with U2 snRNA, assembling the catalytic core. The NTC protein Ecm2 and the RES complex proteins are associated with the Bact complex, highlighting the role of the RES in the remodelling of Bact via Prp2. The RES complex leaves the spliceosome during Bact to B* conversion\u003csup\u003e53,54\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn conclusion, \u003cem\u003eS. cerevisiae\u003c/em\u003e \u003cem\u003eHEH1/SRC1\u003c/em\u003e pre-mRNA is an important substrate for studying alternative splicing through competing and overlapping 5\u0026rsquo;SS. Data presented here, along with the literature, support a plausible mechanism of alternative 5\u0026rsquo;SS selection in the B and Bact spliceosomes orchestrated by U5 and U6 snRNAs as the primary determinants. RNA-RNA interaction is thus central to alternative splicing through competing 5\u0026rsquo;SS. Splicing factors promoting selection of weak 5\u0026rsquo;SS, reported previously (Hub1, Snu66, Prp38, Prp8), and identified in this study (the B complex proteins Prp3, Prp6, Prp31, and Snu66; the RES complex subunits Bud13, Snu17, Pml1 and Urn1, and the NTC complex Ecm2), function from or through the B or Bact spliceosomes. U5 and U6 snRNAs are essential components of both these assemblies. With the help of the trans-acting proteins, U5 and U6 snRNAs pair with target pre-mRNAs for alternative 5\u0026rsquo;SS selection and catalysis.\u003c/p\u003e"},{"header":"STAR METHOD","content":"\u003cp\u003e\u003cstrong\u003eKEY RESOURCE TABLE\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"595\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eReagent\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eResource\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIdentifier\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eRabbit polyclonal anti-c-myc antibody\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eSigma Aldrich\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eC3956\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eRabbit Peroxidase anti-peroxidase soluble antibody\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eSigma Aldrich\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eA0545\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eGoat anti-rabbit IgG peroxidase antibody\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eSigma Aldrich\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eP1291\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eHigh-Capacity cDNA Reverse Transcription Kit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eApplied Biosystem\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003e4368813\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eNuPAGE\u0026trade; Bis-Tris Mini Protein Gels, 4\u0026ndash;12%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eInvitrogen\u0026trade;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eNP0323BOX\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eRQ1 RNase-Free DNase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003ePromega\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eM610A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eZymo-Spin II Columns\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eZymo Research\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eC1008-250\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eVent DNA polymerase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eNEB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eM0254L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 595px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSoftwares\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eFiji-windows 64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eNIH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003ehttps://imagej.net/software/fiji\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eAlphaFold3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eDeepMind (Google)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003ehttps://alphafoldserver.com/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eMETHOD DETAILS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmids, yeast strains, \u003cem\u003eS. cerevisiae\u003c/em\u003e transformation, and growth assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlasmids and strains used in this study are listed in Table 1 and Table 2, respectively. The yJU75 yeast strain, plasmids \u003cem\u003ePRP8\u003c/em\u003e and \u003cem\u003eprp8-101,\u003c/em\u003e and \u003cem\u003eACT1\u0026ndash;CUP1\u0026nbsp;\u003c/em\u003ereporter were kind gifts from C. Guthrie and M. Konarska\u0026apos;s lab. Prp8 mutant strains were made by shuffling out the Ura+ \u003cem\u003ePRP8\u003c/em\u003e wildtype plasmid with the Trp+ \u003cem\u003eprp8\u003c/em\u003e mutants on 5-fluoroorotic acid (FOA) plates. \u003cem\u003eThe S. cerevisiae\u003c/em\u003e deletion strains of splicing factors were obtained from the Euroscarf haploid deletion library. Competent cell preparation and transformation were performed following previously published protocols.\u003csup\u003e55,56\u003c/sup\u003e. Chromosomal tagging for western blot, double knockout strains for genetic interactions, and splicing factors deletion in the yJU75 background were made by using the reported protocol.\u003csup\u003e55,56\u003c/sup\u003e. Protein overexpression was achieved by expressing clones under a strong \u003cem\u003egal promoter\u003c/em\u003e. For growth/spot assays, 5-fold serial dilutions of cells were spotted on the indicated agar plates, and plates were incubated at temperatures indicated in the figure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromosomal fusion of \u003cem\u003ehub1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026ndash;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eD22A\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;to the C-termini of splicing factors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSplicing by Overlap Extension (SOE) PCR was done to make \u003cem\u003ehub1(D22A)\u003c/em\u003e C-terminal fusion to essential splicing factors. This fusion was done based on the published protocol\u003csup\u003e55\u003c/sup\u003e. Three sets of primers were used to amplify three fragments. First set of primers amplified a 200-300 bp fragment of the C termini of the desired gene, excluding the stop codon. The reverse primer of this had an overhang of 20-25 bps that was complementary to the 20-25 bps of \u003cem\u003ehub1(D22A)\u003c/em\u003e N-terminus. The second set of primers amplified the full-length \u003cem\u003ehub1(D22A)\u003c/em\u003e with the Nat antibiotic selection marker cassette from the plasmid clones of pFA6a-\u003cem\u003ehub1(D22A)-natNT2\u003c/em\u003e. The third set of primers amplified a fragment of 200-300 bp from the stop codon of the gene that was going to be tagged. The forward primer for this had an overhang of 20-25 bp with the end of the antibiotic-resistance marker. All three fragments were amplified using Vent DNA polymerase. These fragments were confirmed based on their size on the agarose gel and the correct fragments were gel-purified. All three fragments were mixed and joined by SOE PCR using Vent DNA polymerase using the forward primer of the first fragment and the reverse primer of the third fragment. The joined fragment was confirmed by agarose gel electrophoresis and transformed into the \u003cem\u003eS. cerevisiae\u003c/em\u003e strain. The transformants were selected on agar plates with antibiotic. The chromosomal fusion was confirmed by colony PCR using a forward primer specific to the gene and a reverse primer specific to the antibiotic cassette.