Introns increase gene expression in Caenorhabditis elegans by a notably different mechanism than in plants

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Rose, Aaron Baer, Isaac Shaker, J. Grey Monroe, Ian Korf, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5926918/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 May, 2025 Read the published version in Scientific Reports → Version 1 posted 4 You are reading this latest preprint version Abstract The wide diversity of organisms in which introns stimulate gene expression suggests that this is an ancient phenomenon. However, the mechanisms through which introns boost expression remain poorly understood, and the degree the which the action of introns is evolutionarily conserved is unknown. Here we compared the effect on expression of introns at different positions and tested ten different introns at the same location in a reporter gene in single-copy transgenic nematodes. The introns boosted expression most when near the start of the gene, as previously observed in several organisms. All ten introns tested at the same position increased mRNA accumulation 10- to 17-fold, in contrast to plants where introns vary widely in their effect on expression and relatively few increase mRNA levels 10-fold or more. These results suggest that some aspects of the mechanisms through which introns boost expression are fundamentally different in nematodes and plants. Biological sciences/Genetics/Gene expression Biological sciences/Genetics/Gene regulation Introns gene expression nematode mRNA accumulation intron-mediated enhancement Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Within 2 years of the discovery that protein-coding sequences in eukaryotic genes can be interrupted by intervening sequences (introns)[ 1 ], it was found that introns can increase the expression of the genes in which they are located[ 2 ]. While some introns are known to contain enhancer elements[ 3 ], introns can also have a more general effect on expression with properties that differ from those of enhancers. For many genes, expression declines, in some cases to undetectable levels, if the introns they normally contain are deleted, while the expression of a naturally intronless gene often goes up substantially if an intron from another gene is inserted[ 4 ]. Such findings have been reported in a diverse range of organisms[ 5 – 10 ], suggesting that the way in which introns boost expression is widely conserved. However, this phenomenon has been explored in just a few organisms and is well understood in none. Measuring expression solely as the enzymatic activity or fluorescent protein produced from a reporter gene is common but does not reveal which of the many aspects of gene expression is being influenced by an intron. Therefore, it remains unclear if the actual mechanism through which introns boost expression is the same in all organisms or varies from group to group. Even within a single organism it is risky to assume that all introns affect expression in the same way, or that any intron has only one effect on expression. The known interconnections between many of the various steps of transcription, mRNA processing, export and translation provides multiple opportunities for synergistic effects that increase the overall efficiency of gene expression[ 11 – 16 ]. In mammalian cells, triose phosphate isomerase intron 6 located near the 5’ end of a luciferase reporter gene increases mRNA accumulation roughly 13-fold, while the same intron near the 3’ end of the gene has a 2-fold effect[ 17 ]. Introns also cause a 2- to 4-fold increase in the amount of protein produced per unit of mRNA but have little effect on mRNA stability or nuclear export[ 17 , 18 ]. The increase in mRNA abundance is likely caused by an increase in transcription[ 19 ], as a 5’ splice site enhances the recruitment of transcription initiation factors[ 20 , 21 ] and splicing factors stimulate transcriptional elongation[ 22 – 24 ]. A systematic analysis of the effect of introns on gene expression in plants was first performed by Callis et al. using the Adh1 gene in maize cells[ 5 ]. These authors showed that an intron must be located in transcribed sequences to boost expression, that the intron has the greatest effect when near the 5’ end of the transcript, and that increase in expression is present at the level of mRNA accumulation. Subsequent research confirmed these findings in Arabidopsis[ 25 , 26 ]. In addition, efficiently spliced introns vary widely in their effect on expression, with many having no effect[ 27 , 28 ], and the sequences that stimulate mRNA accumulation are distributed throughout a stimulatory intron[ 28 ]. These observations are not consistent with the idea that the increase in expression caused by plant introns could be due to the act of splicing or involves the conserved sequences at each end of most introns. The need for the intron to be near the promoter to affect expression led to the discovery that promoter-proximal introns in plants are compositionally distinct from other introns[ 28 ]. The location-dependent difference between introns was detected using the frequency distribution of all possible k-mers (usually pentamers or hexamers) in the genomic population of introns separated into two groups based on whether the start of the intron is less or greater than a threshold distance from the start of transcription of that gene. The IMEter algorithm generates a numerical score that describes the degree to which an individual intron is more similar to the k-mer profile of promoter-proximal introns than that of distal introns. The strong correlation between the IMEter score of an intron and its ability to increase mRNA accumulation allows the stimulating ability of an intron to be predicted from its sequence[ 28 ]. Roughly 5–10% of introns in a variety of plants have IMEter scores high enough to indicate that they are likely to have a significant effect on expression[ 29 ]. A motif that is over-represented in Arabidopsis introns with high IMEter scores can convert, in a dose-dependent manner, a non-stimulatory intron into one that strongly boosts expression with the same positional requirements as natural stimulating introns[ 30 , 31 ]. The same motif can increase mRNA accumulation when inserted into exonic sequences of an intronless construct[ 31 ], demonstrating that splicing is not necessary for this motif to boost expression. Remarkably, stimulatory introns in Arabidopsis can boost the expression of a TRP1 gene fusion containing a 303 nt promoter deletion that removes most of the 5’-UTR and all known transcription start sites[ 26 ]. Transcription in those constructs initiates in normally untranscribed intergenic sequences the same distance upstream of the intron as when the promoter is intact. The surprising observation that introns can appear more important than the promoter in controlling the expression of a gene had previously been made in mammals and nematodes[ 32 , 33 ]. In C. elegans , an analysis of the non-coding parts of the unc-54 gene showed that deletion of the introns but leaving the promoter and coding sequences intact nearly eliminates the ability of this gene to rescue an unc-54 mutation in transgenic worms[ 32 ]. In contrast, a construct with the promoter deleted but introns still present is surprisingly functional, with transcription starting in the plasmid sequences that the deletion of the promoter brought next to the unc-54 coding sequences. A version of unc-54 in which the promoter and the introns were simultaneously deleted does not rescue the mutation. A practical response to these findings was the development of intron-containing derivatives of the lacZ and GFP reporter genes that are still widely used in C. elegans . One version of lacZ contained 12 small (51 nt) synthetic introns, as this was reported to yield higher expression than versions with 1, 2, or 4 introns ( https://www.addgene.org/kits/firelab/#protocols-and-resources ). More recently, introns have been inserted into many reporter genes for use in worms[ 34 ]. However, Crane et al.[ 35 ] found that in C. elegans a single intron, either synthetic or natural, increases expression as much as does two or three introns if the lone intron is located near the 5’ end of the gene. The same intron near the 3’ end has no effect. The two synthetic and one natural intron they tested at the same 5’ location cause equivalent increases in expression. The effect of introns varies slightly with different coding sequences and promoters of different strength. The number of introns tested in that study was too small to determine if there are significant differences between natural introns in their ability to boost expression in nematodes as there are in plants. Also, because the effect of introns was measured only as the level of fluorescence in whole transgenic worms containing GFP reporter constructs, it remains unclear which of the many steps that constitute gene expression is most affected by introns in nematodes. Here we report a systematic analysis of the effect of introns on gene expression in transgenic nematodes by inserting the same introns into different locations in a reporter gene, by testing ten different introns at the same position, and by measuring steady state mRNA levels. Introns had the largest effect on expression when placed near the 5’ end of the gene. Introns near the 3’ end had much smaller effects, as did additional introns added to a construct already containing an intron. All ten of the introns tested at the same location boosted mRNA accumulation to a very similar and high degree. This suggests that despite some similarities with plants, key aspects of the underlying mechanism are different in worms and may be related to the mechanism of splicing rather than to specific sequences within introns. Results Testing intron location and number The version of GFP most widely used in the C. elegans community contains three 51 nt synthetic introns in GFP coding sequences. To determine the relative effects on expression of different numbers of introns and intron location, an intronless but otherwise identical derivative of this worm codon-optimized GFP was synthesized. Different combinations of the intron-containing and intronless GFP genes were used to create a series of unc-54:GFP:GFP fusions in which the first, the second, neither or both copies of GFP contained three synthetic introns (Fig. 1a). Transgenic worms containing these constructs were created by Mos1-mediated single copy insertion (MosSCI) and single-copy integration was verified by genomic DNA gel blot analysis. Transgene expression was measured as GFP signal above the endogenous fluorescence of bulk worms using a fluorescent plate reader (Fig. 1b). L1 stage worms were chosen for these measurements because it was possible to generate reasonably synchronized populations of worms at that stage by collecting worms from recently starved plates. Both constructs in which the first GFP contained introns were expressed at a significantly higher level than the constructs lacking introns in the first GFP (Fig. 1b, Supplementary Fig. S1 and Supplementary Table S1, P = 1.92 x 10 -10 ). The presence of introns in the second GFP caused a slight increase in expression whether or not the first GFP contained introns. This suggests that introns near the 5’ end of a gene have a greater effect on expression than the same introns near the 3’ end of the gene, and indicates that intron position is more important than the number of introns in determining expression level. To verify that the same single intron can have different effects on expression depending on its location, an additional set of unc-54:GFP:GFP fusions was created in which the unc-54 intron was tested at its natural location between unc-54 exons 1 and 2, or near the 3’ end of either the first or the second GFP (Fig. 1c). As was observed with the small synthetic introns, the unc-54 intron had a much greater effect on expression when near the 5’ end than in the middle or near the 3’ end of the fusion construct (Fig. 1d and Supplementary Fig. S1). The unc-54 intron alone had a larger effect on expression than the three synthetic introns but it was also located closer to the 5’ end of the gene than the first of the synthetic introns, with first exon lengths in those constructs of 142 nt and 337 nt respectively. Selecting different introns to test To determine if worm introns vary widely in their ability to boost expression, as they do in plants, we attempted to create a worm IMEter for use in selecting introns with different predicted effects on expression. However, we found that the difference in k-mer composition between promoter-proximal and distal introns on which the IMEter is based is too small in C. elegans for the IMEter to function. One way to measure the degree to which the k-mer profiles of proximal and distal introns vary is to sum the absolute value of the differences in frequency of all k-mers in both populations of introns (see methods). The exact value obtained varies depending on the choice of k-mer size and threshold distance used for separating promoter-proximal and distal introns, but the value must be between 0 (if all k-mers have identical frequencies in both populations) and 2 (if all individual k-mers are found exclusively in one population or the other). Using a k-mer size of 5 and a threshold defining promoter-proximal introns as those less than 1000 nt from the start of transcription, the sum of differences of k-mer frequencies in Arabidopsis introns is 0.83, while that of C. elegans introns is only 0.056 (Fig. 2), indicating that the pentamer compositions of proximal and distal introns are highly similar in worms. Therefore, we used an alternative approach to select worm introns that may have different effects on expression. Many of the introns that are known to boost expression in plants and other organisms are found in