RESEARCH ARTICLE Cwf16p Associating with the Nineteen Complex Ensures Ordered Exon Joining in Constitutive Pre-mRNA Splicing in Fission Yeast

Exons are ligated in an ordered manner without the skipping of exons in the constitutive splicing of pre-mRNAs with multiple introns. To identify factors ensuring ordered exon join-ing in constitutive pre-mRNA splicing, we previously screened for exon skipping mutants in Schizosaccharomyces pombe using a reporter plasmid, and characterized three exon skip-ping mutants named ods1 (ordered splicing 1), ods2, and ods3, the responsible genes of which encode Prp2/U2AF59, U2AF23, and SF1, respectively. They form an SF1-U2AF59-U2AF23 complex involved in recognition of the branch and 30 splice sites in pre-mRNA. In the present study, we identified a fourth odsmutant, ods4, which was isolated in an exon-skipping screen. The ods4+ gene encodes Cwf16p, which interacts with the NineTeen Com-plex (NTC), a complex thought to be involved in the first catalytic step of the splicing reac-tion. We isolated two multi-copy suppressors for the ods4-1mutation, Srp2p, an SR protein essential for pre-mRNA splicing, and Tif213p, a translation initiation factor, in S. pombe. The overexpression of Srp2p suppressed the exon-skipping phenotype of all odsmutants, whereas Tif213p suppressed only ods4-1, which has a mutation in the translational start codon of the cwf16 gene. We also showed that the decrease in the transcriptional elonga-tion rate induced by drug treatment suppressed exon skipping in ods4-1. We propose that Cwf16p/NTC participates in the early recognition of the branch and 30 splice sites and coop-erates with the SF1-U2AF59-U2AF23 complex to maintain ordered exon joining