In order to identify alternatively spliced cassette exons in the C. elegans genome, we used the Intronerator [24] to generate an initial set of 1,471 putative alternatively spliced genes. We did this by aligning over 200,000 ESTs and mRNAs to the C. elegans genome and identifying regions where the alignments are consistent with more than one way to process a gene. The program could not distinguish between alternative splicing and alternative promoters, so we analyzed each of these alignments individually to verify alternative splicing. We found 449 examples of genes with strong cDNA evidence for alternative cassette exons, and 454 examples of genes with alternative promoters, which usually had unique first exons that are spliced to common second exons. Of the genes with alternative splicing, 162 also contained alternative promoter usage. The remaining genes in the initial set of putative alternatively spliced genes were mostly due to unusual ESTs in the database that did not fit a gene model. We also saw evidence of ESTs that showed internal deletions at short direct repeats. To the program, these indicated the potential for intron removal, but upon further inspection, these did not meet the criteria of introns (start with the dinucleotides GT or GC and end with AG) and were likely the result of cloning artifacts of the ESTs.

Intronic regions that are highly conserved between C. elegans and C. briggsae are rarely identified by WABA [25]. While analyzing our alternative cassette exon database, we were struck by the fact that we could identify many examples of WABA-defined high homology sequences in introns flanking the alternatively spliced cassette exons, suggesting strong evolutionary conservation. For 142 of the 449 alternatively spliced genes, WABA identified a highly conserved sequence in flanking introns upstream or downstream of 147 alternative cassette exons. Figure 1 shows several examples of screen shots from the Intronerator browser of cDNA-confirmed C. elegans alternatively spliced isoforms (in blue) and a graphical representation of the WABA alignments between C. elegans and C. briggsae (in purple). Dark purple regions, indicating high homology, can be seen in these introns either upstream or downstream of the alternative exon. Sometimes a single conserved element is identified by WABA, while for other cases multiple regions in the introns are conserved.

An important question is whether these 147 C. elegans alternative cassette exons are also alternatively spliced in C. briggsae. Due to a lack of C. briggsae transcript data, we have no direct evidence for alternative splicing of these exons. Therefore, we examined the C. briggsae homologs of these alternative exons for features of functional exons. A previous alignment of 8 MB of the C. briggsae genome with the complete C. elegans genome found that in coding exons there is 79.3% overall nucleotide identity [25]. Consistent with these findings, cross-species alignment of these 147 alternative cassette exons revealed an average nucleotide identity between C. elegans and C. briggsae of 81.8%. Amino acid identity of the open reading frame of these exons was 85.4%. Maintenance of the reading frame for these exons also appears to be well conserved: 57.0% of the 147 exons are the exact same size, 34.3% differ by a multiple of three nucleotides, and only 8.8% differ by a non-multiple of three nucleotides (unpublished data). The high conservation of nucleotide sequence, amino acid sequence, and maintenance of open reading frame is consistent with these being coding exons in C. briggsae.

One feature of alternative exons is that they are often flanked by weak splice sites which allow for splicing regulation (reviewed in [33]). If these exons are alternatively spliced in C. briggsae we would expect that they would be flanked by 3′ and 5′ splice sites of similar strength. We used the UCSC Genome Table Browser (http://www.genome.ucsc.edu) in an attempt to automate rapid alignment of the splice junctions for these 147 exons in C. elegans and C. briggsae. For the majority of these alternative exons, this automated method was successful in identifying C. elegans and C. briggsae 5′ and 3′ splice sites for each exon, and we used these data to compare the similarity of splice sites between the two species. For 5′ splice sites we examined the last three nucleotides of the exon and first six nucleotides of the intron, as these base-pair with U1 small nuclear RNA during initial 5′ splice-site recognition[31]. We found that these nine nucleotides were completely identical for 47.1% of the alternative exons and differed by only one nucleotide in 27.1% of the exons. For the 3′ splice sites, we examined the conservation of the last six nucleotides of the intron and the first nucleotide of the exon, as these are a binding site for the C. elegans homolog of the heterodimeric U2 auxiliary factor (U2AF) involved in initial 3′ splice-site recognition [34]. For 55.4% of the alternative exons examined, these seven nucleotides were completely identical, and for an additional 30.1% they differed by only one nucleotide. This strong conservation of splice-site sequence strength, along with regions of conservation in flanking introns, suggests that these exons may be substrates for alternative splicing in C. briggsae.