abundantly expressed housekeeping genes, such as those encoding ribosomal proteins, actin, tubulin, histones, and translational elongation factors[36]. This led to the hypothesis that stimulatory introns typically drive the expression of that minority of genes whose products are constitutively required in large amounts in all cells, while conventional transcription factors control the expression of most genes whose products are needed in only certain tissue types, at specific times, or in response to a particular stimulus[36]. If introns have variable effects on the expression of the gene in which they are naturally found in worms, the introns from highly expressed should be more likely to stimulate expression than introns from genes expressed at a very low level. Therefore, seven introns were chosen from a variety of genes based on the following criteria. First, C. elegans genes were ranked based on their expression level. The most highly expressed genes had approximately 10 6 RNAseq reads as reported on WormBase (https://wormbase.org). Genes were also selected that had approximately 10 5 , 10 4 , or 10 3 RNAseq reads, forming four expression categories referred to as high, medium, low, and very low. Second, within those categories, genes were selected that have been sufficiently studied to be named, are not in an operon, and do not have multiple alternative splice isoforms. Finally, the introns chosen to test from these genes are between 300 and 600 nt in length and are located less than 500 nt from the start of transcription. An additional intron tested was smu-1 intron 3, which contains poly A/T-rich clusters (PATCs) and was previously shown to prevent the silencing of genes in the germline[37]. The exact mechanism through which PATCs prevent silencing is unclear, and this intron was chosen to explore the possibility PATC-containing introns avoid silencing at least in part by causing an unusually large increase in the expression of the gene in which they are located. The eight new introns were compared to two of the introns tested in the unc-54:GFP:GF P fusions described above, namely unc-54 intron 1 and the 5’-most of the 51 nt synthetic introns. Comparing the stimulating effect of different introns The ten introns were individually inserted into an unc-54:GFP reporter gene at the same location as the natural unc-54 intron 1 (Fig. 1e). Fluorescence was measured in single-copy transgenic worms by photographing individual worms on a fluorescence microscope and measuring fluorescence in the body wall muscles where unc-54 is expressed and endogenous autofluorescence is low, specifically in the head of the worm adjacent to the pharynx (Fig. 3a). The L3 stage was chosen for these experiments because the GFP signal is higher than in L1 worms. The intronless control was expressed at a very low level, generating fluorescence that was slightly but significantly (p < 1x10 -4 ) above the endogenous fluorescence in untransformed worms (Fig. 3a, 3b and 4a, and Supplementary Fig. S1). The effect of each intron on expression is reported as the fold increase in pharynx fluorescence relative to that in the intronless control, although the accuracy of this measure is limited by the very low expression of the intronless control. All ten introns boosted fluorescence 30- to 40-fold relative to the intronless control (p < 1x10 -10 ), and all independent single-copy lines containing the same construct gave very similar results (Figs. 3 and 4a, Table 1 and Supplementary Table S2). Only one single-copy line containing the construct with the unc-54 intron was obtained but the values obtained from this line are supported by a line containing a two-copy insert, which gave roughly twice the fluorescence as the single-copy line. There was considerable variation in fluorescence measurements between individual worms of the same genotype measured at the same time. This variation appeared to be mostly due to slight differences in the ages of the worms examined. Pharynx length, which increases at a steady rate as worms grow[38], correlated with fluorescence (Supplementary Fig. S2), indicating that GFP accumulates as the worms age. Despite the similar mean expression levels and large overlapping standard deviations for the data sets obtained from worms containing the same construct, the combined data for some of the introns were statistically different (p > 0.05). However, an analysis of the source of the variation indicated that differences between lines containing the same construct, observations of the same line taken on different days, and the individual taking the measurements all contributed more to the variation than did the identity of the intron (Supplementary Fig. S1). Indeed, we found no introns for which all independent lines containing that construct were significantly different than the individual lines containing another intron. In contrast, the GFP fluorescence of all intron-containing lines were significantly higher than the intronless control (p < 2 x 10 -10 ). Thus, we conclude that all ten introns cause a similarly large increase in GFP fluorescence, strongly suggesting that all introns in C. elegans boost expression equally well. The synthetic intron and unc-54 intron 1 both increased fluorescence to a very similar degree (36.5 +/- 7.1-fold and 33.6 +/- 11.2-fold respectively, Table 1). This indicates that the higher expression of the unc-54:GFP:GFP construct containing a single unc-54 intron at its normal position compared to that of the three synthetic introns in the first GFP (Fig. 1) was due entirely to the location of the introns, rather than any difference in their stimulating ability. Table 1. The introns tested and their effect on expression. Gene source of intron Intron in native gene Effect on expression Name Encoded protein Expression level Intron size (nt) Intron # Distance from ATG (nt) Fluorescence mRNA unc-54 Myosin heavy chain High 562 1 63 33.6 +/- 11.2 16.7 +/- 2.9 synthetic 51 36.5 +/- 7.1 14.2 +/- 2.9 smu-1 Spliceosomal subunit Medium 305 3 380 31.1 +/- 8.7 9.8 +/- 2.1 rpl-14 Ribosomal protein High 346 1 105 39.1 +/- 12.7 13.8 +/- 2.3 lbp-6 Lipid binding protein High 554 1 207 38.9 +/- 18.1 13.8 +/- 9.0 gpx-1 Glutathione peroxidase Medium 444 2 211 38.5 +/- 12.8 12.4 +/- 2.7 mec-8 mRNA binding protein Medium 528 1 283 38.0 +/- 13.5 13.2 +/- 3.7 daao-1 D-amino acid oxidase Low 406 2 274 32.8 +/- 10.6 13.1 +/- 3.6 gbb-1 GABA B receptor Low 524 1 228 39.6 +/- 12.0 16.1+/- 5.3 cutl-4 Cuticulin-like Very low 354 1 88 34.6 +/- 8.9 15.1 +/- 6.9 The effect on expression is the fold increase in GFP fluorescence or mRNA in lines containing a transgene with that intron relative to the intronless control, mean +/- standard deviation. Introns stimulate mRNA accumulation To determine the level at which expression is primarily affected, total RNA was isolated from partially synchronized populations of worms that were predominantly at the L3 stage. RNA gel blots probed with GFP revealed that all ten of the tested introns caused an increase in GFP mRNA of between 10- and 17-fold relative to the intronless control (Figs. 3c and 4b, Table 1). As with the fluorescence measurements, accurate estimations of the intron-caused fold increase in the level of GFP mRNA are hindered by the very low levels of signal in the intronless control lines. In addition, there is a faint cross-hybridizing band visible even in RNA from untransformed worms that migrates just above the GFP band that also interferes with quantification of GFP mRNA levels in the intronless control. Nevertheless, it is clear that a small amount of GFP mRNA accumulates in lines containing the intronless construct, and that all ten introns cause a similarly large increase in GFP mRNA. The mRNA from all intron-containing constructs comigrates with that seen in the intronless control, with no larger bands visible, indicating that all the introns tested are spliced with similar high efficiency. Thus, the difference in GFP mRNA accumulation accounts for most or possibly all the large difference in fluorescence seen between the intron-containing and intronless lines. Discussion Here we show that introns boost expression in C. elegans most when the intron is closest to the 5’ end of a gene, and that the first intron inserted has the greatest influence on expression, while additional introns have effects that are much smaller and less than additive. These findings confirm and extend observations previously made in worms[35] and other organisms. In addition, we showed that all ten introns tested at the same location caused a similar strong increase in expression, and that the overall effect of an intron on gene expression was due mostly or entirely to differences in mRNA accumulation. The magnitude of the difference in expression of intron-containing and intronless constructs we observed was substantially larger than that reported by Crane et al.[35] (more than 30-fold versus 1.5 to 1.8-fold). The main cause of this difference is the very low level of expression of our intronless unc-54:GFP fusions, while the intronless hsp-90:mCherry , hsp-90:GFP , and vit-2:mCherry fusions used by Crane et al. were expressed at relatively high levels. One potential explanation is that one of the two hsp-90 transcript isoforms (C47E8.5.2) reveals the presence in the hsp-90 5 ’ -UTR of a 1,116 nt intron whose 3 ’ end is 4 nt upstream of the ATG (https://wormbase.org/). Because this entire intron is present in the 2 kb promoter fragment used for all hsp-90 constructs, even those described as intronless, this 5 ’ -UTR intron might be responsible for the relatively high expression of constructs lacking introns in the reporter genes. The introns subsequently added to the reporter gene would have a minor effect since they are not the first intron in the gene. However, there is no evidence for an intron in the 5 ’ -UTR of vit-2 that could explain the high expression of the intronless vit-2:mCherry fusion. Alternatively, it is possible that C. elegans is similar to mammals where the expression of some genes such as b-globin absolutely requires the presence of an intron, while other genes such as dihydrofolate reductase are expressed at detectable levels without an intron even if their expression can be boosted by an intron[4]. Perhaps unc-54 is an example of an intron-dependent gene while hsp-90 and vit-2 are intron-independent. Crane et al.[35] also examined expression in adult worms that have had longer to accumulate GFP from a poorly expressed construct than the L3 worms used here. Other potential sources of variability can be ruled out. The synthetic intron we used was identical to their intron “ia”, the introns were similar distances from the start of transcription, the unc-54 terminator was used in both studies, and the constructs were integrated using the same technique at the same chromosomal location. Several observations suggest that unlike in plants where specific stimulating sequences present only within a minority of introns are responsible for increasing mRNA accumulation, in nematodes it may be a conserved intron structure (such as a splice site) or the act of splicing that boosts expression. First, all ten of the introns tested increased expression to a very similar degree and none failed to have an effect. Second, C. elegans introns lack the significant k-mer compositional differences between promoter-proximal and distal introns seen in plants, even though introns boost expression most when located nearest to the 5’ end of a gene in both plants and nematodes. Third, there is very little room in the 51 nt synthetic intron for sequences that could boost expression to the same degree as introns that are more than ten times its length, such as the 562 nt unc-54 intron 1. In Arabidopsis, the stimulating sequences within introns have additive effects[31]. This may be one reason that first introns, which are the most likely to boost expression, also tend to be longer than other introns[39]. One possible explanation for a splicing related increase in GFP fluorescence is that intron-containing and intronless constructs might yield the same amount of primary transcript but that only spliced mRNAs are efficiently exported to the cytoplasm and translated. The exon junction complex (EJC) proteins that are deposited on the mRNA during splicing are known to promote export[40,41]. However, the RNA used in Fig. 3 was isolated from whole worms and thus would include both nuclear and cytoplasmic RNA. The observation that intron-containing constructs produced 10 to 15 times more GFP mRNA than did the intronless control rules out the possibility that the primary cause of the different expression levels was the subcellular localization of the mRNA. The EJC could have a roughly 2-fold effect on expression because the observed 30- to 40-fold increase in GFP fluorescence caused by introns was roughly twice that seen at the level of mRNA, suggesting that more protein was produced per unit of mRNA when the gene contained an intron. Similar observations have been made in plants where the effect of an intron is consistently twice as large measured at the level of enzyme activity than as mRNA accumulation, which has been attributed to increases in export and translation[25,30,31]. Still, in both worms and plants, the intron-mediated increase in expression can be largely or entirely attributed to increases in mRNA accumulation. The effect of the introns on mRNA accumulation could either be a positive action that increases mRNA production, or the removal of a repression that inhibits expression of intronless constructs. In one example of introns actively stimulating expression, some human genes with weak promoters upstream of alternatively spliced exons have the occupancy of RNA polymerase II boosted by specific splicing factors that recruit the core transcription machinery[42]. In an example of intronless repression in the C. elegans germline, the removal