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSplicing reporters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe splicing reporters used in this study are listed in Table 3. Splicing reporters are modified forms of the conventional \u003cem\u003eACT1\u003c/em\u003e\u003cem\u003e\u0026ndash;\u003c/em\u003e\u003cem\u003eCUP1\u003c/em\u003e\u003csup\u003e33\u003c/sup\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eand \u003cem\u003eRP51\u003c/em\u003e\u003cem\u003e\u0026ndash;\u003c/em\u003e\u003cem\u003eLACZ\u003csup\u003e29\u003c/sup\u003e\u003c/em\u003e. The \u003cem\u003eHEH1S\u0026ndash;CUP1 reporters have a 121 bp segment of the S. cerevisiae HEH1\u0026nbsp;\u003c/em\u003egene (1874-2095 nucleotides), including the intron, using BamHI and KpnI restriction sites. The \u003cem\u003eHEH1S\u003c/em\u003e mRNA is spliced using GCAAGU 5\u0026rsquo;SS, generating in-frame \u003cem\u003eCUP1\u003c/em\u003e mRNA for translation. The methionine initiation codon was introduced by inserting a point mutation at C1881G. Initiation codon and variants of the 5\u0026rsquo;SS were made by site-directed mutagenesis (SDM) using specific primers, and the change was confirmed using Sanger sequencing. For \u003cem\u003eHEH1L\u0026ndash;CUP1\u0026nbsp;\u003c/em\u003ereporter\u003cem\u003e,\u003c/em\u003e a 120 bp segment of the \u003cem\u003eHEH1\u0026nbsp;\u003c/em\u003egene (1874-2094 nucleotides) was used to bring the \u003cem\u003eHEH1L\u003c/em\u003e mRNA spliced using GUGAGU 5\u0026rsquo;SS in-frame with the \u003cem\u003eCUP1\u003c/em\u003e gene. For reporter assays, competent cells from the \u003cem\u003eS. cerevisiae\u0026nbsp;\u003c/em\u003estrain yJU75 were transformed with the reporters, and transformants were selected in media lacking leucine. 1 OD\u003csub\u003e600\u003c/sub\u003e cells were spotted on solid media with different concentrations of CuSO\u003csub\u003e4\u003c/sub\u003e. The plates were incubated for 2-3 days at 30\u0026deg;C. Similarly, for \u003cem\u003eHEH1\u0026ndash;LACZ\u003c/em\u003e reporters, the \u003cem\u003eRP51\u003c/em\u003e part of the \u003cem\u003eRP51\u003c/em\u003e\u003cem\u003e\u0026ndash;\u003c/em\u003e\u003cem\u003eLACZ\u003c/em\u003e reporter (gift from M. Rosbash) was replaced with a \u003cem\u003eHEH1\u0026nbsp;\u003c/em\u003efragment of 399 bp (1792-2191 nucleotides) and 398 bp (1792-2190 nucleotides) to obtain \u003cem\u003eHEH1S\u003c/em\u003e\u003cem\u003e\u0026ndash;\u003c/em\u003e\u003cem\u003eLACZ\u003c/em\u003e and \u003cem\u003eHEH1L\u003c/em\u003e\u003cem\u003e\u0026ndash;\u003c/em\u003e\u003cem\u003eLACZ\u003c/em\u003e reporters\u003cem\u003e,\u003c/em\u003e respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026beta;-galactosidase assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026beta;-galactosidase activity was observed on both solid and liquid media. The X-gal overlay and the ONPG assay were performed by following published protocols\u003csup\u003e57\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA isolation and cDNA synthesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA isolation and cDNA synthesis were done as described previously\u003csup\u003e58\u003c/sup\u003e. Briefly, five OD\u003csub\u003e600\u003c/sub\u003e cells in the log phase were harvested. Total RNA was isolated by the hot acid phenol method using 15 mL phase lock tubes for phase separation. Residual DNA was removed by treating RNA with DNase I for 15 min at room temperature, followed by RNA clean-up using a Zymo‐Spin\u003csup\u003eTM\u003c/sup\u003e II column. cDNA synthesis from 2\u0026mu;g total RNA was done using reverse transcriptase (RT) and random‐hexamer primers at 37\u0026deg;C for 2h. Splicing defects were monitored by detecting intron-containing transcripts or post-splicing mature transcripts using exonic primers across the intron and analysed by agarose gel electrophoresis. For cDNA sequencing, five identical PCR reactions were performed. The PCR products were pooled and kept overnight for precipitation after adding 2.5 times the volume of isopropanol and 1/10\u003csup\u003eth\u003c/sup\u003e the volume of 3M sodium acetate at -20\u0026deg;C. DNA was pelleted by centrifugation for 15 min at 4\u0026deg;C at maximum speed. The pellet was washed twice with 70% ethanol. Dried pellet was dissolved in 30\u0026mu;l of nuclease-free water, and 10\u0026mu;l of the DNA was sequenced by Sanger sequencing using a primer nested inside the forward primer used in amplification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification of \u003cem\u003eHEH1\u003c/em\u003e mRNA isoforms\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHEH1\u003c/em\u003e alternative splicing was analysed using RT-PCR followed by cDNA sequencing. \u003cem\u003eHEH1S\u003c/em\u003e and \u003cem\u003eHEH1L\u003c/em\u003e mRNAs differ in GCAA nucleotides at their exon junctions (\u003cem\u003eHEHL\u003c/em\u003e gains them due to the usage of downstream 5\u0026rsquo;SS), the electropherogram shows mixed peaks corresponding to the two isoforms after the exon-exon junction. The area under the peaks was integrated to find the relative abundance of the two mRNAs. Both common and isoform-specific peaks were analysed within the region corresponding to the 5\u0026rsquo;exon/intron boundary. To understand alternative 5\u0026rsquo;SS usage, common nucleotides after the junction in the two isoforms was excluded from further analysis. For each site, we recorded the nucleotide proportions possible for both the cDNAs and calculated the ratio between the two nucleotides, normalising their combined total to 100%. The average value of both forms is directly related to their respective 5\u0026rsquo;SS usage.