Evidence for Splice Site Pairing via Intron Definition in Schizosaccharomyces pombe

Abstract

Schizosaccharomyces pombe pre-mRNAs are generally multi-intronic and share certain features with premRNAs from Drosophila melanogaster, in which initial splice site pairing can occur via either exon or intron definition. Here, we present three lines of evidence suggesting that, despite these similarities, fission yeast splicing is most likely restricted to intron definition. First, mutating either or both splice sites flanking an internal exon in the S. pombe cdc2 gene produced almost exclusively intron retention, in contrast to the exon skipping observed in vertebrates. Second, we were unable to induce skipping of the internal microexon in fission yeast cgs2, whereas the default splicing pathway excludes extremely small exons in mammals. Because nearly quantitative removal of the downstream intron in cgs2 could be achieved by expanding the microexon, we propose that its retention is due to steric occlusion. Third, several cryptic 5 junctions in the second intron of fission yeast cdc2 are located within the intron, in contrast to their generally exonic locations in metazoa. The effects of expanding and contracting this intron are as predicted by intron definition; in fact, even highly deviant 5 junctions can compete effectively with the standard 5 splice site if they are closer to the 3 splicing signals. Taken together, our data suggest that pairing of splice sites in S. pombe most likely occurs exclusively across introns in a manner that favors excision of the smallest segment possible.Splice site selection has been most extensively studied in higher eukaryotes (reviewed in reference 11), where abundant evidence indicates that the unit initially recognized by the splicing machinery is the exon, as proposed by Robberson et al. nearly a decade ago (53). Particularly compelling in this regard is the observation that the most common effect of a 5Ј splice site mutation is skipping of the preceding exon rather than inclusion of the mutant intron (61; reviewed in reference 6). Moreover, in the subset of cases in which a 5Ј junction mutation causes activation of a cryptic splice site rather than exon skipping, the new exon-intron boundary is almost invariably located within the preceding exon, again supporting the view that communication occurs across the exon rather than the intron. Finally, there are significant constraints on exon length in vertebrate pre-mRNAs, consistent with the proposal that the 3Ј and 5Ј splice sites on opposite sides of the exon must be recognized concurrently. Not only are the vast majority of natural internal exons in vertebrate pre-mRNAs Ͻ300 nucleotides in length (6), but expanding an exon beyond this size causes it to be skipped (53), particularly if it is surrounded by large introns (60). In contrast to the limitations on exon length, the introns in vertebrate pre-mRNAs can be extremely large (tens of kilobases [29]).Although many questions remain to be answered, several components of the machinery responsible for exon definition have been identified. First, UV cross-linking experiments ...

Exonic splicing enhancers in fission yeast: functional conservation demonstrates an early evolutionary origin

“…This effect was reproducible, and was not observed upon mutating the 5Ј-splice site alone. Thus, these results raise the possibility that there may be communication across exon 6 in the srp2 pre-mRNA; this is contrary to our earlier proposal that exon definition does not occur in S. pombe, based on our inability to induce skipping by mutating the splice sites flanking exons that lack purine-rich sequences (Romfo et al 2000).…”

“…Our finding that the SRP2 element, which is found downstream from two micro-exons, functions as a splicing enhancer, combined with our previous demonstration that such pre-mRNAs are inherently inefficient splicing substrates in S. pombe (Romfo et al 2000), suggested a strategy for identifying additional native splicing enhancers. Based on the hypothesis that ESEs may have evolved and been maintained to ensure the splicing of such pre-mRNAs, we examined a subset of the ∼2.5% of S. pombe genes that contain internal micro-exons for downstream purine-rich motifs.…”

“…As exon skipping is commonly observed in metazoans (Berget 1995;Lopez 1998;Reed 2000;Smith and Valcarcel 2000) but not in S. pombe (Romfo et al 2000), it was not possible to use strictly analogous reporters. However, in the course of an earlier study, we demonstrated that that the intron located downstream from the 9-nt micro-exon in fission yeast cgs2 is inefficiently spliced (Romfo et al 2000). Thus, this pre-mRNA provides a natural substrate for analyzing ESE function in S. pombe.…”

“…A similar approach has been used to validate putative ESEs identified by computational methods, with test minigenes that mimic "weak" exons that are skipped unless they contain an enhancer (Fairbrother et al 2002;Zhang and Chasin 2004). As exon skipping is commonly observed in metazoans (Berget 1995;Lopez 1998;Reed 2000;Smith and Valcarcel 2000) but not in S. pombe (Romfo et al 2000), it was not possible to use strictly analogous reporters. However, in the course of an earlier study, we demonstrated that that the intron located downstream from the 9-nt micro-exon in fission yeast cgs2 is inefficiently spliced (Romfo et al 2000).…”

Activation of a cryptic 5′ splice site by U1 snRNA

“…In this report, we present evidence that U1 snRNA is a key trans-acting factor in the activation of an unusual cryptic 59 splice site in the second intron of the cdc2 gene from S. pombe+ In experiments described elsewhere, we have altered the distance between the cryptic 59 junction and the other splicing signals within this intron )+ The results demonstrate that moving the cryptic 59 splice site farther away from the 39 splicing signals dramatically diminished its use, indicating that proximity to the branchpoint is critical for its activation+ Conversely, decreasing the size of the intron allowed the natural 59 splice site to compete much more effectively with the cryptic 59 junction+ These findings are consistent with the prevalence of extremely small introns in S. pombe (Zhang & Marr, 1994;Romfo et al+, 2000)+ An important goal of future work will be to uncover the factor(s) that collaborate with U1 to impose the distance constraints on splice site pairing in S. pombe+ FIGURE 4. A: Potential base pairing of mutant U1 snRNAs to the cryptic 59 splice site+ The aberrant nucleotide within the cryptic junction and the nucleotide mutated in each U1 allele are shown in white against a black background+ The percentage of product corresponding to mRNA spliced at the cryptic junction is taken from the experiment shown in Figure 3B+ B: Effects of two U1 alleles that pair to different extents with the cryptic 59 splice site on the profile of products from cdc2-Int2 mutants+ The experimental design was as in Figure 3+ Because the vast majority of 59 splice sites from diverse species, whether natural or cryptic, contain a U as the second nucleotide (Newman & Norman, 1991;Krawczak et al+, 1992;Kandels-Lewis & Séraphin, 1993;Lesser & Guthrie, 1993;Crispino & Sharp, 1995;Burge et al+, 1999;Spingola et al+, 1999), the presence of an A at position ϩ2 of the cryptic 59 junction in S. pombe cdc2 is unusual+ However, it is not unprecedented, as both a developmentally regulated alternative 59 splice site in the terminal intron of Drosophila melanogaster Antp (Bermingham & Scott, 1988) and a cryptic 59 splice site in mammalian ras (Cohen et al+, 1994) share this anomaly+ How do these 59 junctions circumvent the normal sequence requirements for splicing?…”