of introns from a gene can cause it to be silenced by two distinct pathways, one of which uses argonaute proteins and small RNAs[43]. In the intron-containing versions of the same genes, intron splicing somehow marks transcripts and prevents their recognition as templates for argonaute-mediated silencing. However, the same intronless genes are not silenced in the somatic cells[43] where the unc-54:GFP fusions studied here are predominantly expressed. In summary, there are some similarities and some differences in the effect of introns in worms and plants. In both groups, the effect of the intron on expression was primarily at the level of mRNA accumulation and was larger if the intron was near the 5 ’ end of the gene than if the same intron was located further away from the start. In addition, the remarkable ability of introns to affect expression in the absence of a promoter[26,32] suggests a mechanism that is similar in both groups and distinct from that of enhancer elements, which cannot activate expression in the absence of a promoter and can have effects over much greater distances than introns can. However, a significant difference is that in plants, only an estimated 5-10% of introns boost mRNA accumulation tenfold or more while others have a much smaller effect or none at all[29]. In contrast, all ten of the worm introns tested here had the same strong effect on mRNA accumulation. The observation that a PATC-containing and non-PATC introns boosted mRNA accumulation equally well suggests that the difference in their ability to prevent germline silencing between introns that contain PATCs and other introns is not due to their effect on mRNA production. Perhaps the underlying mechanism in both plants and worms involves introns directing transcript initiation upstream of themselves through interactions between introns and the transcription machinery, explaining the need for introns to be near the 5 ’ end of the gene and the surprising dispensability of promoter sequences. In plants these interactions could be mediated by proteins that bind to sequences present in variable numbers within the body of a minority of introns, while in worms they could involve the spliceosome or other proteins that recognize conserved sequences around the splice sites of all introns. The results from worms, plants, and mammals illustrate that sequences downstream of the transcription start site, especially those near the 5 ’ end of a gene, can have effects on expression that outweigh those of the more familiar regulatory sequences in the promoter around and upstream of the transcription start site[44]. Methods Worms C. elegans strains were maintained at room temperature on MYOB plates with E. coli OP50 as a food source[45,46]. The wild-type strain is N2. Constructs All constructs contain the same 453 nt unc-54 (F11C3.3) promoter fragment, the entire 95 nt unc-54 exon 1 (32 nt of 5 ’ -UTR and 63 nt coding sequence), the first 9 nt of unc-54 exon 2, some version of GFP , and the same 713 nt unc-54 terminator fragment. They vary in the identity of the intron present at the normal location of unc-54 intron 1, the number of GFP genes, and the identity and location of the introns in GFP . The unc-54 promoter fragment was amplified from genomic DNA using primers OAR130 and OAR131, the unc-54 intron was removed by amplification with OAR130 and OAR132, and the unc-54 terminator was amplified with primers OAR135 and OAR136 (primer sequences are found in Supplementary Table S3). An intronless version of GFP was synthesized by Biomatik (Kitchener, Ontario). Derivatives of the various intron-containing and intronless GFP s in which the stop codon was replaced by a 12 nt region encoding a 4 amino acid linker and containing various flanking restriction sites were created by PCR to facilitate the construction of the tandem GFP fusions. The unc-54 intron was inserted by Gibson assembly using the NEBuilder kit (New England Biolabs, Ipswich, MA) near the 3 ’ end of GFP using primers OAR210 and OAR211. In most cases, introns were amplified using high fidelity polymerase directly from wild-type N2 genomic DNA using primers whose 3’ half matches the sequence at the end of the intron and whose 5’ ends match unc-54 sequences flanking the site of intron insertion. For the daao-1 and rpl-14 introns, the template was an intermediate PCR product made using primers OAR237-240 that match exon sequences near the target intron. The 51 nt synthetic intron was made as two complementary primers OAR258 and OAR259 that were annealed to each other. The introns were inserted between unc-54 exons 1 and 2 by Gibson assembly into an unc-54p:unc-54::GFP fusion. The resulting plasmids were sequenced to verify accurate insertion and the absence of mutations before the intron-containing unc-54p:unc-54::GFP fusion was inserted as a Bgl II- Spe I fragment into the vector pCFJ151[47], which contains the unc-119 marker gene. Mos1-mediated single copy insertion ( MosSCI ) The intron-containing unc-54:GFP fusions were integrated into the ttTi5605 site on chromosome 2 in strain EG6699 by MosSCI[47,48]. The injections were performed by InVivo Biosystems (Eugene, OR, formerly known as Knudra Transgenics or NemaMetrix). The resulting worms were screened for progeny that had integrated the unc-119 gene and lost the mCherry markers in the injection mix, and strains homozygous for the transgene insertion were obtained. To verify single-copy integration, DNA from each line was isolated using the DNeasy blood and tissue kit (Qiagen), digested with Spe I and Hin dIII, and subjected to gel blot analysis with a 32 P labeled GFP probe and detected using a Storm phosphoimager. Transgene copy number was estimated by comparing band intensity using ImageQuant software (Molecular Dynamics). All single-copy lines obtained were used and given equal weight in calculations. Quantification of GFP in populations of worms L1 stage worms were collected off starved plates using water, concentrated by brief centrifugation, and 125 mL of resuspended worms were placed in each well of a 96-well plate. Fluorescence was measured on a Perkin Elmer Model 2030-0050 Multilabel Plate Reader at an emission wavelength of 535 nm with an excitation wavelength of 485nm (to measure GFP) or 355 nm (to correct for variation in the number of worms in each well using autofluorescence). For every line, at least six technical replicates were performed on each of three or more separate days. The ratio of the signals obtained with excitation at 485 nm to that at 355 nm was normalized by setting the level for untransformed worms equal to zero and the ratio in transgenic worms containing the intronless control to one. The values for intron-containing constructs represent the average fold increase in above-background fluorescence caused by the intron. Quantif ication of GFP in individual worms Worms from a starved plate were grown on a fresh plate at room temperature for 17-18 hours so that the population was predominantly at the L3 stage. L3 worms were picked and anaesthetized in a 0.25% solution of tetramisole on a 2% agar pad and examined using a UPlan FL 40x, 0.75 numerical aperture objective on an Olympus BX60 compound microscope, with a Hamamatsu Orca 12-bit digital camera and Micromanager software[49]. Single plane images were acquired in brightfield and fluorescent image at 150 ms exposure. The resulting images were then quantified using ImageJ software version 1.53[50] using the segmented line tool with a width of 10 pixels to trace the muscle of each worm starting from the base of the grinder to the tip of the nose and back to the grinder on the opposite side of the worm. The mean reading per pixel for the entire line was corrected for background by subtracting the mean of a line drawn outside the worm parallel to and roughly the same length as the pharynx. Measurements were obtained from at least three biological replicates totaling more than 40 individual worms for each line. To calculate the fold increase in fluorescence caused by each intron, the average reading from untransformed worms was subtracted from the average readings for all transgenic lines containing the same construct, and the individual readings of lines with an intron-containing construct were divided by the average reading of the intronless control lines. Statistics For all tests of significant differences between groups (i.e. lines, introns) we used a linear model in R, lm(value~group) . Model assumptions (e.g. normality of residuals) were validated by visualizing model diagnostic plots. Significant differences (two-sided test) among groups were determined using Tukey Honest Significant Difference ( TukeyHSD function in R) at alpha =0.05, after Bonferroni correction for multiple testing. RNA isolation and RNA gel blots Worms from a starved plate were grown on a fresh plate at room temperature for 17 hours so that the population was predominantly at the L3 stage. One mL of water was used to rinse the worms off 2 plates and pooled in a microfuge tube with the worms from another 2 plates. The tubes were centrifuged at 8,000 rpm (5,000g) for 5 seconds and the supernatant was discarded. The worms were rinsed in 1 ml 0.1% SDS by vortexing and centrifuging as above and the supernatant was removed leaving the worms in a volume of 50 mL or less. The worms were resuspended in 100 mL worm lysis buffer (50 mM KCl, 10 mM TRIS pH 8.3, 2.5 mM MgCl 2 , 0.45% Tween 20) containing proteinase K at a final concentration of 1 mg/mL (30-40 units/mL) and incubated at 55°C for ten minutes with mixing every 2-3 minutes. RNA was isolated using RNeasy Plus kits (Qiagen) following the manufacturers protocol. In short, this involved adding 350 mL buffer RLT Plus containing DTT to the lysed worms, mixing, and pipetting onto a gDNA removal column, which was spun for 30 seconds at 10,000 rpm (8,000g). The flow-through was mixed with 350 mL 70% ethanol and transferred to an RNeasy column, which was spun 15 seconds at 10,000 rpm (8,000g). The column was rinsed once with 700 mL buffer RW1 and twice with 500 mL buffer RPE, then dried by spinning at top speed for 1 minute. RNA was eluted from the column with two washes with 50 mL RNAse free water, which were combined. The yield of RNA (typically 5-15 mg) was determined by spectrophotometer, the RNA was concentrated by ethanol precipitation, and the dried RNA was resuspended in RNAse free water. 2 mg of RNA from each line was used in RNA gel blots performed using reagents from the Northern Max kit (Ambion), hybridized with a 32 P labeled GFP probe, and detected using a Storm phosphoimager. Band intensity was measured using ImageQuant software and corrected for small differences in loading using ethidium bromide stained rRNA bands quantified using BioRad software. Computational Master files containing the sequences of all the introns in C. elegans or Arabidopsis, as well as the distance between the transcription start site and the start of the intron, were extracted from the annotated genomes of those organisms. As in the IMEter algorithm[28], the frequency of occurrence of all possible k-mers was determined separately in the two populations of introns less than or greater than a threshold distance from the transcription start site of the gene in which the intron is located. The frequency of each k-mer is calculated as the count for that k-mer divided by the counts for all k-mers found in that population of sequences. Therefore, the sum of the frequency of all k-mers in each population is 1. The difference between the two frequency distributions was determined as the sum of the absolute values of the difference in frequency of each individual k-mer in the promoter-proximal and distal intron populations. Declarations Additional Information The authors declare no competing interests. Author Contribution A.B.R. designed the experiments, performed experiments, and wrote the manuscript. A.B. and I.S. performed experiments. J.G.M. did statistical analysis. I.K. provided computational input. L.S.R. advised and supplied the facilities for growing the worms and collecting microscopic data. All authors reviewed the manuscript. Acknowledgement Thanks to Alice Pierce, Cindy Bailey, Clarence Reyes, and Hoang Tran, for constructing plasmids, and Jenna Noueihed for help with coding. This work was supported by funds obtained by A.B.R. and L.S.R. teaching first year seminars at UC Davis. Data Availability Statement Expression data and strains are available upon request. The software for comparing kmer frequency distributions is available from GitHub. References Berget, S. M., Moore, C. & Sharp, P. A. Spliced segments at the 5' terminus of adenovirus 2 late mRNA. Proc. Natl. Acad. Sci. USA 74 , 3171-3175 (1977). Gruss, P., Lai, C. J., Dhar, R. & Khoury, G. Splicing as a Requirement for Biogenesis of Functional 16S Messenger-RNA of Simian Virus-40. Proc. Natl. Acad. Sci. USA 76 , 4317-4321 (1979). Panigrahi, A. & O'Malley, B. W. Mechanisms of enhancer action: the known and the unknown. Genome Biol. 22 , 108 (2021). Buchman, A. R. & Berg, P. Comparison of intron-dependent and intron-independent gene expression. Mol. Cell. Biol. 8 , 4395-4405 (1988). Callis, J., Fromm, M. & Walbot, V. Introns increase gene expression in cultured maize cells. Genes & Dev. 1 , 1183-1200 (1987). Chung, S. & Perry, R. Importance of introns for expression of mouse ribosomal protein gene rpL32. Mol. Cell. Biol. 9 , 2075-2082 (1989). Lugones, L. G., Scholtmeijer, K., Klootwijk, R. & Wessels, J. G. Introns are necessary for mRNA accumulation in Schizophyllum commune . Mol. Microbiol. 