{"paper_id":"604a0e28-ef6d-46ee-9317-8dd72edb805d","body_text":"1 \nComprehensive analysis of yeast +1 ribosomal frameshifting unveils \na novel stimulator affirming two distinct frameshifting mechanisms \nDarren A Fenton1,#, Maria Bożko2, Michał I. Świrski2, Gary Loughran1, Martina M Yordanova1, Joanna \nKufel2, John F Atkins1,3, Pavel V Baranov1* \n1School of Biochemistry and Cell Biology, University College Cork, Ireland  \n2Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland  \n3School of Microbiology, University College Cork, Cork T12 K8AF, Ireland. \n#current affiliation: Eunice Kennedy Shriver National Institute of Child Health and Human Development, National \nInstitutes of Health, Bethesda, Maryland 20892, USA \n*Corresponding author: p.baranov@ucc.ie \nAbstract \nRibosomal frameshifting allows the synthesis of a protein from different reading frames. Generally, it involves \na dissociation of the P-site tRNA from its codon and movement to the +1 codon setting the new frame for \nincoming tRNAs. This movement is stabilized by tRNA repairing with the +1 codon. However, in several \noccurrences in the yeast Saccharomyces cerevisiae, P-site tRNA repairing with the +1 codon is impossible. \nOne hypothesis suggests that frameshifting involv es P-site tRNA movement, w ith repairing not being \nessential. In the alternative model the A-site tRNA acceptance at the +1 codon occurs in the absence of P-\nsite tRNA movement. Here we performed a comparative analysis of all known +1 frameshifting sites  in S. \ncerevisiae. In addition , we discovered a novel gene requiring +1 frameshifting for its expression. W e also \nidentified a conserved RNA structure located upstream of  the ABP140 frameshifting site. This structure \nsubstantially increases frameshifting efficiency. Its location suggests that it is formed in mRNA exiting the \nribosome, creating a pulling effect  favouring positioning of the +1 codon in the P-site. We show that  the \nstimulation is limited to frameshifting sites where P-site tRNA repairing is possible, supporting the existence \nof two distinct mechanisms of +1 ribosomal frameshifting.  \nKeywords: Translational Recoding, +1 Ribosomal Frameshifting, mRNA translation, Ty1, ABP140, OAZ1, \nEST3, YPL034W, YJR112W-A, YFS1, YFS2 \n \n \n \n \n  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.644219doi: bioRxiv preprint \n\n 2 \nIntroduction    \n \nTranslating ribosomes are capable of deviating from the “standard” decoding rules in response to specific \nmRNA signals. These deviations are collectively termed “recoding events” (1–3). Ribosomal frameshifting is \na form of recoding where some ribosomes shift their reading frame at a specific site during translation, \nresulting in the creation of a trans -frame protein in addition to the standard translation product.  While \nribosomal frameshifting is common in mobile elements and especially in RNA viruses (4), it is extremely rare \nin chromosomal gene decoding with the exception of ciliates Euplotes where it is considered a feature of its \ngenetic code (5). Normally, frameshifting occurs at a specific combination of (i) codons that allow tRNA \nrepositioning with codons in a new frame and (ii) stimulatory signals that elevate its efficiency through a \nvariety of molecular mechanisms. \nThe first example of a eukaryotic gene requiring frameshifting was discovered in the Saccharomyces \ncerevisiae transposable element Ty1 and was found to utilize efficient +1 ribosomal frameshifting (6–9). \nStrikingly, it was found that a minimal sequence of 7 nucleotides is sufficient to support a remarkably high \n(~40%) frameshifting efficiency in the absence of additional RNA stimulators in the vicinity of the frameshifting \nsite (10). The sequence of the Ty1 frameshifting heptamer is CUU_A.GG_C where underscore indicates \ncodon boundaries in the initial (0) reading frame and dot indicates codon boundaries in the new (+1) frame \n(this notation will be used thereafter). A remarkable feature of this frameshifting site is the high imbalance in \nthe levels of tRNAs recognising the AGG (Arg) and GGC (Gly) codons. The S. cerevisiae genome contains \nonly a single gene copy of Arg-tRNA (CCT), while the number of gene copies of Gly-tRNA (GCC) is up to 18 \ndepending on the strain (11). This suggests a far greater concentration of tRNAs cognate for GGC over \ntRNAs cognate for AGG and subsequently faster decoding of the GGC codon in comparison with the AGG. \nPresumably, slow decoding of AGG allows more time for repairing of the Leu -tRNA in the P-site from CUU \nto UUA while fast decoding of GGC would prevent repairing mRNA in the reverse direction from UUA to CUU \n(10, 12). Subsequently, the same frameshift-inducing heptamer was identified in the genes ABP140 (13) and \nYPL034W/YFS1 (14). Here, we also report its utilization for the expression of YJR112W-A/YFS2. A different \npattern, using the same P -site codon but different A -site codons (CUU_A.GU_U) was found to cause  +1 \nframeshifting in EST3 (15). Albeit different and less efficient, the mechanism of EST3 frameshifting may be \nsimilar. In S. cerevisiae, AGU decoding requires wobble interactions and may be slow, while the number of \ncopies for the cognate