\u003c/p\u003e\n\u003cp\u003eRelative expression of \u003cem\u003eHEH1L\u003c/em\u003e in wild-type and yeast mutants was obtained from the cDNA electropherogram. The areas under 10 common and 10 mixed peaks (before and after the junction, respectively), were integrated and averaged. Similarly, an area under the peaks specific to \u003cem\u003eHEH1L\u003c/em\u003e was integrated and averaged. The ratio of common and \u003cem\u003eHEHL\u003c/em\u003e peaks for different strains was obtained. The fold difference in \u003cem\u003eHEH1L\u003c/em\u003e expression in the mutants was calculated against the wildtype strain by dividing the average of the area under \u003cem\u003eHEH1L\u003c/em\u003e peaks (after the junction) by the average of the area under common peaks (before the junction).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blots\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor protein western blots (immunoblotting), logarithmically growing cells equivalent to 2 OD\u003csub\u003e600\u003c/sub\u003e were harvested. Whole protein was extracted using the trichloroacetic acid (TCA) method\u003csup\u003e55\u003c/sup\u003e. Total proteins used for western blots were denatured by heating cells in a high urea buffer at 65\u0026deg;C for 15 min and then centrifuged. The soluble protein was separated on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) 8% gels, or NuPAGE\u003csup\u003eTM\u003c/sup\u003e 4%-12% Bis-Tris gels (Invitrogen), transferred to a polyvinylidene difluoride (PVDF) membrane, and probed with specific primary and secondary antibodies. Protein bands were quantified using ImageJ following the published method\u003csup\u003e59\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eRESOURCE AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.K.B. or S.K.M. may be contacted for resources used in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Stefan Jentsch (MPI Biochemistry, Martinsried, Germany) for his support. The work in S.K.M. laboratory has been supported by IISER Mohali and the Centre for Protein Science Design and Engineering (CPSDE) of the Ministry of Human Resource and Development (MHRD), Government of India; the Max Planck Society, Germany; and the Wellcome Trust/DBT India Alliance Fellowship/grant awarded to S.K.M. A.K.B. was supported by ICMR fellowship and IISER Mohali. We acknowledge Anupa T. Anil and B. Mohapatra for their inputs and technical guidance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.K.M. initiated the study in the Jentsch laboratory at the MPI Biochemistry, Martinsried, Germany. All authors designed and performed the experiments and analysed the data. A.K.B. and S.K.M. collated the data and prepared the manuscript with inputs from all authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDECLARATION OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWilkinson, M. E., Charenton, C. \u0026amp; Nagai, K. Annual Review of Biochemistry RNA Splicing by the Spliceosome. \u003cstrong\u003e01\u003c/strong\u003e, 46 (2025).\u003c/li\u003e\n\u003cli\u003eFica, S. M. \u0026amp; Nagai, K. 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A. Genome-wide approaches to monitor pre-mrna splicing. \u003cem\u003eMethods Enzymol\u003c/em\u003e \u003cstrong\u003e470\u003c/strong\u003e, 51\u0026ndash;75 (2010).\u003c/li\u003e\n\u003cli\u003eDavarinejad, H. \u003cem\u003eQuantifications of Western Blots with ImageJ\u003c/em\u003e. http://rsb.info.nih.gov/ij/.\u003c/li\u003e\n\u003cli\u003eYan, C., Wan, R., Bai, R., Huang, G. \u0026amp; Shi, Y. Structure of a yeast activated spliceosome at 3.5 \u0026Aring; resolution. \u003cem\u003eScience (1979)\u003c/em\u003e \u003cstrong\u003e353\u003c/strong\u003e, 904\u0026ndash;911 (2016).\u003c/li\u003e\n\u003cli\u003eWan, R., Bai, R., Yan, C., Lei, J. \u0026amp; Shi, Y. Structures of the Catalytically Activated Yeast Spliceosome Reveal the Mechanism of Branching. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e177\u003c/strong\u003e, 339-351.e13 (2019).\u003c/li\u003e\n\u003cli\u003eNguyen, T. H. D. \u003cem\u003eet al.\u003c/em\u003e Cryo-EM structure of the yeast U4/U6.U5 tri-snRNP at 3.7 \u0026Aring; resolution. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e530\u003c/strong\u003e, 298\u0026ndash;302 (2016).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1: Plasmids used in this study.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"604\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eID\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eName\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePlasmids\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDescription\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRef.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e3658\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eWT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC111 pGAL-3MYC\u0026ndash;\u003cem\u003eHEH1\u003c/em\u003e-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. cerevisiae HEH1\u003c/em\u003e gene under GAL promoter with 3MYC tag at the N-terminus and ADH terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eMishra et al. 2011\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1401\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC111 pGAL-3MYC\u0026ndash;heh1 (A1913T)-tADH\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eA1913T mutation pGAL 3MYC\u0026ndash;heh1 tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1402\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC111 pGAL-3MYC\u0026ndash;heh1 (A1915T)-tADH\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eA1915T mutation pGAL 3MYC\u0026ndash;heh1 tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1403\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC111 pGAL-3MYC\u0026ndash;heh1 (G1916T)-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eG1916T mutation pGAL 3MYC\u0026ndash;heh1 tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1404\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC111 pGAL-3MYC\u0026ndash;heh1 (G1916A)-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eG1916A mutation pGAL 3MYC\u0026ndash;heh1 tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1405\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC111 pGAL-3MYC\u0026ndash;heh1 (C1918A)-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eC1918A mutation pGAL 3MYC\u0026ndash;heh1 tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1406\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC111 pGAL-3MYC\u0026ndash;heh1 (C1918T)-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eC1918T mutation pGAL 3MYC\u0026ndash;heh1 tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1407\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC111 pGAL-3MYC\u0026ndash;heh1 (A1920T)-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eA1920T mutation pGAL 3MYC\u0026ndash;heh1 tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1408\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC111 pGAL-3MYC\u0026ndash;heh1 (G1923A)-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eG1923A