32 , 681-689 (1999). Goebels, C. et al. Introns Regulate Gene Expression in Cryptococcus neoformans in a Pab2p Dependent Pathway. PLoS Genetics 9 , e1003686 (2013). Jiang, L. et al. The 5'-UTR intron of the midgut-specific BmAPN4 gene affects the level and location of expression in transgenic silkworms. Insect Biochem. Mol. Biol. 63 , 1-6 (2015). Agarwal, N. & Ansari, A. Enhancement of Transcription by a Splicing-Competent Intron Is Dependent on Promoter Directionality. PLoS Genetics 12 , e1006047 (2016). Lewis, J. D. & Tollervey, D. Like attracts like: getting RNA processing together in the nucleus. Science 288 , 1385-1389 (2000). Proudfoot, N. Connecting transcription to messenger RNA processing. Trends Biochem. Sci. 25 , 290-293 (2000). Kornblihtt, A. R., De La Mata, M., Fededa, J. P., Munoz, M. J. & Nogues, G. Multiple links between transcription and splicing. RNA 10 , 1489-1498 (2004). Pandit, S., Wang, D. & Fu, X. D. Functional integration of transcriptional and RNA processing machineries. Current Opin. Cell Biol. 20 , 260-265 (2008). Heyn, P., Kalinka, A. T., Tomancak, P. & Neugebauer, K. M. Introns and gene expression: cellular constraints, transcriptional regulation, and evolutionary consequences. BioEssays 37 , 148-154 (2015). Saldi, T., Cortazar, M. A., Sheridan, R. M. & Bentley, D. L. Coupling of RNA Polymerase II Transcription Elongation with Pre-mRNA Splicing. J. Mol. Biol. 428 , 2623-2635 (2016). Nott, A., Meislin, S. H. & Moore, M. J. A quantitative analysis of intron effects on mammalian gene expression. RNA 9 , 607-617 (2003). Lu, S. & Cullen, B. R. Analysis of the stimulatory effect of splicing on mRNA production and utilization in mammalian cells. RNA 9 , 618-630 (2003). Furger, A., O'Sullivan, J. M., Binnie, A., Lee, B. A. & Proudfoot, N. J. Promoter proximal splice sites enhance transcription. Genes & Dev. 16 , 2792-2799 (2002). Kwek, K. Y. et al. U1 snRNA associates with TFIIH and regulates transcriptional initiation. Nature Struct. Biol. 9 , 800-805 (2002). Damgaard, C. K. et al. A 5' splice site enhances the recruitment of basal transcription initiation factors in vivo. Mol. Cell 29 , 271-278 (2008). Fong, Y. W. & Zhou, Q. Stimulatory effect of splicing factors on transcriptional elongation. Nature 414 , 929-933 (2001). Lin, S. R., Coutinho-Mansfield, G., Wang, D., Pandit, S. & Fu, X. D. The splicing factor SC35 has an active role in transcriptional elongation. Nature Struct. Mol. Biol. 15 , 819-826 (2008). Mimoso, C. A. & Adelman, K. U1 snRNP increases RNA Pol II elongation rate to enable synthesis of long genes. Mol. Cell 83 , 1264-1279 (2023). Rose, A. B. The effect of intron location on intron-mediated enhancement of gene expression in Arabidopsis . Plant J. 40 , 744-751 (2004). Gallegos, J. E. & Rose, A. B. Intron DNA Sequences Can Be More Important Than the Proximal Promoter in Determining the Site of Transcript Initiation. Plant Cell 29 , 843-853 (2017). Rose, A. B. Requirements for intron-mediated enhancement of gene expression in Arabidopsis . RNA 8 , 1444-1453 (2002). Rose, A. B., Elfersi, T., Parra, G. & Korf, I. Promoter-proximal introns in Arabidopsis thaliana are enriched in dispersed signals that elevate gene expression. Plant Cell 20 , 543-551 (2008). Gallegos, J. E. & Rose, A. B. The enduring mystery of intron-mediated enhancement. Plant Sci 237 , 8-15 (2015). Rose, A. B., Carter, A., Korf, I. & Kojima, N. Intron sequences that stimulate gene expression in Arabidopsis. Plant Mol. Biol. 92 , 337-346 (2016). Gallegos, J. E. & Rose, A. B. An intron-derived motif strongly increases gene expression from transcribed sequences through a splicing independent mechanism in Arabidopsis thaliana . Sci Rep 9 , 13777 (2019). Okkema, P. G., Harrison, S. W., Plunger, V., Aryana, A. & Fire, A. Sequence requirements for myosin gene expression and regulation in Caenorhabditis elegans . Genetics 135 , 385-404 (1993). Virts, E. L. & Raschke, W. C. The role of intron sequences in high level expression from CD45 cDNA constructs. J. Biol. Chem. 276 , 19913-19920 (2001). Fan, X. et al. SapTrap Assembly of Caenorhabditis elegans MosSCI Transgene Vectors. G3 (Bethesda) 10 , 635-644 (2020). Crane, M. M. et al. In vivo measurements reveal a single 5'-intron is sufficient to increase protein expression level in Caenorhabditis elegans . Sci Rep 9 , 9192 (2019). Rose, A. B. Introns as Gene Regulators: A Brick on the Accelerator. Front. Genet. 9 , 672 (2018). Frokjaer-Jensen, C. et al. An Abundant Class of Non-coding DNA Can Prevent Stochastic Gene Silencing in the C. elegans Germline. Cell 166 , 343-357 (2016). Stojanovski, K. et al. Maintenance of appropriate size scaling of the C. elegans pharynx by YAP-1. Nat. Commun. 14 , 7564 (2023). Bradnam, K. R. & Korf, I. Longer first introns are a general property of eukaryotic gene structure. PLoS One 3 , e3093 (2008). Seydoux, G. & Fire, A. Soma-germline asymmetry in the distributions of embryonic RNAs in Caenorhabditis elegans . Development 120 , 2823-2834 (1994). Shiimori, M., Inoue, K. & Sakamoto, H. A specific set of exon junction complex subunits is required for the nuclear retention of unspliced RNAs in Caenorhabditis elegans . Mol. Cell. Biol. 33 , 444-456 (2013). Uriostegui-Arcos, M., Mick, S. T., Shi, Z., Rahman, R. & Fiszbein, A. Splicing activates transcription from weak promoters upstream of alternative exons. Nat. Commun. 14 , 3435 (2023). Makeyeva, Y. V., Shirayama, M. & Mello, C. C. Cues from mRNA splicing prevent default Argonaute silencing in C. elegans . Dev. Cell 56 , 2636-2648 (2021). Voichek, Y., Hristova, G., Molla-Morales, A., Weigel, D. & Nordborg, M. Widespread position-dependent transcriptional regulatory sequences in plants. Nat. Genet. 56 , 2238-2246 (2024). Brenner, S. The genetics of Caenorhabditis elegans . Genetics 77 , 71-94 (1974). Church, D. L., Guan, K. L. & Lambie, E. J. Three genes of the MAP kinase cascade, mek-2, mpk-1/sur-1 and let-60 ras, are required for meiotic cell cycle progression in Caenorhabditis elegans . Development 121 , 2525-2535 (1995). Frokjaer-Jensen, C. et al. Single-copy insertion of transgenes in Caenorhabditis elegans. Nat. Genet. 40 , 1375-1383 (2008). Frokjaer-Jensen, C., Davis, M. W., Ailion, M. & Jorgensen, E. M. Improved Mos1-mediated transgenesis in C. elegans . Nat. Methods 9 , 117-118 (2012). Edelstein, A. D. et al. Advanced methods of microscope control using mManager software. J. Biol. Methods 1 , doi:10.14440/jbm.2014.36 (2014). Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9 , 671-675 (2012). Additional Declarations No competing interests reported. Supplementary Files RoseSupMat.pdf Supplementary Material Fig. S1. Statistical analysis of the constructs shown in Fig. 1. Fig. S2. GFP fluorescence correlates with pharynx length. Table S1. Statistical comparison of fluorescence in worms containing the constructs shown in Figs. 1a and 1c. Table S2. Data for each individual line tested. Table S3. Oligonucleotides used in this study. Cite Share Download PDF Status: Published Journal Publication published 07 May, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 12 Feb, 2025 Editor assigned by journal 12 Feb, 2025 Submission checks completed at journal 30 Jan, 2025 First submitted to journal 29 Jan, 2025 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5926918","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":408974836,"identity":"ae30a748-d13f-457c-b121-183c4f22ffb5","order_by":0,"name":"Alan B. Rose","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIiWNgGAWjYDACCTBpA8QJYMTAcIA4LWmkaznMAFVPhBb+2b0PH/6oOJ/Y3578TOLBHwY5vhsJ+LVI3DlubMxz5nbijDPPzCQS2xiMJQlpYbiRxibN2HY7cYNEgrFBYgND4gZCWuSBWiR//jsH1JL+2SDhD0M9QS0GQC0SvA0HgFpyDB8ksDEkGBDSYnjnGLMxz7Fk4xln3hQ+SGyTMJx55gF+LXK32xgf/qixk+1vT99w8McfG3m+4wRsQQcSpCkfBaNgFIyCUYAdAAANa0nSFblNrQAAAABJRU5ErkJggg==","orcid":"","institution":"University of California, Davis","correspondingAuthor":true,"prefix":"","firstName":"Alan","middleName":"B.","lastName":"Rose","suffix":""},{"id":408974838,"identity":"17094e00-6c39-4fec-9960-256a845706ea","order_by":1,"name":"Aaron Baer","email":"","orcid":"","institution":"University of California, Davis","correspondingAuthor":false,"prefix":"","firstName":"Aaron","middleName":"","lastName":"Baer","suffix":""},{"id":408974840,"identity":"06d6ac08-0be1-4c4a-bef0-37e8350c0bd7","order_by":2,"name":"Isaac Shaker","email":"","orcid":"","institution":"University of California, Davis","correspondingAuthor":false,"prefix":"","firstName":"Isaac","middleName":"","lastName":"Shaker","suffix":""},{"id":408974841,"identity":"bfae8077-bf31-427d-a748-75e94511a34f","order_by":3,"name":"J. Grey Monroe","email":"","orcid":"","institution":"University of California, Davis","correspondingAuthor":false,"prefix":"","firstName":"J.","middleName":"Grey","lastName":"Monroe","suffix":""},{"id":408974842,"identity":"e903e0c4-89ee-4eba-baf2-509bb1ed9cdf","order_by":4,"name":"Ian Korf","email":"","orcid":"","institution":"University of California, Davis","correspondingAuthor":false,"prefix":"","firstName":"Ian","middleName":"","lastName":"Korf","suffix":""},{"id":408974843,"identity":"39880c0f-9691-465a-898d-8d548fd8347f","order_by":5,"name":"Lesilee S. Rose","email":"","orcid":"","institution":"University of California, Davis","correspondingAuthor":false,"prefix":"","firstName":"Lesilee","middleName":"S.","lastName":"Rose","suffix":""}],"badges":[],"createdAt":"2025-01-29 23:38:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5926918/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5926918/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-99739-6","type":"published","date":"2025-05-07T15:57:25+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":75293987,"identity":"583e5e98-2b4b-49d3-9f60-10da85722cf0","added_by":"auto","created_at":"2025-02-03 06:24:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":80142,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect on expression of varying intron location. \u003cstrong\u003ea.\u003c/strong\u003eDiagram of constructs containing small synthetic introns. \u003cstrong\u003eb.\u003c/strong\u003eMicrotiter plate readings of mean fluorescence in single-copy transgenic lines containing the constructs shown in \u003cstrong\u003ea\u003c/strong\u003e. \u003cstrong\u003ec.\u003c/strong\u003e Diagrams of constructs containing \u003cem\u003eunc-54\u003c/em\u003e intron1 in different locations. \u003cstrong\u003ed.\u003c/strong\u003e Microtiter plate readings of mean fluorescence in single-copy transgenic lines containing the constructs shown in \u003cstrong\u003ec\u003c/strong\u003e. In \u003cstrong\u003eb\u003c/strong\u003e and \u003cstrong\u003ed\u003c/strong\u003e, the error bars indicate standard deviations (n.s, not significant, p \u0026gt; 0.05; ** p \u0026lt; 0.01; *** p \u0026lt; 0.0001; see Supplementary Table S1 for specific P values). \u0026nbsp;\u003cstrong\u003ee.\u003c/strong\u003e Diagram of construct for comparing the effect of different introns at the same location.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5926918/v1/3dd26700768ca4b4a905cb5e.png"},{"id":75294343,"identity":"d3c5f1fd-d992-4811-a53d-fc1ec17c564e","added_by":"auto","created_at":"2025-02-03 06:32:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":95956,"visible":true,"origin":"","legend":"\u003cp\u003eComparing the difference in k-mer composition between promoter-proximal and distal introns in \u003cem\u003eA. thaliana\u003c/em\u003e and \u003cem\u003eC. elegans\u003c/em\u003e. Each bar shows the sum of the absolute values of the difference in frequency of all k-mers (size 4, 5, or 6 nt) present in the populations of introns designated promoter-proximal or distal based on a threshold distance (500 or 1000 nt) from the start of transcription to the start of the intron. Higher values indicate greater dissimilarity between promoter-proximal and distal introns.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5926918/v1/1f7cd8294da1018d11137171.png"},{"id":75293989,"identity":"8583fd70-be76-4eb0-bd27-af9bb1787352","added_by":"auto","created_at":"2025-02-03 06:24:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":254363,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of different introns on GFP fluorescence and mRNA accumulation. The lanes in all four panels are vertically aligned and show fluorescence micrographs (\u003cstrong\u003ea\u003c/strong\u003eand \u003cstrong\u003eb\u003c/strong\u003e) or RNA gel blots (\u003cstrong\u003ec\u003c/strong\u003e and \u003cstrong\u003ed\u003c/strong\u003e) using RNA extracted from the same line of untransformed worms or transgenic worms containing \u003cem\u003eunc-54::GFP\u003c/em\u003efusions with no intron or an intron from the indicated gene. Each column under the same heading represents an independent line containing the indicated construct. All are single copy except the line marked with an asterisk, which contains two copies of the transgene. \u003cstrong\u003ea.\u003c/strong\u003e Pharynx of worms. \u003cstrong\u003eb.\u003c/strong\u003e As in \u003cstrong\u003ea\u003c/strong\u003e but at a brighter exposure to reveal the faint fluorescence in the untransformed and intronless lines. \u003cstrong\u003ec.\u003c/strong\u003eRNA gel blot probed with \u003cem\u003eGFP\u003c/em\u003e. \u003cstrong\u003ed.\u003c/strong\u003eThe ethidium bromide-stained gels used in \u003cstrong\u003ec\u003c/strong\u003e to show that each lane contains a similar amount of undegraded RNA.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5926918/v1/b384a19c050d5a443d37ef85.png"},{"id":75293992,"identity":"605ed261-d76c-4e74-a551-ec6e21db007f","added_by":"auto","created_at":"2025-02-03 06:24:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":163365,"visible":true,"origin":"","legend":"\u003cp\u003eQuantification of GFP fluorescence and mRNA accumulation in lines containing different introns. \u003cstrong\u003ea.\u003c/strong\u003eMean fluorescence readings are shown to illustrate the signal relative the endogenous autofluorescence of untransformed worms as well as the intronless control. The fold increase in fluorescence caused by each intron is presented in Supplementary Table S2 and Supplementary Fig. S1. \u003cstrong\u003eb.