There are many examples of regions of introns flanking alternatively spliced exons that function as cis-acting regulators of alternative splicing. We hypothesized that the reason that these high homology WABA-identified intronic elements have been conserved over 100 million years of evolution is that they may regulate alternative splicing. In order to test this hypothesis, we looked at the C. elegans/C. briggsae alignment of the alternatively spliced region of the C. elegans alpha(2) type IV collagen gene let-2. Let-2 has two mutually exclusive alternative exons, exons 9 and 10, and their splicing pattern is evolutionarily conserved as far back as the distantly related parasitic nematode, Ascaris suum [35]. Messages incorporate either exon 9 or exon 10 in a developmentally regulated manner; embryos predominantly use exon 9, adults predominantly use exon 10, and there is a gradual shift in the usage of these two exons during the larval stages [36]. The WABA alignment of this 400-base intron identifies four conserved regions in the intron between exons 10 and 11 (Figure 2). To test the role of these conserved intronic elements in alternative splicing regulation, we employed a splicing reporter construct system containing the alternatively spliced region between exons 8 and 11 that mimics the developmental control of alternative splicing for this region when transformed into C. elegans [37]. We mutated the first conserved element in this intron and monitored the developmental regulation of this splicing. Deletion of this conserved element, in which 28 of 34 bases have been conserved between C. elegans and C. briggsae, results in a major reduction in the usage of exon 10 in L4 animals (unpublished data). The identical effect on splicing of an even smaller deletion within this element, del1.2, is shown in Figure 2. For the L4 time point, the developmental stage at which we should detect maximal exon 10 usage, only minimal splicing of this exon is detected. Since mutation of this element results in only minimal exon 10 inclusion, we hypothesize that either adults produce a splicing factor that interacts with this element to promote exon 10 splicing or that embryos produce a splicing repressor that inhibits exon 10 splicing. In the past, researchers have identified splicing regulatory elements by creating a series of mutations across an intron. In this computational approach, the interspecies genome alignments led us directly to a small element required for the developmentally regulated switch in this splicing.

As demonstrated in the previous section, evolutionarily conserved elements in introns flanking alternatively spliced exons can be important for alternative splicing. We used a computational approach to identify sequences that are more likely to occur in these conserved elements than in total intron sequences. As described above, we have identified 142 alternatively spliced genes in which the introns flanking an alternatively spliced cassette exon contain high homology regions with C. briggsae as defined by WABA. We extracted the highly conserved C. elegans sequences from these introns and put them into a database. The first or last seven nucleotides of introns were excluded from this dataset as these contain conserved signals for the constitutive splicing machinery [38]. This dataset contained 537 conserved elements of average length, 38.5 bases (minimum length, seven bases, longest, 231 bases, and median length, 28 bases) for a total of 20,675 bases. See Table S1 for a list of these elements and the alternatively spliced genes from which they were derived. We also generated a control dataset of all introns annotated in Wormbase genome sequence release WS 120.