mutation pGAL 3MYC\u0026ndash;heh1 tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1409\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC111 pGAL-3MYC\u0026ndash;heh1 (A1924T)-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eA1924T mutation pGAL 3MYC\u0026ndash;heh1 tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1410\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC111 pGAL-3MYC\u0026ndash;heh1 (T1926A)-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eT1926A mutation pGAL 3MYC\u0026ndash;heh1 tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1411\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC111 pGAL-3MYC\u0026ndash;heh1 (A1920T, A1924T)-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eA1920T, A1924T mutation pGAL 3MYC\u0026ndash;heh1 tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1412\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC111 pGAL-3MYC\u0026ndash;heh1 (G1923C, A1924T)-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eG1923C, A1924T mutation pGAL 3MYC\u0026ndash;heh1 tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1413\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC111 pGAL-3MYC\u0026ndash;heh1 (C1918T, A1919C, A1920T)-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eC1918T, A1919C, A1920T mutation pGAL 3MYC\u0026ndash;heh1 tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1414\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC111 pGAL-3MYC\u0026ndash;heh1 (A1920T, G1923A, A1924T)-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eA1920T, G1923A, A1924T mutation pGAL 3MYC\u0026ndash;heh1 tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1415\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC111 pGAL-3MYC\u0026ndash;heh1 (G1923A, A1924T, G1925A)-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eG19223A, A1924T, G1925A mutation pGAL 3MYC\u0026ndash;heh1 tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1416\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC111 pGAL-3MYC\u0026ndash;heh1 (G1923A, A1924T, G1925T, T1926A)-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eG1923A, A1924T, G1925T, T1926A mutation pGAL 3MYC\u0026ndash;heh1 tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1417\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC111 pGAL-3MYC\u0026ndash;heh1 (G1923A, A1924T, G1925A, T1926A, A1927C)-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eG1923A, A1924T, G1925A, T1926A, A1927C mutation pGAL 3MYC\u0026ndash;heh1 tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1418\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC111 pGAL-3MYC\u0026ndash;heh1 (C1918T, A1920T, G1923A, A1924T)-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eC1918T, A1920T, G1923A, A1924T mutation pGAL 3MYC\u0026ndash;heh1 tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1419\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC111 pGAL-3MYC\u0026ndash;heh1 (G1921A)-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eG1921A mutation pGAL 3MYC\u0026ndash;heh1 tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1420\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC111 pGAL-3MYC\u0026ndash;heh1 (G2046C)-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eG2046C mutation pGAL 3MYC\u0026ndash;heh1 tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD897\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYE112 pADH-3myc\u0026ndash;Snu66-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. cerevisiae SNU66\u003c/em\u003e gene under GAL promoter with 3MYC tag at the N-terminus and ADH terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD909\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYE112 pADH-3myc\u0026ndash;snu66-RRAA-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eR16A, R47A mutation pGAL 3MYC\u0026ndash;snu66 tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC333 pHUB1-Hub1-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. cerevisiae HUB1\u0026nbsp;\u003c/em\u003egene under its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1382\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC333 pHUB1-Hub1(R9A)-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eR9A mutation pHUB1-Hub1-tHUB1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1383\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC333 pHUB1-Hub1(K13A)-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eK13A mutation pHUB1-Hub1-tHUB1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1384\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC333 pHUB1-Hub1(R9A, K13A)-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eR9A, K13A mutation pHUB1-Hub1-tHUB1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD739\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC333 pHUB1-Hub1(D22A)-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eD22A mutation pHUB1-Hub1-tHUB1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD740\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC333 pHUB1-Hub1(H63L)-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eH63L mutation pHUB1-Hub1-tHUB1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e3743\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003ePRP8+ WT TRP CEN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003ePRP8 with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1421\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;prp8-161\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eprp8 (P986T) TRP CEN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eprp8 (P986T) with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1422\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;prp8*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eprp8 (P1384L) TRP CEN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eprp8 (P1384L) with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1423\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;syf77\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eprp8 (L1577F) TRP CEN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eprp8 (L1577F) with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1424\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;prp8-107\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eprp8 (F1834L) TRP CEN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eprp8 (F1834L) with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1425\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;prp8-cat\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eprp8 (F1851L) TRP CEN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eprp8 (F1851L) with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1426\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;prp8-cat\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eprp8 (V1860D) TRP