\u003c/strong\u003eRNA gel blot quantification showing the mean fold increase in GFP mRNA relative to the intronless control. Line CB3.10 (marked with an asterisk) contains two copies of the transgene: all other transgenic lines are single-copy. Error bars indicate standard deviation.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5926918/v1/03f7674440344fff4407f967.png"},{"id":82537477,"identity":"8e3d63da-2f09-4b8e-a060-bca8a5030cea","added_by":"auto","created_at":"2025-05-12 16:07:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1567369,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5926918/v1/672b6b98-9eb6-4200-b1a4-fbdbd89d6d1e.pdf"},{"id":75294344,"identity":"a3bb49c1-223a-4028-9413-dace4fd267eb","added_by":"auto","created_at":"2025-02-03 06:32:36","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":267573,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S1.\u003c/strong\u003e Statistical analysis of the constructs shown in Fig. 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S2.\u003c/strong\u003e GFP fluorescence correlates with pharynx length.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S1.\u003c/strong\u003e Statistical comparison of fluorescence in worms containing the constructs shown in Figs. 1a and 1c.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S2.\u003c/strong\u003e Data for each individual line tested.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S3.\u003c/strong\u003e Oligonucleotides used in this study.\u003c/p\u003e","description":"","filename":"RoseSupMat.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5926918/v1/25d60969e98dd9c9d1df1e19.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Introns increase gene expression in Caenorhabditis elegans by a notably different mechanism than in plants","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWithin 2 years of the discovery that protein-coding sequences in eukaryotic genes can be interrupted by intervening sequences (introns)[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], it was found that introns can increase the expression of the genes in which they are located[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. While some introns are known to contain enhancer elements[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], introns can also have a more general effect on expression with properties that differ from those of enhancers. For many genes, expression declines, in some cases to undetectable levels, if the introns they normally contain are deleted, while the expression of a naturally intronless gene often goes up substantially if an intron from another gene is inserted[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Such findings have been reported in a diverse range of organisms[\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], suggesting that the way in which introns boost expression is widely conserved. However, this phenomenon has been explored in just a few organisms and is well understood in none. Measuring expression solely as the enzymatic activity or fluorescent protein produced from a reporter gene is common but does not reveal which of the many aspects of gene expression is being influenced by an intron. Therefore, it remains unclear if the actual mechanism through which introns boost expression is the same in all organisms or varies from group to group. Even within a single organism it is risky to assume that all introns affect expression in the same way, or that any intron has only one effect on expression.\u003c/p\u003e \u003cp\u003eThe known interconnections between many of the various steps of transcription, mRNA processing, export and translation provides multiple opportunities for synergistic effects that increase the overall efficiency of gene expression[\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In mammalian cells, triose phosphate isomerase intron 6 located near the 5\u0026rsquo; end of a luciferase reporter gene increases mRNA accumulation roughly 13-fold, while the same intron near the 3\u0026rsquo; end of the gene has a 2-fold effect[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Introns also cause a 2- to 4-fold increase in the amount of protein produced per unit of mRNA but have little effect on mRNA stability or nuclear export[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The increase in mRNA abundance is likely caused by an increase in transcription[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], as a 5\u0026rsquo; splice site enhances the recruitment of transcription initiation factors[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and splicing factors stimulate transcriptional elongation[\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA systematic analysis of the effect of introns on gene expression in plants was first performed by Callis et al. using the \u003cem\u003eAdh1\u003c/em\u003e gene in maize cells[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. These authors showed that an intron must be located in transcribed sequences to boost expression, that the intron has the greatest effect when near the 5\u0026rsquo; end of the transcript, and that increase in expression is present at the level of mRNA accumulation. Subsequent research confirmed these findings in Arabidopsis[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In addition, efficiently spliced introns vary widely in their effect on expression, with many having no effect[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and the sequences that stimulate mRNA accumulation are distributed throughout a stimulatory intron[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These observations are not consistent with the idea that the increase in expression caused by plant introns could be due to the act of splicing or involves the conserved sequences at each end of most introns.\u003c/p\u003e \u003cp\u003eThe need for the intron to be near the promoter to affect expression led to the discovery that promoter-proximal introns in plants are compositionally distinct from other introns[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The location-dependent difference between introns was detected using the frequency distribution of all possible k-mers (usually pentamers or hexamers) in the genomic population of introns separated into two groups based on whether the start of the intron is less or greater than a threshold distance from the start of transcription of that gene. The IMEter algorithm generates a numerical score that describes the degree to which an individual intron is more similar to the k-mer profile of promoter-proximal introns than that of distal introns. The strong correlation between the IMEter score of an intron and its ability to increase mRNA accumulation allows the stimulating ability of an intron to be predicted from its sequence[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Roughly 5\u0026ndash;10% of introns in a variety of plants have IMEter scores high enough to indicate that they are likely to have a significant effect on expression[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. A motif that is over-represented in Arabidopsis introns with high IMEter scores can convert, in a dose-dependent manner, a non-stimulatory intron into one that strongly boosts expression with the same positional requirements as natural stimulating introns[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The same motif can increase mRNA accumulation when inserted into exonic sequences of an intronless construct[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], demonstrating that splicing is not necessary for this motif to boost expression. Remarkably, stimulatory introns in Arabidopsis can boost the expression of a \u003cem\u003eTRP1\u003c/em\u003e gene fusion containing a 303 nt promoter deletion that removes most of the 5\u0026rsquo;-UTR and all known transcription start sites[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Transcription in those constructs initiates in normally untranscribed intergenic sequences the same distance upstream of the intron as when the promoter is intact.\u003c/p\u003e \u003cp\u003eThe surprising observation that introns can appear more important than the promoter in controlling the expression of a gene had previously been made in mammals and nematodes[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In \u003cem\u003eC. elegans\u003c/em\u003e, an analysis of the non-coding parts of the \u003cem\u003eunc-54\u003c/em\u003e gene showed that deletion of the introns but leaving the promoter and coding sequences intact nearly eliminates the ability of this gene to rescue an \u003cem\u003eunc-54\u003c/em\u003e mutation in transgenic worms[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In contrast, a construct with the promoter deleted but introns still present is surprisingly functional, with transcription starting in the plasmid sequences that the deletion of the promoter brought next to the \u003cem\u003eunc-54\u003c/em\u003e coding sequences. A version of \u003cem\u003eunc-54\u003c/em\u003e in which the promoter and the introns were simultaneously deleted does not rescue the mutation.\u003c/p\u003e \u003cp\u003eA practical response to these findings was the development of intron-containing derivatives of the \u003cem\u003elacZ\u003c/em\u003e and \u003cem\u003eGFP\u003c/em\u003e reporter genes that are still widely used in \u003cem\u003eC. elegans\u003c/em\u003e. One version of \u003cem\u003elacZ\u003c/em\u003e contained 12 small (51 nt) synthetic introns, as this was reported to yield higher expression than versions with 1, 2, or 4 introns (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.addgene.org/kits/firelab/#protocols-and-resources\u003c/span\u003e\u003cspan address=\"https://www.addgene.org/kits/firelab/#protocols-and-resources\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). More recently, introns have been inserted into many reporter genes for use in worms[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, Crane et al.[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] found that in \u003cem\u003eC. elegans\u003c/em\u003e a single intron, either synthetic or natural, increases expression as much as does two or three introns if the lone intron is located near the 5\u0026rsquo; end of the gene. The same intron near the 3\u0026rsquo; end has no effect. The two synthetic and one natural intron they tested at the same 5\u0026rsquo; location cause equivalent increases in expression. The effect of introns varies slightly with different coding sequences and promoters of different strength. The number of introns tested in that study was too small to determine if there are significant differences between natural introns in their ability to boost expression in nematodes as there are in plants. Also, because the effect of introns was measured only as the level of fluorescence in whole transgenic worms containing \u003cem\u003eGFP\u003c/em\u003e reporter constructs, it remains unclear which of the many steps that constitute gene expression is most affected by introns in nematodes.\u003c/p\u003e \u003cp\u003eHere we report a systematic analysis of the effect of introns on gene expression in transgenic nematodes by inserting the same introns into different locations in a reporter gene, by testing ten different introns at the same position, and by measuring steady state mRNA levels. Introns had the largest effect on expression when placed near the 5\u0026rsquo; end of the gene. Introns near the 3\u0026rsquo; end had much smaller effects, as did additional introns added to a construct already containing an intron. All ten of the introns tested at the same location boosted mRNA accumulation to a very similar and high degree. This suggests that despite some similarities with plants, key aspects of the underlying mechanism are different in worms and may be related to the mechanism of splicing rather than to specific sequences within introns.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eTesting intron location and number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe version of \u003cem\u003eGFP\u003c/em\u003e most widely used in the \u003cem\u003eC. elegans\u003c/em\u003e community contains three 51 nt synthetic introns in \u003cem\u003eGFP\u003c/em\u003e coding sequences. To determine the relative effects on expression of different numbers of introns and intron location, an intronless but otherwise identical derivative of this worm codon-optimized \u003cem\u003eGFP\u003c/em\u003e was synthesized. Different combinations of the intron-containing and intronless \u003cem\u003eGFP\u003c/em\u003e genes were used to create a series of \u003cem\u003eunc-54:GFP:GFP\u003c/em\u003e fusions in which the first, the second, neither or both copies of \u003cem\u003eGFP\u003c/em\u003e contained three synthetic introns (Fig. 1a). Transgenic worms containing these constructs were created by Mos1-mediated single copy insertion (MosSCI) and single-copy integration was verified by genomic DNA gel blot analysis. Transgene expression was measured as GFP signal above the endogenous fluorescence of bulk worms using a fluorescent plate reader (Fig. 1b). L1 stage worms were chosen for these measurements because it was possible to generate reasonably synchronized populations of worms at that stage by collecting worms from recently starved plates.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBoth constructs in which the first \u003cem\u003eGFP\u003c/em\u003e contained introns were expressed at a significantly higher level than the constructs lacking introns in the first \u003cem\u003eGFP\u003c/em\u003e (Fig. 1b, Supplementary Fig. S1 and Supplementary Table S1, P = 1.92 x 10\u003csup\u003e-10\u003c/sup\u003e). The presence of introns in the second \u003cem\u003eGFP\u003c/em\u003e caused a slight increase in expression whether or not the first \u003cem\u003eGFP\u003c/em\u003e contained introns. This suggests that introns near the 5\u0026rsquo; end of a gene have a greater effect on expression than the same introns near the 3\u0026rsquo; end of the gene, and indicates that intron position is more important than the number of introns in determining expression level. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo verify that the same single intron can have different effects on expression depending on its location, an additional set of \u003cem\u003eunc-54:GFP:GFP\u003c/em\u003e fusions was created in which the \u003cem\u003eunc-54\u003c/em\u003e intron was tested at its natural location between \u003cem\u003eunc-54\u003c/em\u003e exons 1 and 2, or near the 3\u0026rsquo; end of either the first or the second \u003cem\u003eGFP\u003c/em\u003e (Fig. 1c). As was observed with the small synthetic introns, the \u003cem\u003eunc-54\u003c/em\u003e intron had a much greater effect on expression when near the 5\u0026rsquo; end than in the middle or near the 3\u0026rsquo; end of the fusion construct (Fig. 1d and Supplementary Fig. S1). The \u003cem\u003eunc-54\u003c/em\u003e intron alone had a larger effect on expression than the three synthetic introns but it was also located closer to the 5\u0026rsquo; end of the gene than the first of the synthetic introns, with first exon lengths in those constructs of 142 nt and 337 nt respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSelecting different introns to test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine if worm introns vary widely in their ability to boost expression, as they do in plants, we attempted to create a worm IMEter for use in selecting introns with different predicted effects on expression. However, we found that the difference in k-mer composition between promoter-proximal and distal introns on which the IMEter is based is too small in \u003cem\u003eC. elegans\u003c/em\u003e for the IMEter to function. One way to measure the degree to which the k-mer profiles of proximal and distal introns vary is to sum the absolute value of the differences in frequency of all k-mers in both populations of introns (see methods). The exact value obtained varies depending on the choice of k-mer size and threshold distance used for separating promoter-proximal and distal introns, but the value must be between 0 (if all k-mers have identical frequencies in both populations) and 2 (if all individual k-mers are found exclusively in one population or the other). Using a k-mer size of 5 and a threshold defining promoter-proximal introns as those less than 1000 nt from the start of transcription, the sum of differences of k-mer frequencies in Arabidopsis introns is 0.83, while that of \u003cem\u003eC. elegans\u003c/em\u003e introns is only 0.056 (Fig. 2), indicating that the pentamer compositions of proximal and distal introns are highly similar in worms. Therefore, we used an alternative approach to select worm introns that may have different effects on expression.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMany of the introns that are known to boost expression in plants and other organisms are found in abundantly expressed housekeeping genes, such as those encoding ribosomal proteins, actin, tubulin, histones, and translational elongation factors[36]. This led to the hypothesis that stimulatory introns typically drive the expression of that minority of genes whose products are constitutively required in large amounts in all cells, while conventional transcription factors control the expression of most genes whose products are needed in only certain tissue types, at specific times, or in response to a particular stimulus[36]. If introns have variable effects on the expression of the gene in which they are naturally found in worms, the introns from highly expressed should be more likely to stimulate expression than introns from genes expressed at a very low level. Therefore, seven introns were chosen from a variety of genes based on the following criteria. First, \u003cem\u003eC. elegans\u003c/em\u003e genes were ranked based on their expression level. The most highly expressed genes had approximately 10\u003csup\u003e6\u003c/sup\u003e RNAseq reads as reported on WormBase (https://wormbase.org). Genes were also selected that had approximately 10\u003csup\u003e5\u003c/sup\u003e, 10\u003csup\u003e4\u003c/sup\u003e, or 10\u003csup\u003e3\u0026nbsp;\u003c/sup\u003eRNAseq reads, forming four expression categories referred to as high, medium, low, and very low. Second, within those categories, genes were selected that have been sufficiently studied to be named, are not in an operon, and do not have multiple alternative splice isoforms. Finally, the introns chosen to test from these genes are between 300 and 600 nt in length and are located less than 500 nt from the start of transcription. An additional intron tested was \u003cem\u003esmu-1\u003c/em\u003e intron 3, which contains poly A/T-rich clusters (PATCs) and was previously shown to prevent the silencing of genes in the germline[37]. The exact mechanism through which PATCs prevent silencing is unclear, and this intron was chosen to explore the possibility PATC-containing introns avoid silencing at least in part by causing an unusually large increase in the expression of the gene in which they are located. The eight new introns were compared to two of the introns tested in the \u003cem\u003eunc-54:GFP:GF\u003c/em\u003eP fusions described above, namely \u003cem\u003eunc-54\u003c/em\u003e intron 1 and the 5\u0026rsquo;-most of the 51 nt synthetic introns. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparing the stimulating effect of different introns\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ten introns were individually inserted into an \u003cem\u003eunc-54:GFP\u003c/em\u003e reporter gene at the same location as the natural \u003cem\u003eunc-54\u003c/em\u003e intron 1 (Fig. 1e). Fluorescence was measured in single-copy transgenic worms by photographing individual worms on a fluorescence microscope and measuring fluorescence in the body wall muscles where \u003cem\u003eunc-54\u003c/em\u003e is expressed and endogenous autofluorescence is low, specifically in the head of the worm adjacent to the pharynx (Fig. 3a). The L3 stage was chosen for these experiments because the GFP signal is higher than in L1 worms. The intronless control was expressed at a very low level, generating fluorescence that was slightly but significantly (p \u0026lt; 1x10\u003csup\u003e-4\u003c/sup\u003e) above the endogenous fluorescence in untransformed worms (Fig. 3a, 3b and 4a, and Supplementary Fig. S1). The effect of each intron on expression is reported as the fold increase in pharynx fluorescence relative to that in the intronless control, although the accuracy of this measure is limited by the very low expression of the intronless control. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll ten introns boosted fluorescence 30- to 40-fold relative to the intronless control (p \u0026lt; 1x10\u003csup\u003e-10\u003c/sup\u003e), and all independent single-copy lines containing the same construct gave very similar results (Figs. 3 and 4a, Table 1 and Supplementary Table S2). Only one single-copy line containing the construct with the \u003cem\u003eunc-54\u003c/em\u003e intron was obtained but the values obtained from this line are supported by a line containing a two-copy insert, which gave roughly twice the fluorescence as the single-copy line. There was considerable variation in fluorescence measurements between individual worms of the same genotype measured at the same time. This variation appeared to be mostly due to slight differences in the ages of the worms examined. Pharynx length, which increases at a steady rate as worms grow[38], correlated with fluorescence (Supplementary Fig. S2), indicating that GFP accumulates as the worms age. Despite the similar mean expression levels and large overlapping standard deviations for the data sets obtained from worms containing the same construct, the combined data for some of the introns were statistically different (p \u0026gt; 0.05). However, an analysis of the source of the variation indicated that differences between lines containing the same construct, observations of the same line taken on different days, and the individual taking the measurements all contributed more to the variation than did the identity of the intron (Supplementary Fig. S1). Indeed, we found no introns for which all independent lines containing that construct were significantly different than the individual lines containing another intron. In contrast, the GFP fluorescence of all intron-containing lines were significantly higher than the intronless control (p \u0026lt; 2 x 10\u003csup\u003e-10\u003c/sup\u003e). Thus, we conclude that all ten introns cause a similarly large increase in GFP fluorescence, strongly suggesting that all introns in \u003cem\u003eC. elegans\u003c/em\u003e boost expression equally well.\u003cu\u003e\u0026nbsp;\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eThe synthetic intron and \u003cem\u003eunc-54\u003c/em\u003e intron 1 both increased fluorescence to a very similar degree (36.5 +/- 7.1-fold and 33.6 +/- 11.2-fold respectively, Table 1). This indicates that the higher expression of the \u003cem\u003eunc-54:GFP:GFP\u003c/em\u003e construct containing a single \u003cem\u003eunc-54\u003c/em\u003e intron at its normal position compared to that of the three synthetic introns in the first GFP (Fig. 1) was due entirely to the location of the introns, rather than any difference in their stimulating ability.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. The introns tested and their effect on expression.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"99%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" valign=\"bottom\" style=\"width: 39px;\"\u003e\n \u003cp\u003eGene source of intron\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"bottom\" style=\"width: 32px;\"\u003e\n \u003cp\u003eIntron in native gene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 27px;\"\u003e\n \u003cp\u003eEffect on expression\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003eName\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e\n \u003cp\u003eEncoded protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003eExpression level\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003eIntron size (nt)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003eIntron #\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003eDistance from ATG (nt)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e\n \u003cp\u003eFluorescence\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003emRNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cem\u003eunc-54\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e\n \u003cp\u003eMyosin heavy chain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e562\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e33.6 +/- 11.2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e16.7 +/- 2.9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003esynthetic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e36.5 +/- 7.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e14.2 +/- 2.9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cem\u003esmu-1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e\n \u003cp\u003eSpliceosomal subunit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003eMedium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e305\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e380\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e31.1 +/- 8.7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e9.8 +/- 2.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cem\u003erpl-14\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e\n \u003cp\u003eRibosomal protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e346\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e105\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e39.1 +/- 12.7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e13.8 +/- 2.3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cem\u003elbp-6\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e\n \u003cp\u003eLipid binding protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e554\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e207\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e38.9 +/- 18.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e13.8 +/- 9.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cem\u003egpx-1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e\n \u003cp\u003eGlutathione peroxidase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003eMedium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e444\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e211\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e38.5 +/- 12.8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e12.4 +/- 2.7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cem\u003emec-8\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e\n \u003cp\u003emRNA binding protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003eMedium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e528\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e283\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e38.0 +/- 13.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e13.2 +/- 3.7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cem\u003edaao-1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e\n \u003cp\u003eD-amino acid oxidase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003eLow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e406\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e274\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e32.8 +/- 10.6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e13.1 +/- 3.6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cem\u003egbb-1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e\n \u003cp\u003eGABA B receptor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003eLow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e524\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e228\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e39.6 +/- 12.