Val-tRNA is high reaching up to 21 (11). \nA more different +1 frameshifting heptamer (GCG_A.GU_U) was identified in the Ty3 transposable element \n(16). While the A-site component of this pattern is identical to EST3 frameshifting, the P-site codon (GCG) is \ndifferent and seemingly does not support repairing in the +1 reading frame. Importantly, while the optimal \nAla-tRNA (CGU) for GCG is present , it is rare. The same codon can be decoded by a near cognate \nisoacceptor tRNA with anticodon CGI which is apparently responsible for frameshifting since the oversupply \nof Ala-tRNA(CGU) inhibits frameshifting (17). The same P-site codon is used in the S. cerevisiae antizyme \n(OAZ1/YPL052W) frameshifting heptamer (GCG_U.GA_C) where frameshifting is driven by competition \nbetween termination at a stop codon in poor context with incorporation of a tRNA at the +1 codon. The \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.644219doi: bioRxiv preprint \n\n 3 \nefficiency of antizyme frameshifting is sensitive to polyamine levels (18–20). The discovery of an unusual P-\nsite codon in the Ty3 frameshifting heptamer prompted a subsequent investigation of the repertoire of P-site \ncodons supporting +1 ribosomal frameshifting . It revealed several P-site codons supporting above -\nbackground levels of +1 frameshifting in A.GU_U or A.GG_C A -site contexts with little P -site repairing \npotential with the +1 codon (21). This led to the suggestion that certain tRNAs, when in the P site, possess \nproperties that allow ribosomes to incorporate A-site tRNAs in the +1 frame without prior P-site tRNA slippage \n(22). To unify these observations into a single parsimonious mechanism, kinetic considerations have been \nused to argue that it is the dissociation of the P-site tRNA from its zero-frame codon that is the limiting step. \nEven short-lived unstable P -site tRNA int eractions with the +1 codon may be sufficient for high efficien cy \nframeshifting given the high imbalance between cognate in - and out -of-frame tRNAs in the A -site (12). \nHowever, until now, little experimental evidence has been provided to resolve this conundrum. It has \nremained unclear whether one or two different mechanisms are responsible for +1 ribosomal frameshifting in \nyeast at different P-site codons (Fig. 1). \n \n \nFigure 1. Models of +1 Frameshifting Mechanisms in Saccharomyces cerevisiae. Top model: Peptidyl-\ntRNA slippage and repairing to the overlapping UUA codon in the +1 frame is followed by A-site recognition \nof the tRNA decoding the +1-frame GGC codon. The middle and bottom depict two alternative mechanisms \nproposed to explain frameshifting when tRNA repairing with the +1 codon in the P-site could not occur. Middle \nmodel: Peptidyl-tRNA slippage, with no repairing, followed by A -site recognition of the tRNA deco ding the \n+1-frame GUU codon. Bottom model: +1 frameshifting involves no slippage or repairing of the peptidyl-tRNA. \nInstead, frameshifting occurs due to direct recognition of tRNA to the +1 codon in the A-site. \n \nIn this study, our initial motivation was to carry out a systematic comparative study of naturally occurring +1 \nframeshifting sites, with a goal to provide a reliable reference for +1 frameshifting efficiencies in yeast. This \nwas due, since prior frameshif ting measurements were made in different research groups using different \nreporter systems that may be subject to various technical artifacts (23), and thus could not be directly \ncompared. For this purpose, we have developed a new vector designed for studying recoding signals in \nyeast, based on the StopGo reporter system previously developed for mammalian cell lines (24). Using this \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.644219doi: bioRxiv preprint \n\n 4 \nreporter system, we measured ribosomal frameshifting at all the above-mentioned cases with and without \ntheir surrounding mRNA context. The exploration of the contexts revealed the existence of a previously \nunknown +1 frameshifting stimulator within the ABP140 mRNA, located upstream of the frameshifting site \nheptamer. This stimulator increases +1 frameshifting efficiency from 40% to 60%. Phylogenetic analysis of \nthe corresponding sequence combined with site-directed mutagenesis strongly suggest that this stimulator is \nan RNA structure whose formation pushes the ribosome downstream into the +1 frame. We tested the effect \nof this stimulator on different frameshifting heptamers. This enabled us to discriminate frameshifting occurring \ndue to P-site tRNA slippage or direct out-of-frame A-site tRNA recognition. \nIn addition to resolving a long -standing mechanistic conundrum, our study provides a reference to all S. \ncerevisiae +1 frameshifting cases reported so far, including a novel case we discovered while exploring \navailable ribosome profiling data. Finally, ribosome profiling data was used as an orthogonal approach for \nestimating endogenous levels of ribosomal frameshifting. \n \nResults \n \nA new reporter to test recoding efficiencies in S. cerevisiae.  \nMeasurements of frameshifting efficiencies are obtained using vectors that contain candidate frameshifting \nsequences fused between two expression reporters. Previously used reporters in yeast frameshifting studies \nincluded β-galactosidase, luciferase, β-galactosidase-luciferase fusions, and dual-luciferase assays (7, 25, \n26). Luciferase assays are attractive as they allow sensitive readings, especially for cases where a low \nrecoding signal needs to be measured. An in-frame control (i.e. both reporters in the same frame) is used to \nestablish the \"100% efficiency\" signal, allowing comparison with out -of-frame reporters to determine \nframeshifting efficiency (23). However, the protein sequence encoded by the test sequence could affect the \nactivities of one or both reporters , leading to alterations in measured activities  and distorting accurate \nmeasurements of frameshifting (23). To mitigate this issue, the original dual luciferase reporter vector (pDLuc) \ndeveloped for use in mammalian  models (27, 28) was modified to incorporate StopGo/2A on both sides of \nthe test sequence  in pSGDLuc vector  (24). The StopGo/2A peptide motif results in the synthesis of two \nseparated protein products (encoded upstream and downstream of it) because the translating ribosome fails \nto form a peptide bond at a specific position (29) These motifs were originally found in viruses where they \nare responsible for the production of distinct products from the same translated ORF  (30). Therefore, most \nreporter proteins produced from the in -frame and test sequence are identical, allowing a more accurate \ndetermination of the frameshifting efficiencies. Indeed the use of StopGo/2A-containg dual luciferase reporter \nled to identification of a false positive frameshifting case in a human gene due to limitations of the previous \nreporter (31).  \nWe adapted the mammalian pSGDLuc reporter plasmid for use in S. cerevisiae with three modifications. 1). \nWe optimised Renilla and Firefly luciferase coding sequences by introducing synonymous codon \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.644219doi: bioRxiv preprint \n\n 5 \nsubstitutions based on yeast codon usage (Methods). 2). As the StopGo/2A motif used in the mammalian \nreporter (F2A, derived from foot -and-mouth disease virus) is only ~50% efficient in S. cerevisiae , we \nsubstituted it with a more efficient variant, E2A (derived from the equine rhinitis A virus) which is ~90% \nefficient (32). The new vector backbone encodes both leucine and G418 selection markers (Fig. 2A). 3). We \nmodified a multiple cloning site to incorporate the NheI and NdeI restriction enzyme sites to allow cloning via \nT4 ligation or Gibson assembly reactions. The new ve ctor termed pYSGDLuc (Fig. 2A) can be used for \nvarious types of recoding studies in yeast. \n \n \nFigure 2. Uniform assessment of frameshifting efficiency at known frameshifting cases. A,  A \nschematic representation of the pYSGDLuc. LEU2 represents the Leucine biosynthesis gene and AmpR and \nKanR represent ampicillin (for E. coli cloning) and kanamycin resistance (for yeast G418 selection) genes. \nB, Schematic of frameshifting reporters for testing the Ty1 heptamer sequence, including WT and mutant in-\nframe control sequences. C, Representation of natural frameshifting cassettes tested in this study, grey bars \nindicate the length of tested sequences flanking heptamers. D, Frameshifting efficiencies determined with \nluciferase assays. Cases are clustered together based on the frameshifting heptamer (for example, ABP140, \nYFS1, YFS2 and Ty1 frameshifting at CUU_A.GG_C). Error bars represent standard deviation (n=8). E, \nWestern Blotting of replicate cell extracts derived from cultures expressing the Ty1 heptamer (CUU_A.GG_C) \nreporters, using anti-Renilla and anti-Firefly antibodies. Densitometry analyses estimate ~45% frameshifting. \n \nComparative analysis of S. cerevisiae  frameshifting cassettes  using pYSGDLuc  and publicly \navailable ribosome profiling data. \nUsing pYSGDLuc, we performed a comparative study of all known naturally occurring cases of efficient +1 \nframeshifting in S. cerevisiae . Measuring these cases in the same reporter system under the same \nenvironments (e.g. yeast strain, media, equipment and reagents), allows objective comparison of \nframeshifting efficiencies, that are not distorted by the differences in environmental parameters. Each tested \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.644219doi: bioRxiv preprint \n\n 6 \ncase included both an in-frame control and frameshifting test sequence as recommended by newly developed \nguidelines (23), see Fig. 2B. For all cases, we tested the minimum heptamer sequence (e.g. CUU_A.GG_C) \nrequired for frameshifting that includes the P-site and A-site codons before and after frameshifting (Fig. 2C). \nThis provided the basal rate of frameshifting  at corresponding heptamers independent of their natural \nsurrounding nucleotide context. To assess the effect of the frameshifting heptamer within the native mRNA \ncontext, we included a ~100 nt flanking region. The frameshifting site for YSF1 is located close (~42 nt) to \nthe 5’ end of the mRNA transcript, therefore, a shorter upstream test sequence was included . The \nframeshifting efficiencies calculated as a ratio of luciferases ratios between test sequences and controls are \nshown in Figure 2D as percentages. \nTo test if there may be unexpected products produced from these reporters, we carried out western blotting \nto confirm product sizes for Renilla and Firefly reporter proteins (Fig. 2E, left ). Densitometry analyses \nestimated similar frameshifting efficiencies as the luciferase assays (Fig. 2E, right). \nThe measured differences between four frameshifting heptamer -only reporters appeared to be consistent \nwith previous reports, CUU_A.GG_C ~40% > CUU_A.GU_U ~15% > GCG_A.GU_U ~10% > GCG_U.GA_C \n~3% (Fig. 2D). Testing frameshifting  efficiencies at these heptamers demonstrated a certain degree of \ncontext dependence. The inclusion of natural sequence contexts of Ty3 , YFS1 and YFS2 (Fig. 2C) did not \nalter basal levels of frameshifting at their corresponding frameshifting heptamers. However, frameshifting in \nthe ABP140 context was increased 1.5-fold to ~60% and unsurprisingly, near 3-fold in OAZ1 natural context \nto ~8%. The Ty1 and EST3 contexts had opposite effects, with TY1 reducing frameshifting efficiency 2-fold \nto ~20% and 1.5-fold to 10% in EST3.  \nInclusion of the 200 nt surrounding sequence into pYSGDLuc cassette does not entirely recreate the \nparameters of natural frameshifting at least for two reasons. First, sequence elements at large distances from \nthe frameshifting site may alter its efficiencies  (33), second the efficiency of frameshifting may depend on \nparameters of mRNA translation not directly linked to the surrounding sequence, such as ribosome load ing \n(34). Therefore, to assess the natural levels of frameshifting we used publicly available ribosome profiling \ndata and infer red frameshifting efficiency from a change in ribosome footprint density downstream of \nframeshifting sites, similarly to previous work on viral frameshifting (35), see Methods. A global aggregate of \nthese ribosome profiling data from different studies for ABP140, EST3 and OAZ1 is presented in Figure 3A. \nFor most cases, we found a strong concordance between frameshifting efficiencies measured with these two \nmethods (Fig. 3B), suggesting that our pYSGD-Luc reporter provides accurate measurements of  \nframeshifting efficiencies and the absence of relevant context outside of the 200 nt proximal window. We \nnote that for YFS1 and YJR112W-A/YFS2, the short length of coding region upstream of frameshifting sites \nlimits the accuracy of this method in comparison with ABP140, EST3 and OAZ1. Nonetheless, for ABP140, \nEST3 and OAZ1, we find a strong concordance in frameshifting efficiencies between luciferase assays and \nribosome profiling. \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.644219doi: bioRxiv preprint \n\n 7 \n \nFigure 3. Analysis of publicly available ribosome profiling data. A. Subcodon ribosomal profiles for ABP140, \nEST3 and OAZ1 genes. For each gene top plots show aggregated ribosome profiling data differentially \ncoloured based on the supported reading frame. The colours are matched to the reading frames in the ORF \nplot below where black and white dashes represent stop and ATG codons in each reading frame respectively. \nThe position of the frameshifting sites are denoted with black arrows, stop codons of the zero frame ORFs \nare indicated with grey vertical lines. B. Ribosomal frameshifting inferred from 85 individual ribosome profiling \ndatasets based on the relative ratio of footprint densities in the +1 ORF downstream of the zero frame ORF \nare shown as green dots. Blue dots indicate frameshifting efficiencies obtained with dual luciferase assays \nfor each replicate (n=8 -12). The parameters of distribution for both types of data are show with boxplots \nwhere centre line represent s the median, box limits indicate the 25th and 75th percentiles and whiskers \nextend 1.5 times the interquartile range from the 25th and 75th percentiles. C. Same as in A, but for novel \ninstance of frameshifting discovered in this study during translation of YJR112W-A/YFS2 mRNA. Note the \nfull coding regions are not displayed. D. Codon alignment of the 0-frame ORF of YJR112W-A/YFS2 orthologs \nin Saccharomyces genus with the universally conserved CUU_A.GG_C indicated. Yellow codons represent \nsynonymous changes and orange codons represent non-synonymous changes \n \nA novel case of cellular +1 frameshifting at the YJR112W-A/YFS2 locus  \nAn independent m anual analysis of ribosome profiling data revealed an unusual distribution of ribosome \nprotected fragments (RPFs) in the YJR112W-A locus in S. cerevisiae (Fig. 3C). YJR112W-A is annotated as \nan intron containing gene (see Supplementary Fig. S1). However, publicly available RNA-seq data in GWIPS-\nviz (36) suggest that the annotated intron is a part of mRNA as RNA-seq density is uniform across intronic \nand exonic regions of this gene (Supplementary Fig. S1). The highest RPF density matches an ORF initiated \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.644219doi: bioRxiv preprint \n\n 8 \nwith the most 5’ end AUG codon and spans the region annotated as intron and a part of the second annotated \nexon (Supplementary Fig. S1 ). The examination of this ORF sequence revealed the presence of the \nCUU_A.GG_C heptamer suggesting that ribosomes