Because many alternative splicing factors have binding preferences for relatively short sequence motifs [11,13,18,28–32], we decided to search the conserved intronic element dataset for short sequence motifs that appear more frequently here than in total introns. In order to identify sequences that are more prevalent in the conserved intronic regions bordering alternative exons, we counted the number of occurrences of every possible hexamer or pentamer motif in both our conserved intron element and total intron datasets. For each motif in the conserved element dataset, we determined the observed frequency, which was the count of each motif divided by the total number of possible motifs of that length in that dataset. We also determined an expected frequency based on the number of counts of each motif in the total intron dataset divided by the total number of possible motifs in that dataset. We then calculated an observed over expected (obs/exp) ratio for each motif, which was the observed frequency of each motif in the conserved element dataset divided by the expected frequency of each motif based on the total intron dataset. Table 1 shows the 40 top scoring pentamer and hexamer motifs in the conserved element dataset as determined by the obs/exp ratio value.

Several previously identified mammalian splicing regulatory sequences are present in our lists of high-scoring hexamers and pentamers. This is encouraging because many of the known mammalian splicing factors have C. elegans homologs. For example, the sequence TGCATG and its subset pentamer GCATG have been shown to be important intronic regulators of splicing for the mammalian fibronectin and calcitionin/CGRP genes [14,15,39]. The mammalian FOX-1 protein selected the GCAUG sequence in a SELEX experiment, and it has been shown that the FOX-1 protein alters alternative splicing of several GCAUG-containing genes in transient transfection assays [13]. FOX-2, a homolog of FOX-1, has also been shown to regulate splicing of transcripts containing UGCAUG in neuronal cell culture [12]. FOX-1 was originally identified in C. elegans as a splicing factor of the transcript for the sex determination gene, xol-1 [40]. Another well studied mammalian motif is that of intronic GU dinucleotide repeats, which have been shown to regulate splicing of the human CFTR gene [41] and serve as a binding site for the ETR-3 splicing regulatory factor [42]. CU dinucleotide repeats serve as a binding site for the PTB family members in the intronic regions that regulate inclusion of the N1 exon of c-src [43]. While targets for C. elegans alternative splicing factors might be inferred from their homology to mammalian proteins, very little experimental evidence exists for nematode splicing-factor binding sites. The C. elegans muscle-specific splicing factor SUP-12 regulates unc-60 alternative splicing. This protein interacts with a GU-rich region, similar to the GU-rich motifs identified in Table 1, of an unc-60 intron [44]. (For a review of other mammalian splicing factors with C. elegans homologs see [33].) While many of the high-scoring conserved motifs that we identified match known mammalian splicing regulatory sequences, we have also identified new motifs that may also be involved in pre-mRNA splicing regulation in mammals as well as nematodes.

One important aspect of C. elegans introns is that they are shorter than introns in vertebrates. Half of C. elegans introns are below 60 nucleotides in length, too short to be spliced in vertebrates [38]. However, many C. elegans introns are long and resemble vertebrate introns. In our alternatively spliced intron dataset, the median intron size is 263 bases (shortest is 43, longest is 10,719, and average is 561), indicating that these regulated introns generally belong in the larger intron class. We asked whether a similar analysis of the pentamers and hexamers found in the full introns flanking alternatively spliced exons would yield similar high-scoring motifs as our analysis of the conserved sequence subset of these introns. We did a pentamer and hexamer motif count of the full introns flanking the 147 alternatively spliced exons from which we previously extracted evolutionarily conserved elements. We counted the occurrence of every pentamer and hexamer in these full-length alternative introns. We ranked these motifs by the obs/exp ratio when we compared the occurrence of these motifs to their prevalence in our total intron dataset. The top 40 pentamer and hexamer motifs ranked by their obs/exp ratios are shown in Table 2. Many of the top scoring pentamers and hexamers that are overrepresented in conserved elements in introns flanking alternatively spliced exons are also overrepresented in total introns flanking alternatively spliced exons (compare Table 1 with Table 2). However, the obs/exp scores for the motifs in the alternative splicing introns is 2- to 3-fold lower than in the conserved element dataset extracted from these introns. For example, TGCATG has an observed to expected ratio of 3.35 in the whole introns that flank alternative exons but 8.87 for the conserved elements within those introns. TCTATC has a ratio of 2.55 in the total alternatively spliced intron analysis and 6.96 in the conserved element dataset analysis. The same holds true for high-scoring pentamers. CTATC goes from 1.85 for the observed to expected ratio in the introns flanking alternative exon dataset up to 4.23 in the conserved element dataset. From this analysis, the sequences of the introns flanking alternatively spliced exons can yield some data about potential pentamers and hexamers involved in alternative splicing regulation. However, limiting this analysis to WABA-conserved regions between C. elegans and C. briggsae can substantially improve the signal to help us identify splicing regulatory elements.

