Evidence for Splice Site Pairing via Intron Definition in Schizosaccharomyces pombe

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 5′ 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 5′ 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 3′ and 5′ 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 <300 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 revealed that binding of the U1 snRNP to the downstream 5′ splice site stabilizes the association of U2AF65 with the polypyrimidine tract of the upstream intron (32). Likely candidates to form a bridge between these components were identified by protein-protein interaction assays, which indicated that the 70,000-Da protein of the U1 snRNP binds to members of the serine-arginine-rich (SR) family of splicing factors, which in turn bind to the small subunit of the U2AF heterodimer (5, 38, 70). The U1-70K/SR/U2AF35/U2AF65 network has also been proposed to play a role in communication across introns (70). However, because one of these components (U2AF35) is absent in Saccharomyces cerevisiae and two others (U2AF65 and SR proteins) are not highly conserved, this mode of connecting splice sites may not be ubiquitous (1, 2). A distinct network of intron-spanning interactions forms at an early stage of the splicing pathway in yeast and most likely in mammals as well (2, 7). In addition to this network, which extends from the large subunit of U2AF to the branchpoint bridging protein to a different component of the U1 snRNP, Prp40p, recent work with Drosophila melanogaster points to a third set of early intron-bridging interactions involving a divergent member of the SR protein family, SRp54 (36). The relationships among these networks of protein-protein interactions remain to be elucidated. Extremely small exons also pose recognition problems for the vertebrate splicing machinery, leading to a default splicing pattern in which the microexon is skipped (e.g., 9, 18, 59). This phenomenon was originally proposed to result from steric interference between closely juxtaposed 3′ and 5′ splice sites (9), but it is now attributed primarily to a lack of positive interactions across the small exon (10, 13, 59). In the three examples studied most extensively, incorporation of the microexon is promoted by complex enhancer elements located in the downstream intron (10, 13, 66). In the case of c-src, it has been shown that a large assemblage of proteins, including hnRNP F (44), K-SRP (45), and hnRNP H (14), binds to the intronic enhancer and regulates microexon inclusion, possibly by promoting use of the abutting 5′ splice site. In both budding yeast and fission yeast, as well as other unicellular eukaryotes, small introns predominate, and exon size does not appear to be constrained (15, 56, 72). These observations prompted Talerico and Berget (62) to propose that, in simple eukaryotes, the intron rather than the exon serves as the initial unit of recognition during spliceosome assembly (62; reviewed in reference 6). Consistent with this proposal, alternative exon usage has not yet been demonstrated in either yeast species. However, two well-documented instances of regulated splicing have been described in S. cerevisiae, both utilizing intron retention as an on-off switch for protein expression (19, 20). The situation is less clear in Schizosaccharomyces pombe, but a similar form of regulation at the level of splicing has been proposed for mes1 pre-mRNA during meiosis (37). While small introns are also common in certain metazoa including Caenorhabditis elegans and D. melanogaster, these species contain large vertebratelike introns as well (22, 46). In the fruit fly, there is experimental evidence for initial splice site pairing via “intron definition,” since expansion of small introns leads either to their retention or to activation of a cryptic 3′ splice site (27, 62). On the other hand, several examples of exon skipping have been reported in Drosophila, both naturally occurring, as in the sex determination regulatory cascade (reviewed in reference 41) and experimentally induced (e.g., 47, 57), consistent with splice site pairing via exon definition. In S. cerevisiae, only a handful of pre-mRNAs harbor more than one intron (58), and the trans-acting factors implicated in exon-spanning interactions are either absent or highly divergent (1, 2, 8). In contrast, S. pombe contains all of the factors implicated in forming bridges between exons, including at least two canonical SR proteins and highly conserved homologs of both subunits of U2AF (26, 42, 49, 67). This fact, together with the ability of the Drosophila splicing machinery to utilize both the exon and intron definition modes, prompted us to ask whether communication can occur across exons in S. pombe. To address this question, we first engineered constructs containing splice site mutations which, in mammals, would produce the outcome that is most diagnostic for this mode of initial splice site pairing, namely, exon skipping. In S. pombe, mutating the downstream 5′ splice site produced exclusively intron retention, and even in a pre-mRNA carrying severe mutations in both flanking splice sites, exon skipping was rare. To address the possibility that the lack of skipping was due to the large size of the internal exon, we turned to a different S. pombe pre-mRNA which contains a microexon. Again, the profile of products was as predicted by the intron definition model. A final indication that splice site pairing proceeds via intron definition in fission yeast is the location of several cryptic 5′ splice sites within an intron. The competition between these and the natural 5′ junction provided an opportunity to explore parameters that influence splice site pairing in S. pombe. In alleles containing deletions and insertions within the intron, as well as those with wild-type splice site spacing, we found that the pattern of cryptic splice site usage not only conformed to the predictions of the intron definition model but suggested that the fission yeast splicing machinery has a strong preference for excising the smallest intron possible.