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e16.1+/- 5.3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cem\u003ecutl-4\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 15px;\"\u003e\n \u003cp\u003eCuticulin-like\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003eVery low\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e354\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e34.6 +/- 8.9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e15.1 +/- 6.9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe effect on expression is the fold increase in \u003cem\u003eGFP\u003c/em\u003e fluorescence or mRNA in lines containing a transgene with that intron relative to the intronless control, mean +/- standard deviation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntrons stimulate mRNA accumulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the level at which expression is primarily affected, total RNA was isolated from partially synchronized populations of worms that were predominantly at the L3 stage. RNA gel blots probed with\u0026nbsp;\u003cem\u003eGFP\u003c/em\u003e revealed that all ten of the tested introns caused an increase in\u0026nbsp;\u003cem\u003eGFP\u003c/em\u003e mRNA of between 10- and 17-fold relative to the intronless control (Figs. 3c and 4b, Table 1). As with the fluorescence measurements, accurate estimations of the intron-caused fold increase in the level of\u0026nbsp;\u003cem\u003eGFP\u003c/em\u003e mRNA are hindered by the very low levels of signal in the intronless control lines. In addition, there is a faint cross-hybridizing band visible even in RNA from untransformed worms that migrates just above the\u0026nbsp;\u003cem\u003eGFP\u003c/em\u003e band that also interferes with quantification of\u0026nbsp;\u003cem\u003eGFP\u003c/em\u003e mRNA levels in the intronless control. Nevertheless, it is clear that a small amount of GFP mRNA accumulates in lines containing the intronless construct, and that all ten introns cause a similarly large increase in GFP mRNA. The mRNA from all intron-containing constructs comigrates with that seen in the intronless control, with no larger bands visible, indicating that all the introns tested are spliced with similar high efficiency. Thus, the difference in GFP mRNA accumulation accounts for most or possibly all the large difference in fluorescence seen between the intron-containing and intronless lines.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere we show that introns boost expression in \u003cem\u003eC. elegans\u003c/em\u003e most when the intron is closest to the 5\u0026rsquo; end of a gene, and that the first intron inserted has the greatest influence on expression, while additional introns have effects that are much smaller and less than additive. These findings confirm and extend observations previously made in worms[35] and other organisms. In addition, we showed that all ten introns tested at the same location caused a similar strong increase in expression, and that the overall effect of an intron on gene expression was due mostly or entirely to differences in mRNA accumulation.\u003c/p\u003e\n\n\u003cp\u003eThe magnitude of the difference in expression of intron-containing and intronless constructs we observed was substantially larger than that reported by Crane et al.[35] (more than 30-fold versus 1.5 to 1.8-fold). The main cause of this difference is the very low level of expression of our intronless \u003cem\u003eunc-54:GFP\u003c/em\u003e fusions, while the intronless \u003cem\u003ehsp-90:mCherry\u003c/em\u003e, \u003cem\u003ehsp-90:GFP\u003c/em\u003e, and \u003cem\u003evit-2:mCherry\u003c/em\u003e fusions used by Crane et al. were expressed at relatively high levels. One potential explanation is that one of the two \u003cem\u003ehsp-90\u003c/em\u003e transcript isoforms (C47E8.5.2) reveals the presence in the \u003cem\u003ehsp-90\u003c/em\u003e 5\u003cspan dir=\"RTL\"\u003e\u0026rsquo;\u003c/span\u003e-UTR of a 1,116 nt intron whose 3\u003cspan dir=\"RTL\"\u003e\u0026rsquo; \u003c/span\u003eend is 4 nt upstream of the ATG (https://wormbase.org/). Because this entire intron is present in the 2 kb promoter fragment used for all \u003cem\u003ehsp-90\u003c/em\u003e constructs, even those described as intronless, this 5\u003cspan dir=\"RTL\"\u003e\u0026rsquo;\u003c/span\u003e-UTR intron might be responsible for the relatively high expression of constructs lacking introns in the reporter genes. The introns subsequently added to the reporter gene would have a minor effect since they are not the first intron in the gene. However, there is no evidence for an intron in the 5\u003cspan dir=\"RTL\"\u003e\u0026rsquo;\u003c/span\u003e-UTR of \u003cem\u003evit-2\u003c/em\u003e that could explain the high expression of the intronless \u003cem\u003evit-2:mCherry\u003c/em\u003e fusion.\u003c/p\u003e\n\n\u003cp\u003eAlternatively, it is possible that \u003cem\u003eC. elegans\u003c/em\u003e is similar to mammals where the expression of some genes such as b-globin absolutely requires the presence of an intron, while other genes such as dihydrofolate reductase are expressed at detectable levels without an intron even if their expression can be boosted by an intron[4]. Perhaps \u003cem\u003eunc-54\u003c/em\u003e is an example of an intron-dependent gene while \u003cem\u003ehsp-90\u003c/em\u003e and \u003cem\u003evit-2\u003c/em\u003e are intron-independent. Crane et al.[35] also examined expression in adult worms that have had longer to accumulate GFP from a poorly expressed construct than the L3 worms used here. Other potential sources of variability can be ruled out. The synthetic intron we used was identical to their intron \u0026ldquo;ia\u0026rdquo;, the introns were similar distances from the start of transcription, the \u003cem\u003eunc-54\u003c/em\u003e terminator was used in both studies, and the constructs were integrated using the same technique at the same chromosomal location.\u003c/p\u003e\n\n\u003cp\u003eSeveral observations suggest that unlike in plants where specific stimulating sequences present only within a minority of introns are responsible for increasing mRNA accumulation, in nematodes it may be a conserved intron structure (such as a splice site) or the act of splicing that boosts expression. First, all ten of the introns tested increased expression to a very similar degree and none failed to have an effect. Second, \u003cem\u003eC. elegans\u003c/em\u003e introns lack the significant k-mer compositional differences between promoter-proximal and distal introns seen in plants, even though introns boost expression most when located nearest to the 5\u0026rsquo; end of a gene in both plants and nematodes. Third, there is very little room in the 51 nt synthetic intron for sequences that could boost expression to the same degree as introns that are more than ten times its length, such as the 562 nt \u003cem\u003eunc-54\u003c/em\u003e intron 1. In Arabidopsis, the stimulating sequences within introns have additive effects[31]. This may be one reason that first introns, which are the most likely to boost expression, also tend to be longer than other introns[39].\u003c/p\u003e\n\n\u003cp\u003eOne possible explanation for a splicing related increase in GFP fluorescence is that intron-containing and intronless constructs might yield the same amount of primary transcript but that only spliced mRNAs are efficiently exported to the cytoplasm and translated. The exon junction complex (EJC) proteins that are deposited on the mRNA during splicing are known to promote export[40,41]. However, the RNA used in Fig. 3 was isolated from whole worms and thus would include both nuclear and cytoplasmic RNA. The observation that intron-containing constructs produced 10 to 15 times more GFP mRNA than did the intronless control rules out the possibility that the primary cause of the different expression levels was the subcellular localization of the mRNA. The EJC could have a roughly 2-fold effect on expression because the observed 30- to 40-fold increase in GFP fluorescence caused by introns was roughly twice that seen at the level of mRNA, suggesting that more protein was produced per unit of mRNA when the gene contained an intron. Similar observations have been made in plants where the effect of an intron is consistently twice as large measured at the level of enzyme activity than as mRNA accumulation, which has been attributed to increases in export and translation[25,30,31]. Still, in both worms and plants, the intron-mediated increase in expression can be largely or entirely attributed to increases in mRNA accumulation. \u003c/p\u003e\n\n\u003cp\u003eThe effect of the introns on mRNA accumulation could either be a positive action that increases mRNA production, or the removal of a repression that inhibits expression of intronless constructs. In one example of introns actively stimulating expression, some human genes with weak promoters upstream of alternatively spliced exons have the occupancy of RNA polymerase II boosted by specific splicing factors that recruit the core transcription machinery[42]. In an example of intronless repression in the \u003cem\u003eC. elegans\u003c/em\u003e germline, the removal of introns from a gene can cause it to be silenced by two distinct pathways, one of which uses argonaute proteins and small RNAs[43]. In the intron-containing versions of the same genes, intron splicing somehow marks transcripts and prevents their recognition as templates for argonaute-mediated silencing. However, the same intronless genes are not silenced in the somatic cells[43] where the \u003cem\u003eunc-54:GFP\u003c/em\u003e fusions studied here are predominantly expressed.\u003c/p\u003e\n\n\u003cp\u003eIn summary, there are some similarities and some differences in the effect of introns in worms and plants. In both groups, the effect of the intron on expression was primarily at the level of mRNA accumulation and was larger if the intron was near the 5\u003cspan dir=\"RTL\"\u003e\u0026rsquo; \u003c/span\u003eend of the gene than if the same intron was located further away from the start. In addition, the remarkable ability of introns to affect expression in the absence of a promoter[26,32] suggests a mechanism that is similar in both groups and distinct from that of enhancer elements, which cannot activate expression in the absence of a promoter and can have effects over much greater distances than introns can. However, a significant difference is that in plants, only an estimated 5-10% of introns boost mRNA accumulation tenfold or more while others have a much smaller effect or none at all[29]. In contrast, all ten of the worm introns tested here had the same strong effect on mRNA accumulation. The observation that a PATC-containing and non-PATC introns boosted mRNA accumulation equally well suggests that the difference in their ability to prevent germline silencing between introns that contain PATCs and other introns is not due to their effect on mRNA production.\u003c/p\u003e\n\n\u003cp\u003ePerhaps the underlying mechanism in both plants and worms involves introns directing transcript initiation upstream of themselves through interactions between introns and the transcription machinery, explaining the need for introns to be near the 5\u003cspan dir=\"RTL\"\u003e\u0026rsquo; \u003c/span\u003eend of the gene and the surprising dispensability of promoter sequences. In plants these interactions could be mediated by proteins that bind to sequences present in variable numbers within the body of a minority of introns, while in worms they could involve the spliceosome or other proteins that recognize conserved sequences around the splice sites of all introns. The results from worms, plants, and mammals illustrate that sequences downstream of the transcription start site, especially those near the 5\u003cspan dir=\"RTL\"\u003e\u0026rsquo; \u003c/span\u003eend of a gene, can have effects on expression that outweigh those of the more familiar regulatory sequences in the promoter around and upstream of the transcription start site[44].\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cu\u003eWorms\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eC. elegans\u003c/em\u003e strains were maintained at room temperature on MYOB plates with \u003cem\u003eE. coli\u003c/em\u003e OP50 as a food source[45,46]. The wild-type strain is N2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eConstructs\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eAll constructs contain the same 453 nt \u003cem\u003eunc-54\u003c/em\u003e (F11C3.3) promoter fragment, the entire 95 nt \u003cem\u003eunc-54\u003c/em\u003e exon 1 (32 nt of 5\u003cspan dir=\"RTL\"\u003e\u0026rsquo;\u003c/span\u003e-UTR and 63 nt coding sequence), the first 9 nt of \u003cem\u003eunc-54\u0026nbsp;\u003c/em\u003eexon 2, some version of \u003cem\u003eGFP\u003c/em\u003e, and the same 713 nt \u003cem\u003eunc-54\u003c/em\u003e terminator fragment. They vary in the identity of the intron present at the normal location of \u003cem\u003eunc-54\u003c/em\u003e intron 1, the number of \u003cem\u003eGFP\u003c/em\u003e genes, and the identity and location of the introns in \u003cem\u003eGFP\u003c/em\u003e. The \u003cem\u003eunc-54\u003c/em\u003e promoter fragment was amplified from genomic DNA using primers OAR130 and OAR131, the \u003cem\u003eunc-54\u003c/em\u003e intron was removed by amplification with OAR130 and OAR132, and the \u003cem\u003eunc-54\u003c/em\u003e terminator was amplified with primers OAR135 and OAR136 (primer sequences are found in Supplementary Table S3). An intronless version of \u003cem\u003eGFP\u003c/em\u003e was synthesized by Biomatik (Kitchener, Ontario). Derivatives of the various intron-containing and intronless \u003cem\u003eGFP\u003c/em\u003es in which the stop codon was replaced by a 12 nt region encoding a 4 amino acid linker and containing various flanking restriction sites were created by PCR to facilitate the construction of the tandem \u003cem\u003eGFP\u003c/em\u003e fusions. The \u003cem\u003eunc-54\u003c/em\u003e intron was inserted by Gibson assembly using the NEBuilder kit (New England Biolabs, Ipswich, MA) near the 3\u003cspan dir=\"RTL\"\u003e\u0026rsquo;\u0026nbsp;\u003c/span\u003eend of \u003cem\u003eGFP\u003c/em\u003e using primers OAR210 and OAR211.