We also used the database of introns flanking alternatively spliced exons to identify potential differences in the appearance of pentamer and hexamer motifs in the introns upstream or downstream of alternative exons. We grouped the introns upstream of the alternative exons and the introns downstream of the alternative exons separately into datasets and did motif counts and determined the obs/exp ratio for each when compared to the total intron dataset. These results are shown in Tables 3 and 4, with the top scoring pentamers and hexamers in the introns upstream and downstream of alternative exons ranked by their obs/exp ratios. Some of the high- scoring motifs show very little preference for the upstream or downstream intron. For example, high-scoring hexamer TGCATG has an obs/exp ratio of 3.90 in the downstream intron and 3.06 in the upstream intron. Others show a strong preference. CTCTCT and TCTCTC, likely binding sites for PTB, have obs/exp ratios of 5.15 and 4.84, respectively, in upstream introns, but 1.36 and 1.30 in downstream introns. TCTATC, which will be analyzed below, also has a preference for upstream introns, with an obs/exp ratio of 3.25 in upstream introns and 1.79 in downstream introns. Conversely, CCAACC has a strong preference for downstream introns, with an obs/exp ratio of 3.17 in the downstream intron and 1.29 in the upstream intron. In addition to identifying potential binding sites for splicing regulatory factors, these differences in appearance in introns upstream or downstream of alternatively spliced exons may hold some keys to function.

To determine whether these pentamers and hexamers are found in introns flanking all alternative cassette exons or are specific for those introns with WABA-conserved elements, another dataset was constructed from the introns flanking the alternative cassette exons of 307 genes lacking WABA-defined evolutionarily conserved intronic elements. A motif search similar to that described in the previous paragraph was conducted by dividing the new, non-conserved intron dataset into two sets: introns upstream of alternative exons and those downstream. Motifs were counted for each of the sets and compared to the total intron dataset to generate an obs/exp ratio and corresponding ranking for each element. It can be seen from Table 4 that the obs/exp scores of all of the top 30 hexamer motifs identified in conserved introns drop in the analysis of non-conserved introns, suggesting the motifs are indeed specific to introns containing conserved regions. For example, our top ranking hexamer from the conserved intron analysis, TGCATG, has an obs/exp score of 3.9 in downstream introns and 3.06 upstream. This same motif only scores 1.75 in downstream and 1.93 in upstream in non-conserved alternative introns (Table 4). For CTCTCT and TCTCTC, our two top hexamer upstream motifs in introns with WABA-conserved elements, the obs/exp ratio drops over 3-fold in this analysis, and they rank as 579 and 688, respectively, in the upstream non-conserved intron set (Table 4). Conversely, top scoring motifs from the non-conserved intron database are not highly represented in the conserved intron dataset. For example, GGCCAC, with an obs/exp ratio of 4.36 (Table S2) was the top scoring motif in the upstream non-conserved dataset. Strikingly, this same motif has an obs/exp score of 0.11 in total introns that contain WABA-conserved elements upstream of an alternatively spliced exon (unpublished data). This dramatically different representation of top scoring pentamers and hexamers in introns containing WABA-conserved elements versus those that do not suggests that the presence or absence of intronic WABA-defined conservation may define two distinct classes of alternatively spliced exons with distinct splicing regulatory mechanisms.