translating this ORF undergo +1 ribosomal frameshifting. \nIndeed, the ribosome footprint density is present downstream of this ORF matching the +1 reading frame, \nalbeit at a lower density than at the zero frame ORF. We conclude that the current S. cerevisiae reference \nannotation of YJR112W-A CDS is incorrect, and that YJR112W-A does not contain introns, instead its CDS \nis comprised of two overlapping ORFs translated via ribosomal frameshifting. We named this gene YFS2 \n(Yeast Frameshifting 2), following naming convention from a previous manuscript that reported a similar \ndiscovery of new +1 frameshifting case (14). The sequence alignments of YFS2/YJR112W-A orthologs from \nother Saccharomyces demonstrated the universal conservation of CUU_A.GG_C pattern in these species \n(Fig 3 D) indicating that it evolves under purifying selection suggesting functional importance of this \nframeshifting site for these species’ fitness. When tested in its native context, YFS2 frameshifting is approx. \n40% efficient, like the CUU_A.GG_C heptamer efficiency. Therefore, it is unlikely this novel case contains \nproximal stimulating or attenuating sequence elements as tested in our luciferase system. We have named \nthis gene YFS2 (Yeast Frame Shift 2), following the more recent discovery of YFS1 frameshifting (14). Both \nYFS1 and YFS2 contain the CUU_A.GG_C frameshift ing heptamer close to the 5’ end of the mRNA \ntranscripts. \n \nThe ABP140 mRNA contains a frameshifting stimulator 5’ of its shift site \nSurprisingly, ABP140 +1 frameshifting is 60% efficient despite it containing the same frameshifting heptamer, \nCUU_A.GG_C, whose frameshifting  efficiency is ~40% in other contexts , suggesting a stimulatory \nframeshifting element within the flanking 200 nt native sequence. The unusually high efficiency of \nframeshifting in endogenous ABP140 context is further supported by ribosome profiling data (Fig. 3A,B). We \nused RNAfold (37) to predict potential RNA secondary structures within flanking regions and identified a \npotential stable RNA stem loop 6 nt upstream of the frameshifting heptamer (Fig. 4A,B). An alignment of \nABP140 sequences from multiple Saccharomyces species shows conservation of sequence specifying the \npredicted stem loop at this position with several compensatory substitutions that maintain potential base \npairing (Fig. 3E). To analyse whether there is an increased purifying selection acting on synonymous \npositions within  ABP140 which would be expected in the presence of RNA secondary structure  under \npurifying evolutionary selection, we used Synplot2 (38). It reveals a statistically significant decrease in the \nsynonymous substitution rates in comparison with regions outside of the potential RNA secondary structure \n(Fig. 3C,D). \nIntroduction of synonymous changes that would disrupt the predicted RNA  secondary structure in the \npYSGDLuc bearing the ABP140 frameshifting cassette reduced frameshifting levels to ~40% . This level is \nsimilar to what is obtained with the frameshifting heptamer alone (Fig. 5C), suggesting that the sequence \nencoding the structure functions as a stimulator of frameshifting . Note that another possibility could be that \nABP140 frameshifting is stimulated by the nascent peptide within the ribosome peptide tunnel, as discovered \nin fungal antizyme sequences (39). To test this possibility, we generated mutations that did not change the \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.644219doi: bioRxiv preprint \n\n 9 \nsequence of the encoded protein but altered the mRNA structure (Fig. 5B). This also removed the stimulatory \neffect suggesting that the stimulation occurs by the RNA structure and not the encoded peptide sequence. \nReplacing the ABP140 stem loop structure with a shorter variant found in Kluyveromyces marxianus ortholog \n(Supplementary Fig. 2) reduced frameshifting to below 50%, but it remains well above 40% in the absence \nof a stem loop structure at this position (Fig. 5C). Progressive truncations from the 5’ end of the S. cerevisiae \nABP140 test sequence also showed that stem loop disruption reduced frameshifting efficiency, and the entire \nstructure is needed for the stimulation. Shortening the structure by 7 nts removed its stimulatory effect (Fig. \n5D). Collectively these data provide strong support for the proposition of the upstream RNA structure as a \nframeshifting stimulator. \n \n \nFigure 4. Conserved RNA stem-loop in ABP140 upstream of the frameshifting site. A. RNA secondary \nstructure (minimum free energy, partition function and centroid values)  and entropy calculations across the \nABP140 test sequence  as calculated via RNAfold.  B. RNA secondary diagram, base substitution in \northologous sequences Base substitutions from the Saccharomyces are denoted with arrows. C. Analysis of \nthe rate of synonymous substitutions with Synplot2 for the ABP140 protein coding region. Observed/expected \nratio of substitutions is shown at the bottom track with corresponding p-values above. Dotted line represents \np < 0.05 threshold. Below is a zoomed region for the frameshifting cassette. Synplot2 data are visualized \nfrom a 17-codon sliding window. E. Codon alignment of the ABP140 coding sequence with S. cerevisiae as \na reference.  \n \nIn theory, in addition to frameshifting, the second luciferase could also be expressed via ribosome reinitiation \ndownstream of the first ORF by non-frameshifted ribosomes that terminated at the in-frame stop codon. To \nexclude this possibility, we removed the second StopGo (E2A) site in the vector (between test sequence and \nFirefly) and caried out W estern Blotting Analysis of yeast cells expressing this reporter . It showed only a \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.644219doi: bioRxiv preprint \n\n 10 \nsingle Firefly luciferase product  and no shorter products that would be expected in case of reinitiation  \n(Supplementary Fig. 3). The frameshifting efficiency measured with luciferase assays remained the same \n(Fig. 5C). \n \n \nFigure 5. The stimulatory mode of upstream RNA structure affirms two distinct mechanisms of +1 \nribosomal frameshifting. A. The proposed model of +1 ribosomal frameshifting by the upstream RNA \nstructure stimulator. B. Tested RNA structures, WT, mutants (via synonymous codon substitutions) and the \nstem loop of Kluyveromyces marxianus. C. Luciferase results showing frameshifting on the ABP140 test \nsequence for wildtype and mutants. E2A represents deletion of the second E2A sequence positioned \nbetween the test sequence and firefly luciferase coding sequence. D. Luciferase assay results showing \ntruncations of the 5’ mRNA context of the ABP140 test sequence. As a control, the Ty1 heptamer and the \noriginal ABP140 test sequence was tested alongside the truncations. E. Luciferase assay results showing \nwildtype frameshifting efficiencies of the heptamers (blue bars) and when these frameshifting heptamers \nare placed in the context of ABP140 (orange bars).  \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.644219doi: bioRxiv preprint \n\n 11 \nThe stimulatory RNA structure promotes P-site tRNA slippage and affirms existence of two distinct \nmechanisms of +1 ribosomal frameshifting.  \nMost RNA structures stimulating ribosomal frameshifting have been found downstream of frameshifting \nsites promoting -1 ribosomal frameshifting (4, 40, 41). In SARS-CoV-2 and likely other instances of \nribosomal frameshifting, the stimulation is achieved as a result of the tensions in mRNA between the \ndecoding centre of the ribosome (A and P-sites) and mRNA tunnel entrance where the stimulatory structure \nis located. This tension prevents triplet movement of mRNA together with tRNAs during the translocation \n(42). However, in bacteria frameshifting is often stimulated by Shine-Dalgarno (SD) and anti-SD \ninteractions. This is likely based on a similar principle of tensions within mRNA caused by these \ninteractions. A short distance between the SD and decoding centre pulls mRNA out of the decoding centre \npromoting +1 frameshifting while a long distance pushes mRNA towards decoding centre promoting -1 \nframeshifting (43, 44). Furthermore, a somewhat similar phenomenon occurs during transcription in \nbacteria. Transcription terminators consist of RNA secondary structures upstream of polyU in RNA:DNA \nhybrid within the RNA polymerase bubble and termination involves pulling polyU towards the structure \nduring its formation (45). Transcriptional slippage occurring due to the realignment of RNA relative to its \nDNA template is also known to be stimulated by RNA secondary structures upstream (46). Inspired by \nthese examples we proposed a model of how the RNA stem-loop stimulator discovered in this study may \nstimulate +1 frameshifting during decoding of the ABP140 mRNA (Fig. 5A). When mRNA is exiting the \nribosome, it starts forming the RNA secondary structure eventually pulling mRNA out of the ribosome, thus \npromoting forward movement of the P-site tRNA and its realignment from the zero frame CUU codon with \nthe +1 UUA codon. \nIf this model is correct the RNA secondary structure should only promote +1 frameshifting that involves P -\nsite tRNA movement relative to mRNA (top two mechanisms in Figure 1, but not the bottom one). Thus, we \nexplored the ABP140 frameshifting stimulator within the context of other known frameshifting heptamers. To \nachieve this, we substituted the native CUU _A.GG_C frameshifting site with the frameshift ing heptamers \nfrom EST3, Ty3, and OAZ1. If the model is correct and we observe a significant increase in frameshifting \nefficiency, it would imply that all frameshifting sites involve the repairing of peptidyl-tRNA to the overlapping \n+1 frame codon. We observed a substantial increase in +1 frameshifting from 15% to 40% for the EST3 \nheptamer CUU_A.GU_U, but no significant increase for the Ty3 (GCG_A.GU_U) and OAZ1 (GCG_U.GA_C) \nheptamers (Fig. 5E). Therefore, these results strongly sug gest that ribosomal frameshifting on heptamers \nwith GCG in the P-site do not involve tRNA repairing with the +1 codon as originally suggested by (22) and \ninstead, a distinct +1 frameshifting mechanism is present. \n \nDISCUSSION \n \nPrior work on ribosomal frameshifting in S. cerevisiae provided widely varying estimates of frameshifting \nefficiencies, likely because of high variability in assays and the frameshifting cassettes used (47–50). \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.644219doi: bioRxiv preprint \n\n 12 \nTherefore, to be able to compare different cases of frameshifting, it is important to measure them uniformly. \nIn this work, we set out to explore all known instances of +1 ribosomal frameshifting in S. cerevisiae using a \ncombination of approaches: reporter assays, ribosome profiling and comparative sequenc e analysis. While \nthe aim of the study was to provide a comprehensive and uniform characterisation of frameshifting cassettes \nacross different S. cerevisiae genes utilizing ribosomal frameshifting in their expression, several unexpected \nfindings arose.  \nFirst, we found a gene , YJR112W-A, that is misannotated in  the S. cerevisiae genome annotation as an \nintron-containing gene, while it is apparently  transcribed into  an intronless mRNA expressing a protein \nencoded in two ORFs , that are decoded as a single protein via +1 ribosomal frameshifting. This finding \nillustrates that even nowadays, the protein coding catalogues of eukaryotic genes are not compete even in \nspecies with comparatively low frequencies of splicing such as S. cerevisiae. It demonstrates that the current \nannotation pipelines do not adequately capture the complexity of mRNA decoding mechanisms and fail to \ndetect rare decoding strategies. This finding also demonstrates the utility of ribosome profiling data at \novercoming these limitations in identifying novel translated regions.  \nSecond, unexpectedly we identified a  novel mechanism of ribosomal frameshifting stimulation. Hitherto \nframeshifting stimulatory structures were identified downstream of frameshifting sites and are believed to act \nupon elongating ribosomes by slowing down its move ment because of the requirement to unwind these \nstructures (42). Here , we identified a stimulatory structure upstream of frameshifting site in ABP140, \nsuggesting that it acts on the ribosome downstream by promoting its forward movement.  Such a possibility \nis supported by a previous study, in which a stem -loop was shown to stimulate +1 frameshifting when \nartificially placed upstream of the Ty1 frameshift ing heptamer in S. cerevisiae (51). It is likely that such a \nmode of frameshifting stimulation is not limited to S. cerevisiae and may work in other organisms. \nThird, we utilized this novel stimulator to address a long -standing question in the ribosomal frameshifting \nfield. How can  +1 frameshifting work in the absence of apparent repairing of the P-site tRNA with an \noverlapping P -site codon ? Placing the structure upstream of different frameshifting sites revealed its \nspecificity to th ose frameshifting sites that allow for P -site tRNA repairing. It provided no stimulation at \nframeshifting sites where repairing is seemingly impossible, suggesting that in these cases, frameshifting \ndoes not involve repositioning of the P-site codon from the zero to the +1 frame. This finding argues against \na unified model of ribosomal frameshifting in which the forward movement of the P-site tRNA is required \nirrespective of its ability to repair with the +1 codon (12). On the contrary, it suggests that a distinct mechanism \nexists enabling A-site tRNA acceptance at the +1 codon that does not require P -site tRNA repositioning as \nsuggested earlier (22). The existence of two such mechanisms may not be specific to yeast, frameshifting \nsites with seemingly impossible P-site codon repairing have been reported in glass sponge mitochondria (52) \nand under severe limitations of specific amino acids in E. coli (53, 54). \nFinally, while in this study, we focused on the ABP140 stem-loop, other cases of frameshifting also showed \nintriguing results. Both the Ty1 and EST3 test sequences with native contexts showed a decrease in \nframeshifting efficiency suggesting that these contexts may have attenuator elements. Surprisingly, EST3 \nsupposedly contains a stimulatory element (50) surrounding the frameshifting heptamer and we do not \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.644219doi: bioRxiv preprint \n\n 13 \nobserve any such effect with both experimental reporters and publicly available ribosome profiling data \nanalysis. \n \nMethods \n \nCloning \nSnapGene (www.snapgene.com) was used to codon optimise  (based on codon usage)  Renilla and Firefly \nluciferase sequences from pSGDLuc (24) and synthesized as a gblock from IDT (Integrated DNA \nTechnologies, Leuven, Belgium). The reporter cassette was inserted into the pGREG-505 plasmid which was \nlinearized with SalI (NEB, #R3138S) and size selected to remove the HIS3 coding sequence. For insertion \nof short frameshifting heptamers, annealed oligos were ligated into the NdeI (NEB, #R0111S) and NheI (NEB, \n#R3131S) digested vector with T4 DNA ligase (NEB, #M0202S) and incubated overnight at room temperature \nin a 5 µL reaction. For insertion of longer frameshifting cassettes, 60 ng of NdeI and NheI digested plasmid \nand ~7 ng of ~200 bp inserts were added to 15 µL of homemade Gibson assembly master mix (55) and \nincubated at 50 oC for 1 hour in a thermocycler. 