CEN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eprp8 (V1860D) with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1427\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;prp8-201\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eprp8 (T1861P) TRP CEN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eprp8 (T1861P) with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1428\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;prp8-cat\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eprp8 (V1862D) TRP CEN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eprp8 (V1862D) with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1429\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;prp8-D143\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eprp8 (K1864E) TRP CEN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eprp8 (K1864E) with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1430\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eprp8 (T1865K) TRP CEN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eprp8 (T1865K) with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1431\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;prp8-162\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eprp8 (V1870N) TRP CEN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eprp8 (V1870N) with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e3745\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;prp8-101\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eprp8 (E1960K) TRP CEN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eprp8 (E1960K) with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1432\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;prp8-153\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eprp8 (E1982A) TRP CEN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eprp8 (E1982A) with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1433\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;prp8-161, prp8-101\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eprp8 (P986T) TRP CEN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eprp8 (P986T, E1960K) with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1434\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;prp8-162\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eprp8 (V1870N) TRP CEN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003eprp8 (F1834L, E1960K) with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1435\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC111 pGAL-\u003cem\u003eSNU17\u003c/em\u003e-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. cerevisiae SNU17\u003c/em\u003e gene under GAL promoter and ADH terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1436\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC111 pGAL-\u003cem\u003eECM2\u003c/em\u003e-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. cerevisiae ECM2\u003c/em\u003e gene under GAL promoter and ADH terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1437\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eYC111 pGAL-\u003cem\u003eURN1\u003c/em\u003e-tADH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. cerevisiae URN1\u003c/em\u003e gene under GAL promoter and ADH terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1438\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eU1\u0026bull;{WT}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003epYE195 SNR19-WT +\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. cerevisiae\u0026nbsp;\u003c/em\u003eSNR19-WT+ gene with its own promoter(1000bp) and terminator(310bp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1439\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eU1\u0026bull;{A7G}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003epYE195 snr19 (A7G)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. cerevisiae\u0026nbsp;\u003c/em\u003esnr19 (A7G)\u003cem\u003e\u0026nbsp;\u003c/em\u003egene with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1440\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eU5\u0026bull;{WT}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003epYE195 SNR7-L-WT +\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. cerevisiae\u0026nbsp;\u003c/em\u003eSNR 7-L-WT+ gene with its own promoter(100bp) and terminator(294bp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1441\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eU5\u0026bull;{U(98)C}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003epYE195 snr7-L(U98C)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. cerevisiae\u0026nbsp;\u003c/em\u003esnr7-L (U98C) gene with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1442\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eU5\u0026bull;{U(98)G}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003epYE195 snr7-L(U98G)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. cerevisiae\u0026nbsp;\u003c/em\u003esnr7-L (U98G) gene with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1443\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eU5\u0026bull;{U(98)A}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003epYE195 snr7-L(U98A)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. cerevisiae\u0026nbsp;\u003c/em\u003esnr7-L (U98A) gene with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1444\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eU6\u0026bull;{WT}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003epYE195 SNR6-WT +\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. cerevisiae\u0026nbsp;\u003c/em\u003eSNR6-WT+ gene with its own promoter(1000bp) and terminator(374bp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1445\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eU6\u0026bull;{A51G}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003epYE195 snr6(A51G)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. cerevisiae\u0026nbsp;\u003c/em\u003esnr6 (A51G) gene with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1446\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eU6\u0026bull;{A51U}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003epYE195 snr6(A51U)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. cerevisiae\u0026nbsp;\u003c/em\u003esnr6 (A51U) gene with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1447\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eU6\u0026bull;{G50U}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003epYE195 snr6(G50U)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. cerevisiae\u0026nbsp;\u003c/em\u003esnr6 (G50U) gene with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1448\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eU6\u0026bull;{G50C}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003epYE195 