Mutations in the SF1-U2AF59-U2AF23 complex cause exon skipping in Schizosaccharomyces pombe.
To identify genes involved in the mechanism to ensure ordered 5' to 3' exon joining in constitutively spliced pre-mRNAs, we screened for mutants that cause exon skipping in the fission yeast Schizosaccharomyces pombe using a reporter plasmid, which contains the ura4+ gene with the nda3 intron 1-exon 2-intron 2 sequence. The reporter plasmid was designed to produce the functional ura4+ mRNA, when the central nda3 exon is skipped during the splicing reaction. We mutagenized cells harboring the plasmid by UV irradiation and isolated 34 ura+ mutants that grew on minimal medium. Of those, eight mutants were found to be temperature sensitive (ts) for growth. Complementation analyses revealed that the ts mutants belong to three distinct complementation groups named ods (ordered splicing) 1, 2, and 3. RT-PCR analyses showed that products of exon skipping were actually generated in the ods mutants. We cloned the genes responsible for the ods mutations, and found that ods1+, ods2+, and ods3+ encode splicing factors Prp2p/U2AF59, U2AF23, and SF1, respectively, which form a SF1-U2AF59-U2AF23 complex involved in recognition of the branch-point and 3' splice site sequences in a pre-mRNA. We also showed that mutations in the SF1-U2AF59-U2AF23 binding sequences in the reporter plasmid result in exon skipping in wild-type S. pombe cells. In addition, drugs that decrease the rate of transcription elongation were found to suppress the exon skipping in the ods mutants. These results suggest that co-transcriptional recognition of a nascent pre-mRNA by the SF1-U2AF59-U2AF23 complex is essential for ordered exon joining in constitutive splicing in S. pombe.
Haraguchi
Noriko
N
Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto 860-8555, Japan.
Andoh
Tomoko
T
Frendewey
David
D
Tani
Tokio
T
D001483:Base Sequence; D005091:Exons; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics; D012322:RNA Precursors / Q000235:genetics; D012326:RNA Splicing; D000072260:RNA Splicing Factors; D012331:RNA, Fungal / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012261:Ribonucleoproteins / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D012568:Schizosaccharomyces / Q000235:genetics*; D000072668:Splicing Factor U2AF
C409040:MSL5 protein, S cerevisiae; D009687:Nuclear Proteins; D012322:RNA Precursors; D000072260:RNA Splicing Factors; D012331:RNA, Fungal; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D029701:Saccharomyces cerevisiae Proteins; D000072668:Splicing Factor U2AF