5 µL of the Gibson assembly or T4 ligation reaction was \ntransformed into chemically competent E. coli DH5a cells. \nYeast culturing and transformation \n5 mL of YPD  broth was inoculated with  a single colony of  S. cerevisiae (strain BY4741) and incubated \novernight at 30 oC and 200 RPM. The next day, 200-400 ng of plasmid was transformed  into cells via the \nLithium acetate/Salmon sperm carrier method (56). Cultures were plated onto SC 2% glucose media with an \namino acid drop out -Leucine mix (Formedium, #DSCK052) agar plates and incubated at 30oC for 2-3 days \nuntil colonies formed. \nLuciferase Assays \nTwo independent yeast colonies were inoculated in 5 mL 2% glucose -Leucine medium in a 50 mL  Falcon \ntube and incubated overnight at 30oC and 200 rpm. The next day, the A600 (O.D. 600) values were measured \nand the culture was diluted to an A600 of ~0.1. For the 96 well plate assay, 200 µL of culture was plated into \neach well (a total of six wells per biological replicate). The plate was sealed using gas -permeable tissue \nculture seals (4titude, #4ti-0516/384), placed on a plate shaker inside a 30oC incubator and incubated for ~6 \nhours. 50 µL of the culture was then transfer red to a white full area 96 well plate and 50 µL of 2X Passive \nLysis Buffer (Promega) was added to each well and incubated for 50 -60 minutes with shaking at room \ntemperature to allow lysis. 50 µL of homemade LAR (substrate for Firefly luciferase) and 50 µL homemade \nStopGlow (substrate for Renilla luciferase) were injected into each well (57). \nWestern Blotting \nYeast transformants were inoculated in 5 mL -Leucine media in a 50 mL falcon tube and incubated overnight \nat 30oC with 200 RPM. A600 values were measured and ~10 A600 units were transferred to a 1.5 mL tube. \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.644219doi: bioRxiv preprint \n\n 14 \nCells were washed once in sterile water and boiled in 280 µL of 1X SDS-sample buffer for 3 minutes. 5-10 \nµL was transferred onto NuPage protein gels  (Invitrogen) and run for 200V for ~24 minutes  with 1X MES \nrunning buffer (Invitrogen). Proteins were transferred to a Nitrocellulose membrane using a BioRad Transblot. \nMembranes were blocked with 5% low fat milk in 1X PBS-T for 45 minutes at room temperature with gentle \nagitation. The membranes were treated with a primary antibody solution consisting of mouse a nti-Renilla \n(Millipore) and goat anti-Firefly (Promega) in 1% BSA and PBS-T and was incubated overnight at 4oC. The \nmembrane was washed three times in PBS-T for 5 minutes each before and after secondary antibodies (anti-\nmouse red and anti -goat green).  Membranes were visualized using an Odyssey imager.  Densitometry \nanalyses were carried out using imageJ.  \nRibosome Profiling Analysis \nData from each study was obtained via the Ribocrypt browser  (https://ribocrypt.org/). To determine the +1 -\nframeshifting efficiency, the number of RPFs on each frame was normalized by the number of codons. The \nnormalized number of RPFs in the +1 frame was divided by the number in the 0-frame and multiplied by 100 \nto obtains the % frameshifting. \nBioinformatic and Computational Analyses \nABP140 homologs were found using TBLASTN (58) against the genomes of budding yeast species. To \nproduce in-frame alignments for homologs that do not contain frameshifting, the +1 -frame was fused to the \n0-frame by replacing CTTAGGC with CTTGGC. RNAfold was used to determine RNA secondary structures \n(37). Orthologous sequences were aligned using MUSCLE (59) and converted to a codon alignment using \nPAL2NAL (60). A codon alignment viewer was generated with python. The rate of synonymous across \nmultiple sequence alignment was analysed with Synplot2 (38), using a window size of 17 codons. \n \nAuthor Contributions \nDAF and MB carried out experiments. DAF and MS carried out bioinformatic analyses. DAF, JFA and PVB \nconceived the study. D AF, GL and M MY designed experiments and analysed results.  DAF, JFA and PVB \ndrafted the manuscript and all authors participating in manuscript editing. PVB, JK and JFA secured funding \nand provided supervision. \n \nAcknowledgements \nWe thank Yousuf Khan (Stanford University) and Sinéad O’Loughlin for technical advice in the early stages \nof this project. We thank Javier Valera (University College Cork) for supplying the pGREG-505 plasmid. This \nresearch was funded in whole or in part by the Wellcome Trust [210692/Z/18/] and Taighde Éireann – \nResearch Ireland [20/FFP-A/8929] to P.V.B. For the purpose of Open Access, the author has applied a CC \nBY public copyright licence to any Author Accepted Manuscript (AAM) version arising from this su bmission. \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 19, 2025. ; https://doi.org/10.1101/2025.03.19.644219doi: bioRxiv preprint \n\n 15 \nThis work was also supported by grants from Poland National Science Centre [UMO-2021/41/B/NZ2/03036] \nto J.K. and Irish Research Council [IRCLA/2019/74] to J.F.A.  \n \nConflict of Interests \nP.V.B. and G.L. are cofounders and shareholders of EIRNABio Ltd. \n \nReferences \n1. Gesteland,R.F., Weiss,R.B. and Atkins,J.F. (1992) Recoding: reprogrammed genetic decoding. Science \n(New York, N.Y.), 257, 1640–1641. \n2. Baranov,P. V, Atkins,J.F. and Yordanova,M.M. 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