snr6(G50C)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. cerevisiae\u0026nbsp;\u003c/em\u003esnr6 (G50C) gene with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1449\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eU6\u0026bull;{A49U}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003epYE195 snr6(A49U)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. cerevisiae\u0026nbsp;\u003c/em\u003esnr6 (A49U) gene with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1450\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eU6\u0026bull;{U46C}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003epYE195 snr6(U46C)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. cerevisiae\u0026nbsp;\u003c/em\u003esnr6 (U46C) gene with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1451\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eU6\u0026bull;{A45U}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003epYE195 snr6(A45U)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. cerevisiae\u0026nbsp;\u003c/em\u003esnr6 (A45U) gene with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1452\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eU6\u0026bull;{A45G}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003epYE195 snr6(A45G)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. cerevisiae\u0026nbsp;\u003c/em\u003esnr6 (A45G) gene with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003eD1453\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eU6\u0026bull;{T46C, A45T}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003epYE195 snr6(T46C, A45T)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. cerevisiae\u0026nbsp;\u003c/em\u003esnr6 (T46C, A45T) gene with its own promoter and terminator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2: Yeast strains used in this study.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"606\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrain\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenotype\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRef.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYJU75 \u0026nbsp;(SC11)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eMATa ade2 cup1\u003c/em\u003e\u003cem\u003eD\u003c/em\u003e\u003cem\u003e::ura3 his3 leu2 lys2 prp8\u003c/em\u003e\u003cem\u003eD\u003c/em\u003e\u003cem\u003e::LYS2 trp1; pJU169 (PRP8 URA3 CEN ARS)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eGift from C. Guthrie\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eSC87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003eYJU75\u003cem\u003e\u0026nbsp;hub1::\u003c/em\u003eKanMX6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eSC196\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003eYJU75 PRP8 TRP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eSC197\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003eYJU75 \u003cem\u003eprp8-101 TRP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eSC198\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003eYJU75 PRP8 TRP \u003cem\u003ehub1::\u003c/em\u003eKanMX6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eSC200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eYJU75 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eSC214\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eYJU75 prp8-101P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eSC224\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eYJU75 hub1::KanMX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eSC297\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003eSC200 \u003cem\u003enam8::\u003c/em\u003eKanMX6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eSC321\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003eSC11\u003cem\u003e.\u0026nbsp;\u003c/em\u003eDib1\u003cem\u003e\u0026ndash;\u003c/em\u003e\u003cem\u003ehub1(D22A)\u003c/em\u003e\u003cem\u003e:: natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eSC322\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003eSC11\u003cem\u003e.\u0026nbsp;\u003c/em\u003ePrp31\u003cem\u003e\u0026ndash;\u003c/em\u003e\u003cem\u003ehub1(D22A)\u003c/em\u003e\u003cem\u003e:: natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eSC323\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003eSC11\u003cem\u003e.\u0026nbsp;\u003c/em\u003eSad1\u003cem\u003e\u0026ndash;\u003c/em\u003e\u003cem\u003ehub1(D22A)\u003c/em\u003e\u003cem\u003e:: natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eSC324\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003eSC87\u003cem\u003e.\u0026nbsp;\u003c/em\u003eDib1\u003cem\u003e\u0026ndash;\u003c/em\u003e\u003cem\u003ehub1(D22A)\u003c/em\u003e\u003cem\u003e:: natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eSC325\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003eSC87\u003cem\u003e.\u0026nbsp;\u003c/em\u003ePrp31\u003cem\u003e\u0026ndash;\u003c/em\u003e\u003cem\u003ehub1(D22A)\u003c/em\u003e\u003cem\u003e:: natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eSC326\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003eSC87\u003cem\u003e.\u0026nbsp;\u003c/em\u003eSad1\u003cem\u003e\u0026ndash;\u003c/em\u003e\u003cem\u003ehub1(D22A)\u003c/em\u003e\u003cem\u003e:: natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cem\u003eBY4741\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eMATa his3\u0026Delta;1 leu2\u0026Delta;0 met15\u0026Delta;0 ura3\u0026Delta;0\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM601\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eBY4741 hub1 ::KanMX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM602\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eBY4741 lea1::KanMX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM603\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eBY4741 ecm2::KanMX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM604\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eBY4741 bud13::KanMX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eBY4741 nup60::KanMX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM606\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eBY4741 lsm1::KanMX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM607\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eBY4741 sky1::KanMX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM608\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eBY4741 snu17::KanMX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM609\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eBY4741 pml1::KanMX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM610\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eBY4741 urn1::KanMX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM611\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eBY4741 mlp2::KanMX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM612\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eBY4741 