To ensure that the motifs identified in the conserved element dataset were truly overrepresented in this region and not a consequence of WABA creating bias towards GC-rich regions in the AT-rich nematode genomes, we analyzed the base composition of our datasets. The GC content of our WABA-defined conserved element dataset was 39%, the total intron dataset that contained these conserved elements was 34%, and the total intron reference dataset was 32%. While this slight variation in GC content might suggest that the WABA HMM may have created a bias, two arguments can be made against this. The first is that the high-scoring motifs in the WABA-conserved region were also highly represented in the total introns containing these motifs (Table 2), consistent with these results not being derived from bias in GC content. The second is that GC enrichment bias is not observed in many of our highest scoring motifs. For example, TCTATC has a GC content of 33%, yet is highly enriched in conserved elements.

The C. elegans unc-52 gene is a homolog of the mammalian extracellular matrix protein perlecan and contains 37 exons. Exons 16, 17, and 18 are alternatively spliced in a complex and regulated manner. These three exons each encode an Ig protein motif and can be included or skipped in any number of combinations in the final unc-52 mRNA transcript [45]. RT-PCR analysis shows unc-52 is also alternatively spliced in C. briggsae, suggesting the splicing pattern may be controlled by similar methods (unpublished data). The use of a subset of these alternatively spliced forms is controlled by the splicing regulatory protein, MEC-8 [46,47]. The genetic regulation of unc-52 alternative splicing makes this gene an attractive model for studying splicing regulation. Most importantly for this analysis, the introns flanking either side of alternative exon 16 contain regions of high nucleotide conservation as identified by WABA. A number of the top scoring pentamers and hexamers from our conserved element motif analysis are found in these conserved regions (Figure 3A).

To test the role of these motifs in alternative splicing of unc-52, we created a muscle-expressed alternative splicing reporter transgene derived from this region (Figure 3B). Expression studies using the native unc-52 promoter to drive GFP expression have indicated that this extracellular matrix protein is expressed in the muscle and hypodermis [47,48]. However, studies of the unc-52 splicing regulator mec-8 have suggested that mec-8 function on unc-52 alternative splicing is mostly focused in hypodermal tissues [47]. Our intention is to mimic the native gene's splicing as closely as possible, so we tested whether the alternative splicing of our muscle-specific unc-52 reporter could be regulated by mec-8. RT-PCR analysis of our wild-type unc-52 alternative splicing reporter in mec-8(+) (wild-type) and mec-8(e398) (mutant) backgrounds indicates a dramatic difference in splicing (Table S3). When the reporter is spliced in a mec-8(e398) mutant that lacks its RNA-binding domain, an increase in exon 18-containing transcripts is observed, as well as a 75% drop in abundance of 15-16-19 and 15–19 isoforms. This closely mimics the effects of mec-8(e398) on native unc-52 splicing [46]. These results demonstrate that the tissue-specific expression of our unc-52 reporter is an adequate representation of the alternative splicing of the native gene, and that it is also under the control of the splicing factor MEC-8.

In order to test the role of evolutionarily conserved intronic elements and motifs in the regulation of unc-52 alternative splicing, we created unc-52 alternative splicing reporter constructs with different combinations of deletions in these sequences and monitored their in vivo splicing by RT-PCR. Due to the similar size of exons 17 and 18, a restriction digest with BamHI was performed on the RT-PCR products. Spliced isoforms containing exon 18 would be cleaved by BamHI and thus run at smaller sizes on the gel (Figures 3B and 4A). A summary of these results is shown in Figure 4. All quantitation is based on the analysis of a minimum of three different RNA isolations from the indicated strains. In general, splicing reporters with deletions of the conserved intronic sequences flanking either side of exon 16 seemed to have the largest effect on three specific isoforms of mature unc-52 mRNA, those containing exons 15-16-19, 15-16-18–19, and 15-18-19 (Figure 4B).

