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 pre-mRNAs fromDrosophila 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 incgs2 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 yeastcdc2 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 inS. pombe most likely occurs exclusively across introns in a manner that favors excision of the smallest segment possible.