Abstract
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 joining in constitutive pre-mRNA splicing, we previously screened for exon skipping mutants in Schizosaccharomyces pombe using a reporter plasmid, and characterized three exon skipping 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 3′ splice sites in pre-mRNA. In the present study, we identified a fourth ods mutant, ods4, which was isolated in an exon-skipping screen. The ods4 + gene encodes Cwf16p, which interacts with the NineTeen Complex (NTC), a complex thought to be involved in the first catalytic step of the splicing reaction. We isolated two multi-copy suppressors for the ods4-1 mutation, 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 ods mutants, 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 elongation 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 3′ splice sites and cooperates with the SF1-U2AF59-U2AF23 complex to maintain ordered exon joining.
Highlights
In the processing of eukaryotic messenger RNA precursors, removal of introns from pre-mRNAs is essential for gene expression
To understand the molecular mechanism that ligates successive exons orderly in constitutive pre-mRNA splicing, we previously screened for exon-skipping mutants in S. pombe and identified three temperature-sensitive mutations that caused exon skipping in S. pombe [12]
We found that the ods4+ gene encodes Cwf16p, which associates with the NTC (Prp19-associated NineTeen Complex)
Summary
In the processing of eukaryotic messenger RNA precursors (pre-mRNAs), removal of introns from pre-mRNAs (pre-mRNA splicing) is essential for gene expression. SR proteins that contain one or two N-terminal RRMs (RNA-recognition motifs) and a C-terminal RS (arginine/serine-rich) domain were previously reported to bind to ESE (exonic splicing enhancer) sequences in order to regulate alternative and constitutive pre-mRNA splicing in coordination with other splicing factors [8]. We previously screened for mutants that caused exon skipping in fission yeast Schizosaccharomyces pombe using a pURA4β reporter plasmid to identify genes that maintain ordered exon joining in constitutive pre-mRNA splicing (Fig 1) [12]. The genes responsible for the mutations encode the subunits of the SF1-U2AF59-U2AF23 complex, suggesting that the initial recognition of the branch and 30 splice sites is important for ensuring ordered exon joining in S. pombe [12]. Our results suggested that Cwf16p/NTC is involved in the initial co-transcriptional recognition of pre-mRNA to ensure ordered exon-joining in constitutive pre-mRNA splicing
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