Our pentamer and hexamer motif analysis of conserved intronic elements flanking alternatively spliced exons identified GCATG and TGCATG as high-scoring motifs likely to be found in introns flanking either side of an alternative exon. Deletion of the GCATG upstream of alternative exon 16 from the unc-52 splicing reporter resulted in nearly a doubling of the proportion of reporter transcripts containing exon 16 and a drop in the proportion using 15-18-19. This suggests that GCATG may play a role in repressing the inclusion of exon 16 (Figure 4B).

A conserved intronic element just downstream of exon 16 contains another of our top scoring motifs, (T)CTATC. A deletion of this TCTATC had the opposite effect of deleting the upstream intronic GCATG; the proportion of exon 16-containing transcripts decreased while the amount of 15-18-19 increased. A larger deletion of this downstream motif (Deletion B) or a replacement of the motif with the reverse complement sequence (Mutation B) amplified this effect as summarized in Figure 4B. Specifically, an approximately 75% reduction in exon 16- containing transcripts relative to wild-type can be seen when this conserved region is altered in the Mutation B construct. A similar drop of almost 85% is seen when this conserved element is deleted in the Deletion B reporter construct. This indicates that TCTATC is part of a regulatory element that enhances the inclusion of exon 16, and its loss by mutation leads to a decrease in exon 16 inclusion.

Perhaps most interesting was the effect produced by the double deletion of two conserved motifs flanking exon 16: the upstream GCATG and the downstream TCTATC. Individual deletions of these two motifs suggested they work in opposition to include or exclude exon 16. However, the double mutant causes a shift in splice pattern similar to, but much more dramatic, than that seen in the ΔGCATG construct (Figure 4B). The double mutant had such a dramatic increase in 15-16-19 levels that it became the new predominant isoform, making up 61% of the mRNAs. This result suggests that the conserved motifs on either side of exon 16 are responsible for the inclusion of this exon and that they work in collaboration and as part of a larger regulatory network to produce a multilayered regulation of unc-52 splicing. In general, changes to the evolutionarily conserved intronic elements flanking exon 16 did not alter the relative amounts of the 15-17-19, 15-17-18–19, and 15–19 isoforms dramatically. These elements flanking exon 16, for the most part, appear to regulate the decision to form either the 15-16-19 isoform or the 15-18-19 isoform (Figure 4B).

A control dataset of introns was obtained by downloading from the UCSC Genome Browser (http://www.genome.ucsc.edu) all of the introns in the C. elegans genome as annotated in Wormbase release WS120. From the sequences of these 118,492 introns, we removed the first and last seven nucleotides to avoid constitutive splicing signals. We then counted the number of occurrences of every possible hexamer or pentamer motif in the database by using the EMBOSS Compseq program [54]. For each motif in the database, we assigned a frequency score that was the number of occurrences of each motif divided by the total number of possible occurrences of motifs of that length in the dataset. This score for each possible motif in the control set was used in our analysis of both the introns that flank alternatively spliced exons and the conserved elements in these introns as our expected score. In order to obtain the obs/exp ratio for each motif in a test dataset, we divided the observed frequency score for each motif in that dataset by the expected frequency score for each motif based on the total intron control dataset.

C. elegans and C. briggsae alignments for the 147 alternative spliced exons with intronic conservation were obtained from the UCSC Genome Table Browser conservation track [55]. The mean nucleotide percentage identity was computed from this alignment along with the number of insertions and deletions. In order to obtain the mean amino acid identity, all the 147 exons were compared to the complete C. briggsae genome (WormBase cb25.agp8) using TBLASTX [56], and the mean amino acid identity was then calculated from these alignments.

We have previously described the development of an in vivo splicing reporter for the alternatively spliced region of the let-2 gene fused to GFP and expressed in muscle cells [37]. Site-directed mutagenesis of the wild-type reporter for let-2 alternative splicing from that paper was used to delete the core of the first conserved element, indicated in Figure 2, and replace it with GAA. Methods for detecting alternative splicing of this transcript with ³²P-labeled primers using RT-PCR, denaturing polyacrylamide gel electrophoresis, and autoradiography have been previously described [37].