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn most cases, introns were amplified using high fidelity polymerase directly from wild-type N2 genomic DNA using primers whose 3\u0026rsquo; half matches the sequence at the end of the intron and whose 5\u0026rsquo; ends match \u003cem\u003eunc-54\u003c/em\u003e sequences flanking the site of intron insertion. For the \u003cem\u003edaao-1\u003c/em\u003e and \u003cem\u003erpl-14\u003c/em\u003e introns, the template was an intermediate PCR product made using primers OAR237-240 that match exon sequences near the target intron. The 51 nt synthetic intron was made as two complementary primers OAR258 and OAR259 that were annealed to each other. The introns were inserted between \u003cem\u003eunc-54\u003c/em\u003e exons 1 and 2 by Gibson assembly into an \u003cem\u003eunc-54p:unc-54::GFP\u003c/em\u003e fusion. The resulting plasmids were sequenced to verify accurate insertion and the absence of mutations before the intron-containing \u003cem\u003eunc-54p:unc-54::GFP\u003c/em\u003e fusion was inserted as a \u003cem\u003eBgl\u003c/em\u003eII-\u003cem\u003eSpe\u003c/em\u003eI fragment into the vector pCFJ151[47], which contains the \u003cem\u003eunc-119\u003c/em\u003e marker gene.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eMos1-mediated single copy insertion (\u003c/u\u003e\u003cu\u003eMosSCI\u003c/u\u003e\u003cu\u003e)\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eThe intron-containing \u003cem\u003eunc-54:GFP\u003c/em\u003e fusions were integrated into the ttTi5605 site on chromosome 2 in strain EG6699 by MosSCI[47,48]. The injections were performed by InVivo Biosystems (Eugene, OR, formerly known as Knudra Transgenics or NemaMetrix). The resulting worms were screened for progeny that had integrated the \u003cem\u003eunc-119\u003c/em\u003e gene and lost the \u003cem\u003emCherry\u003c/em\u003e markers in the injection mix, and strains homozygous for the transgene insertion were obtained. To verify single-copy integration, DNA from each line was isolated using the DNeasy blood and tissue kit (Qiagen), digested with \u003cem\u003eSpe\u003c/em\u003eI and \u003cem\u003eHin\u003c/em\u003edIII, and subjected to gel blot analysis with a \u003csup\u003e32\u003c/sup\u003eP labeled GFP probe and detected using a Storm phosphoimager. Transgene copy number was estimated by comparing band intensity using ImageQuant software (Molecular Dynamics). All single-copy lines obtained were used and given equal weight in calculations. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eQuantification of GFP in populations of worms\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eL1 stage worms were collected off starved plates using water, concentrated by brief centrifugation, and 125 mL of resuspended worms were placed in each well of a 96-well plate. Fluorescence was measured on a Perkin Elmer Model 2030-0050 Multilabel Plate Reader at an emission wavelength of 535 nm with an excitation wavelength of 485nm (to measure GFP) or 355 nm (to correct for variation in the number of worms in each well using autofluorescence). For every line, at least six technical replicates were performed on each of three or more separate days. The ratio of the signals obtained with excitation at 485 nm to that at 355 nm was normalized by setting the level for untransformed worms equal to zero and the ratio in transgenic worms containing the intronless control to one. The values for intron-containing constructs represent the average fold increase in above-background fluorescence caused by the intron.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eQuantif\u003c/u\u003e\u003cu\u003eication of\u003c/u\u003e\u003cu\u003e\u0026nbsp;GFP in\u0026nbsp;\u003c/u\u003e\u003cu\u003eindividual worms\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eWorms from a starved plate were grown on a fresh plate at room temperature for 17-18 hours so that the population was predominantly at the L3 stage. L3 worms were picked and anaesthetized in a 0.25% solution of tetramisole on a 2% agar pad and examined using a UPlan FL 40x, 0.75 numerical aperture objective on an Olympus BX60 compound microscope, with a Hamamatsu Orca 12-bit digital camera and Micromanager software[49]. Single plane images were acquired in brightfield and fluorescent image at 150 ms exposure. The resulting images were then quantified using ImageJ software version 1.53[50] using the segmented line tool with a width of 10 pixels to trace the muscle of each worm starting from the base of the grinder to the tip of the nose and back to the grinder on the opposite side of the worm. The mean reading per pixel for the entire line was corrected for background by subtracting the mean of a line drawn outside the worm parallel to and roughly the same length as the pharynx. Measurements were obtained from at least three biological replicates totaling more than 40 individual worms for each line. To calculate the fold increase in fluorescence caused by each intron, the average reading from untransformed worms was subtracted from the average readings for all transgenic lines containing the same construct, and the individual readings of lines with an intron-containing construct were divided by the average reading of the intronless control lines.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eStatistics\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eFor all tests of significant differences between groups (i.e. lines, introns) we used a linear model in R, \u003cem\u003elm(value~group)\u003c/em\u003e. Model assumptions (e.g. normality of residuals) were validated by visualizing model diagnostic plots. Significant differences (two-sided test) among groups were determined using Tukey Honest Significant Difference (\u003cem\u003eTukeyHSD\u003c/em\u003e function in R) at \u003cem\u003ealpha\u003c/em\u003e=0.05, after Bonferroni correction for multiple testing.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eRNA isolation and RNA gel blots\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eWorms from a starved plate were grown on a fresh plate at room temperature for 17 hours so that the population was predominantly at the L3 stage. One mL of water was used to rinse the worms off 2 plates and pooled in a microfuge tube with the worms from another 2 plates. The tubes were centrifuged at 8,000 rpm (5,000g) for 5 seconds and the supernatant was discarded. The worms were rinsed in 1 ml 0.1% SDS by vortexing and centrifuging as above and the supernatant was removed leaving the worms in a volume of 50 mL or less. The worms were resuspended in 100 mL worm lysis buffer (50 mM KCl, 10 mM TRIS pH 8.3, 2.5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.45% Tween 20) containing proteinase K at a final concentration of 1 mg/mL (30-40 units/mL) and incubated at 55\u0026deg;C for ten minutes with mixing every 2-3 minutes. RNA was isolated using RNeasy Plus kits (Qiagen) following the manufacturers protocol. In short, this involved adding 350 mL buffer RLT Plus containing DTT to the lysed worms, mixing, and pipetting onto a gDNA removal column, which was spun for 30 seconds at 10,000 rpm (8,000g). The flow-through was mixed with 350 mL 70% ethanol and transferred to an RNeasy column, which was spun 15 seconds at 10,000 rpm (8,000g). The column was rinsed once with 700 mL buffer RW1 and twice with 500 mL buffer RPE, then dried by spinning at top speed for 1 minute. RNA was eluted from the column with two washes with 50 mL RNAse free water, which were combined. The yield of RNA (typically 5-15 mg) was determined by spectrophotometer, the RNA was concentrated by ethanol precipitation, and the dried RNA was resuspended in RNAse free water.\u003c/p\u003e\n\u003cp\u003e2 mg of RNA from each line was used in RNA gel blots performed using reagents from the Northern Max kit (Ambion), hybridized with a \u003csup\u003e32\u003c/sup\u003eP labeled GFP probe, and detected using a Storm phosphoimager. Band intensity was measured using ImageQuant software and corrected for small differences in loading using ethidium bromide stained rRNA bands quantified using BioRad software.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eComputational\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eMaster files containing the sequences of all the introns in \u003cem\u003eC. elegans\u003c/em\u003e or Arabidopsis, as well as the distance between the transcription start site and the start of the intron, were extracted from the annotated genomes of those organisms. As in the IMEter algorithm[28], the frequency of occurrence of all possible k-mers was determined separately in the two populations of introns less than or greater than a threshold distance from the transcription start site of the gene in which the intron is located. The frequency of each k-mer is calculated as the count for that k-mer divided by the counts for all k-mers found in that population of sequences. Therefore, the sum of the frequency of all k-mers in each population is 1. The difference between the two frequency distributions was determined as the sum of the absolute values of the difference in frequency of each individual k-mer in the promoter-proximal and distal intron populations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eAdditional Information\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.B.R. designed the experiments, performed experiments, and wrote the manuscript. A.B. and I.S. performed experiments. J.G.M. did statistical analysis. I.K. provided computational input. L.S.R. advised and supplied the facilities for growing the worms and collecting microscopic data. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThanks to Alice Pierce, Cindy Bailey, Clarence Reyes, and Hoang Tran, for constructing plasmids, and Jenna Noueihed for help with coding. This work was supported by funds obtained by A.B.R. and L.S.R. teaching first year seminars at UC Davis.\u003c/p\u003e\u003ch2\u003eData Availability Statement\u003c/h2\u003e \u003cp\u003eExpression data and strains are available upon request. The software for comparing kmer frequency distributions is available from GitHub.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBerget, S. M., Moore, C. \u0026amp; Sharp, P. A. Spliced segments at the 5\u0026apos; terminus of adenovirus 2 late mRNA. \u003cem\u003eProc. Natl. Acad. Sci. USA\u003c/em\u003e \u003cstrong\u003e74\u003c/strong\u003e, 3171-3175 (1977).\u003c/li\u003e\n\u003cli\u003eGruss, P., Lai, C. J., Dhar, R. \u0026amp; Khoury, G. 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Methods\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 671-675 (2012).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Introns, gene expression, nematode, mRNA accumulation, intron-mediated enhancement","lastPublishedDoi":"10.21203/rs.3.rs-5926918/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5926918/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe wide diversity of organisms in which introns stimulate gene expression suggests that this is an ancient phenomenon. However, the mechanisms through which introns boost expression remain poorly understood, and the degree the which the action of introns is evolutionarily conserved is unknown. Here we compared the effect on expression of introns at different positions and tested ten different introns at the same location in a reporter gene in single-copy transgenic nematodes. The introns boosted expression most when near the start of the gene, as previously observed in several organisms. All ten introns tested at the same position increased mRNA accumulation 10- to 17-fold, in contrast to plants where introns vary widely in their effect on expression and relatively few increase mRNA levels 10-fold or more. These results suggest that some aspects of the mechanisms through which introns boost expression are fundamentally different in nematodes and plants.\u003c/p\u003e","manuscriptTitle":"Introns increase gene expression in Caenorhabditis elegans by a notably different mechanism than in plants","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-03 06:24:31","doi":"10.21203/rs.3.rs-5926918/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-02-12T10:18:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-02-12T10:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-01-30T08:03:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-01-29T23:35:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"417020d5-26f9-49fa-8e76-3d0685444548","owner":[],"postedDate":"February 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":43609377,"name":"Biological sciences/Genetics/Gene expression"},{"id":43609378,"name":"Biological sciences/Genetics/Gene regulation"}],"tags":[],"updatedAt":"2025-05-12T16:01:19+00:00","versionOfRecord":{"articleIdentity":"rs-5926918","link":"https://doi.org/10.1038/s41598-025-99739-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-05-07 15:57:25","publishedOnDateReadable":"May 7th, 2025"},"versionCreatedAt":"2025-02-03 06:24:31","video":"","vorDoi":"10.1038/s41598-025-99739-6","vorDoiUrl":"https://doi.org/10.1038/s41598-025-99739-6","workflowStages":[]},"version":"v1","identity":"rs-5926918","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5926918","identity":"rs-5926918","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}