mlp1::KanMX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM613\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eBY4741 pml39::KanMX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM614\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eBY4741 brr1::KanMX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM615\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eBY4741 mud2::KanMX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM616\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eBY4741 mud1::KanMX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM617\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eBY4741 prp17::KanMX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM618\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eBY4741 npl3::KanMX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM619\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eBY4741 cwc15::KanMX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM620\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eBY4741 cwc21::KanMX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM621\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eBY4741 cwc27::KanMX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM622\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eBY4741 ntc20::KanMX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM623\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eBY4741 ntc30::KanMX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM624\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eBY4741 ntc31::KanMX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cem\u003eW303\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eade2-1 his3-11, 15 leu2-3, 112 \u0026nbsp; \u0026nbsp; ura3 trp1-1 ssd1 can1-100\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eGift from K. Nasmyth\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM625\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eW303a P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::KanMX6\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM626\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003eW303a\u003cem\u003e\u0026nbsp;P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::KAN Prp8\u0026ndash;hub1(D22A)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM627\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003eW303a\u003cem\u003e\u0026nbsp;P\u003csub\u003eCUP1-\u003c/sub\u003e\u003c/em\u003e\u003cem\u003e\u003csub\u003e1\u003c/sub\u003e\u003c/em\u003e\u003cem\u003eTAP\u0026ndash;\u003c/em\u003e\u003cem\u003eSRC1::KAN Snu66\u003c/em\u003e\u003cem\u003e\u0026ndash;\u003c/em\u003e\u003cem\u003ehub1(D22A)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM628\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003eW303a\u003cem\u003e\u0026nbsp;P\u003csub\u003eCUP1-\u003c/sub\u003e\u003c/em\u003e\u003cem\u003e\u003csub\u003e1\u003c/sub\u003e\u003c/em\u003e\u003cem\u003eTAP\u0026ndash;\u003c/em\u003e\u003cem\u003eSRC1::KAN Prp38\u003c/em\u003e\u003cem\u003e\u0026ndash;\u003c/em\u003e\u003cem\u003ehub1(D22A)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM629\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003eW303a\u003cem\u003e\u0026nbsp;P\u003csub\u003eCUP1-\u003c/sub\u003e\u003c/em\u003e\u003cem\u003e\u003csub\u003e1\u003c/sub\u003e\u003c/em\u003e\u003cem\u003eTAP\u0026ndash;\u003c/em\u003e\u003cem\u003eSRC1::KAN Snu66\u003c/em\u003e\u003cem\u003e\u0026ndash;\u003c/em\u003e\u003cem\u003ehub1(D22A) Prp38\u003c/em\u003e\u003cem\u003e\u0026ndash;\u003c/em\u003e\u003cem\u003ehub1(D22A)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM630\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eW303a hub1::HIS3MX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::KanMX6\u003c/em\u003e \u003cem\u003eSNU17\u0026ndash;\u003c/em\u003e\u003cem\u003ehub1(D22A)\u003c/em\u003e\u003cem\u003e:: natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM631\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eW303a hub1::HIS3MX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::KanMX6\u003c/em\u003e \u003cem\u003ePRP4\u0026ndash;\u003c/em\u003e\u003cem\u003ehub1(D22A)\u003c/em\u003e\u003cem\u003e:: natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM632\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eW303a hub1::HIS3MX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::KanMX6\u003c/em\u003e \u003cem\u003eBRR2\u0026ndash;\u003c/em\u003e\u003cem\u003ehub1(D22A)\u003c/em\u003e\u003cem\u003e:: natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM633\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eW303a hub1::HIS3MX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::KanMX6\u003c/em\u003e \u003cem\u003ePRP6\u0026ndash;\u003c/em\u003e\u003cem\u003ehub1(D22A)\u003c/em\u003e\u003cem\u003e:: natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM634\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eW303a hub1::HIS3MX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::KanMX6\u003c/em\u003e \u003cem\u003ePRP3\u0026ndash;\u003c/em\u003e\u003cem\u003ehub1(D22A)\u003c/em\u003e\u003cem\u003e:: natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM635\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eW303a hub1::HIS3MX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::KanMX6\u003c/em\u003e \u003cem\u003eLEA1\u0026ndash;\u003c/em\u003e\u003cem\u003ehub1(D22A)\u003c/em\u003e\u003cem\u003e:: natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM636\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eW303a hub1::HIS3MX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::KanMX6\u003c/em\u003e \u003cem\u003ePRP9\u0026ndash;\u003c/em\u003e\u003cem\u003ehub1(D22A)\u003c/em\u003e\u003cem\u003e:: natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM637\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eW303a hub1::HIS3MX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::KanMX6\u003c/em\u003e \u003cem\u003ePRP11\u0026ndash;\u003c/em\u003e\u003cem\u003ehub1(D22A)\u003c/em\u003e\u003cem\u003e:: natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM638\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eW303a hub1::HIS3MX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::KanMX6\u003c/em\u003e \u003cem\u003eMUD1\u0026ndash;\u003c/em\u003e\u003cem\u003ehub1(D22A)\u003c/em\u003e\u003cem\u003e:: natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eYSKM639\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eW303a hub1::HIS3MX6 P\u003csub\u003eCUP1-1\u003c/sub\u003eTAP\u0026ndash;SRC1::KanMX6\u003c/em\u003e \u003cem\u003eNAM8\u0026ndash;\u003c/em\u003e\u003cem\u003ehub1(D22A)\u003c/em\u003e\u003cem\u003e:: natNT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003eSC186\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 445px;\"\u003e\n \u003cp\u003e\u003cem\u003eW303a snu66::HIS3MX6\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3: Splicing reporters used in this study.