The exon 15–19 region of unc-52 was PCR-amplified from wild-type (N2) C. elegans genomic DNA using primers 5′-GGAATTCGATGAGTACATCTGTATCGC and 5′-GGAATTCACATCTGAACTGATGTCGCTC for cloning into the vector pPD96.02, developed by Andrew Fire's lab. This vector contains the unc-54 body wall muscle-specific promoter driving expression of a green fluorescent protein (GFP)/β galactosidase (lacZ) fusion protein with an N-terminal nuclear localization signal derived from SV40 T antigen. The final 127 nucleotides of exon 15 through the first 173 nucleotides of exon 19 of unc-52 and all the genomic sequence in between were cloned into a unique EcoRI site, 16 codons before the end of the lacZ open reading frame in the final exon of the fusion protein. The Kunkel method of site-directed mutagenesis was used to create the larger deletion and mutations for the plasmid constructs Mutation B and Deletion B [57]. Pentamer and hexamer deletion constructs of the conserved unc-52 intron sequence were created using the Quickchange site-directed mutagenesis kit (Stratagene, La Jolla, California, United States) according to the manufacturer's instructions. Animals carrying these constructs as extra-chromosomal arrays were generated by standard injection/transformation [58]. Transformed N2 animals were identified by GFP-expression in nuclei of body wall muscle cells.

C. elegans strains were grown on NGM agar plates using standard methods [59]. RNA was extracted from worms as previously described [60]. Strain CB938 carrying the mec-8(e398) mutant allele was obtained from the C. elegans Genetics Center, a National Institutes of Health funded Center for Research Resources at the University of Minnesota.

cDNAs of the unc-52 splicing reporters were made in 25-μl reaction mixtures. The annealing step contained 3 μg total RNA and 25-pmol oligodeoxynucleotide primer complementary to the lacZ construct (5′- GTTGAAGAGTAATTGGACTTA-3′ for C. elegans and 5′- AACTGGTGTCGCTCTCCT-3′ for C. briggsae) in 17-μl final volume. These were heated to 94 °C for 2 min and cooled to room temperature for 10 min. The rest of the reverse transcription reaction components were added to reach a final volume of 25 μl. The final reaction mixtures consisted of 1 mM each of dATP, dCTP, dGTP, and dTTP; 1 U RNA Guard (Promega, Madison, Wisconsin, United States); 1 × AMV RT buffer (Promega); and 10 U AMV reverse transcriptase (Promega). Reactions were incubated at 37 °C for 1.5 h and stored at −20 °C.

1.0 μl of the cDNA reaction mixture was used as the template in 25-μl PCR reaction mixtures. 1.0 pmoles of the same oligonucleotide used in the reverse transcription reactions was added to PCR reaction mixtures along with ³²P-labeled 5′ lacZ vector-specific primer (5′-CTGGAGCCCGTCAGTATCGGC-3′ for C. elegans and 5′-ATTCGTTGCTGGGTCCCAGG-3′ for C. briggsae). The reaction mixtures also contained 1 × PCR buffer, 0.25 mM of each of the four dNTPs, and Taq DNA polymerase. The 5′ oligo was labeled with (γ-³²P) ATP by T4 polynucleotide kinase. Reaction mixtures were incubated for 25 cycles at 94 °C for 1.0 min, 59 °C for 1.0 min, and 72 °C for 1.0 min. 2.0 μl of PCR product were digested with the restriction enzyme BamHI. There is a unique BamH1 site in unc-52 exon 18, and this digestion step allows us to distinguish the different alternatively spliced isoforms more clearly on the gel. Digested PCR products were separated on 40-cm long, 0.4-mm thick, 5% or 6% polyacrylamide urea gels in TBE buffer. Gels were dried onto filter paper. These were then visualized using a Molecular Dynamics PhosphorImager (Sunnyvale, California, United States). Relative splice-site usage was quantified using ImageQuant software as previously described [37].