Charles M. Romfo

Functional recognition of the 3' splice site AG by the splicing factor U2AF35

In metazoans, spliceosome assembly is initiated through recognition of the 5′ splice site by U1 snRNP and the polypyrimidine tract by the U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor, U2AF (refs 1, 2). U2AF is a heterodimer comprising a large subunit, U2AF65, and a small subunit, U2AF35 (ref. 3). U2AF65 directly contacts the polypyrimidine tract and is required for splicing in vitro. In comparison, the role of U2AF35 has been puzzling: U2AF35 is highly conserved and is required for viability, but can be dispensed with for splicing in vitro. Here we use site-specific crosslinking to show that very early during spliceosome assembly U2AF35 directly contacts the 3′ splice site. Mutational analysis and in vitro genetic selection indicate that U2AF35 has a sequence-specific RNA-binding activity that recognizes the 3′-splice-site consensus, AG/G. We show that for introns with weak polypyrimidine tracts, the U2AF35–3′-splice-site interaction is critical for U2AF binding and splicing. Our results demonstrate a new biochemical activity of U2AF35, identify the factor that initially recognizes the 3′ splice site, and explain why the AG dinucleotide is required for the first step of splicing for some but not all introns.

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.

Functional recognition of the 3|[prime]| splice site AG by the splicing factor U2AF35

In metazoans, spliceosome assembly is initiated through recognition of the 5′ splice site by U1 snRNP and the polypyrimidine tract by the U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor, U2AF (refs 1, 2). U2AF is a heterodimer comprising a large subunit, U2AF65, and a small subunit, U2AF35 (ref. 3). U2AF65 directly contacts the polypyrimidine tract and is required for splicing in vitro. In comparison, the role of U2AF35 has been puzzling: U2AF35 is highly conserved and is required for viability, but can be dispensed with for splicing in vitro. Here we use site-specific crosslinking to show that very early during spliceosome assembly U2AF35 directly contacts the 3′ splice site. Mutational analysis and in vitro genetic selection indicate that U2AF35 has a sequence-specific RNA-binding activity that recognizes the 3′-splice-site consensus, AG/G. We show that for introns with weak polypyrimidine tracts, the U2AF35–3′-splice-site interaction is critical for U2AF binding and splicing. Our results demonstrate a new biochemical activity of U2AF35, identify the factor that initially recognizes the 3′ splice site, and explain why the AG dinucleotide is required for the first step of splicing for some but not all introns.

Functional recognition 3' splice site AG by the splicing factor U2AF35

In metazoans, spliceosome assembly is initiated through recognition of the 5′ splice site by U1 snRNP and the polypyrimidine tract by the U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor, U2AF (refs 1, 2). U2AF is a heterodimer comprising a large subunit, U2AF65, and a small subunit, U2AF35 (ref. 3). U2AF65 directly contacts the polypyrimidine tract and is required for splicing in vitro. In comparison, the role of U2AF35 has been puzzling: U2AF35 is highly conserved and is required for viability, but can be dispensed with for splicing in vitro. Here we use site-specific crosslinking to show that very early during spliceosome assembly U2AF35 directly contacts the 3′ splice site. Mutational analysis and in vitro genetic selection indicate that U2AF35 has a sequence-specific RNA-binding activity that recognizes the 3′-splice-site consensus, AG/G. We show that for introns with weak polypyrimidine tracts, the U2AF35–3′-splice-site interaction is critical for U2AF binding and splicing. Our results demonstrate a new biochemical activity of U2AF35, identify the factor that initially recognizes the 3′ splice site, and explain why the AG dinucleotide is required for the first step of splicing for some but not all introns.