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"604\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSplicing Reporters\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 472px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDescription\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cem\u003eACT1\u003c/em\u003e\u0026ndash;\u003cem\u003eCUP1\u0026nbsp;\u003c/em\u003ereporter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 472px;\"\u003e\n \u003col style=\"list-style-type: lower-roman;\"\u003e\n \u003cli\u003e\u0026nbsp;5\u0026rsquo;SS GUAUGU (Gift from C. Guthrie)\u003c/li\u003e\n \u003cli\u003e5\u0026rsquo;SS GCAUGU (D1454)\u003c/li\u003e\n \u003cli\u003e5\u0026rsquo;SS GCAAGU (D1455)\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cem\u003eHEH1L\u003c/em\u003e\u0026ndash;\u003cem\u003eCUP1\u003c/em\u003e reporter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 472px;\"\u003e\n \u003cp\u003e\u003cem\u003eACT1\u003c/em\u003e exon1-intron-exon2 in\u003cem\u003e\u0026nbsp;ACT1\u003c/em\u003e\u0026ndash;\u003cem\u003eCUP1\u003c/em\u003e reporter is replaced with \u003cem\u003eHEH1\u003c/em\u003e exon1-intron-exon2 (nucleotides 1874-2094) to keep \u003cem\u003eHEH1L\u003c/em\u003e 5\u0026rsquo;SS GUGAGU in frame with\u003cem\u003e\u0026nbsp;CUP1.\u003c/em\u003e Its variants were made using SDM.\u003c/p\u003e\n \u003col style=\"list-style-type: lower-roman;\"\u003e\n \u003cli\u003e5\u0026rsquo;SS \u003cu\u003eGUGAGU\u003c/u\u003eAC (D1456)\u003c/li\u003e\n \u003cli\u003e5\u0026rsquo;SS \u003cu\u003eGUGAGA\u003c/u\u003eAC (D1457)\u003c/li\u003e\n \u003cli\u003e5\u0026rsquo;SS \u003cu\u003eGUAUAU\u003c/u\u003eAC (D1458)\u003c/li\u003e\n \u003cli\u003e5\u0026rsquo;SS \u003cu\u003eGUAUAA\u003c/u\u003eAC (D1459)\u003c/li\u003e\n \u003cli\u003e5\u0026rsquo;SS \u003cu\u003eGUAUGU\u003c/u\u003eAC (D1460)\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cem\u003eHEH1S\u003c/em\u003e\u0026ndash;\u003cem\u003eCUP1\u003c/em\u003e reporter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 472px;\"\u003e\n \u003cp\u003e\u003cem\u003eACT1\u003c/em\u003e exon1-intron-exon2 in \u003cem\u003eACT1\u003c/em\u003e\u0026ndash;\u003cem\u003eCUP1\u003c/em\u003e reporter is replaced with \u003cem\u003eHEH1\u003c/em\u003e exon1-intron-exon2 (nucleotide 1874-2095) to keep \u003cem\u003eHEH1S\u003c/em\u003e 5\u0026rsquo;SS GCAAGU in frame with\u003cem\u003e\u0026nbsp;CUP1.\u0026nbsp;\u003c/em\u003eIts variants were made using SDM.\u003c/p\u003e\n \u003col style=\"list-style-type: lower-roman;\"\u003e\n \u003cli\u003e5\u0026rsquo;SS \u003cu\u003eGCAAGU\u003c/u\u003eGUGAGUAC (D1461)\u003c/li\u003e\n \u003cli\u003e5\u0026rsquo;SS \u003cu\u003eGCAAGU\u003c/u\u003eGUGAGAAC (D1462)\u003c/li\u003e\n \u003cli\u003e5\u0026rsquo;SS \u003cu\u003eGCAACU\u003c/u\u003eGUAUAUAC (D1463)\u003c/li\u003e\n \u003cli\u003e5\u0026rsquo;SS \u003cu\u003eGCAAGU\u003c/u\u003eGUAUAAAC (D1464)\u003c/li\u003e\n \u003cli\u003e5\u0026rsquo;SS \u003cu\u003eGCAAGU\u003c/u\u003eGUAUGUAC (D1465)\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cem\u003eHEH1L\u003c/em\u003e\u0026ndash;\u003cem\u003eLACZ\u0026nbsp;\u003c/em\u003ereporter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 472px;\"\u003e\n \u003cp\u003e\u003cem\u003eRP51\u003c/em\u003e exon1-intron-exon2 is replaced with \u003cem\u003eHEH1\u003c/em\u003e exon1-intron-exon2 (nucleotide 1792-2190) is fused with the \u003cem\u003eLACZ\u0026nbsp;\u003c/em\u003egene to keep \u003cem\u003eHEH1L\u003c/em\u003e 5\u0026rsquo;SS GUGAGU in frame with\u003cem\u003e\u0026nbsp;LACZ.\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cem\u003eHEH1S\u003c/em\u003e\u0026ndash;\u003cem\u003eLACZ\u0026nbsp;\u003c/em\u003ereporter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 472px;\"\u003e\n \u003cp\u003e\u003cem\u003eRP51\u003c/em\u003e exon1-intron-exon2 is replaced with \u003cem\u003eHEH1\u003c/em\u003e exon1-intron-exon2 (nucleotide 1792-2191) is fused with the \u003cem\u003eLACZ\u0026nbsp;\u003c/em\u003egene to keep \u003cem\u003eHEH1S\u003c/em\u003e 5\u0026rsquo;SS GCAAGU in frame with\u003cem\u003e\u0026nbsp;LACZ.\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Alternative 5’ splice site, SRC1, Prp8, RES complex, Ecm2, U5 snRNA, U6 snRNA, B complex, Bact complex","lastPublishedDoi":"10.21203/rs.3.rs-7007499/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7007499/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlternative splicing of precursor-messenger RNAs (pre-mRNA) bearing introns with competing and overlapping 5’ splice sites (5’SS) produces more mRNAs. We investigated the mechanism of alternative splicing via competing 5’SS by monitoring RNA and protein products of the yeast \u003cem\u003eSRC1/HEH1\u003c/em\u003e gene in different mutant strains. \u003cem\u003eHEH1\u003c/em\u003e alternative splicing requires a sixteen-nucleotide pre-mRNA segment spanning its two 5’SS. The nucleotides are decoded by U5 and U6 small nuclear RNAs (snRNA), supported by specific proteins of the spliceosomal B and Bact complexes, including Prp8. \u003cem\u003eHEH1\u003c/em\u003e alternative splicing became independent of the supporting proteins following recalibration of the pre-mRNA-snRNA base pairings through changes in the 5’SS or U5 and U6 snRNAs. Assisted by proteins that stabilize low-fidelity, high-efficiency spliceosome conformations, the competing \u003cem\u003eHEH1\u003c/em\u003e 5’SS are marked for alternative splicing by U5 and U6 snRNAs during B to Bact transition of the spliceosome.\u003c/p\u003e","manuscriptTitle":"Mechanism of alternative splicing through competing 5’ splice sites","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-18 07:30:21","doi":"10.21203/rs.3.rs-7007499/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5e948c40-fc07-42eb-8899-a8864546a655","owner":[],"postedDate":"July 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":51731284,"name":"Biological sciences/Molecular biology/RNA metabolism/Alternative splicing"},{"id":51731285,"name":"Biological sciences/Biochemistry/RNA"}],"tags":[],"updatedAt":"2026-01-24T08:06:50+00:00","versionOfRecord":{"articleIdentity":"rs-7007499","link":"https://doi.org/10.1038/s41467-025-67659-8","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-12-16 05:00:00","publishedOnDateReadable":"December 16th, 2025"},"versionCreatedAt":"2025-07-18 07:30:21","video":"","vorDoi":"10.1038/s41467-025-67659-8","vorDoiUrl":"https://doi.org/10.1038/s41467-025-67659-8","workflowStages":[]},"version":"v1","identity":"rs-7007499","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7007499","identity":"rs-7007499","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}