Abstract
The genome of the fission yeast Schizosaccharomyces pombe encodes 17 kinases that are essential for cell growth. These include the cell-cycle regulator Cdc2, as well as several kinases that coordinate cell growth, polarity, and morphogenesis during the cell cycle. In this study, we further characterized another of these essential kinases, Prp4, and showed that the splicing of many introns is dependent on Prp4 kinase activity. For detailed characterization, we chose the genes res1 and ppk8, each of which contains one intron of typical size and position. Splicing of the res1 intron was dependent on Prp4 kinase activity, whereas splicing of the ppk8 intron was not. Extensive mutational analyses of the 5’ splice site of both genes revealed that proper transient interaction with the 5’ end of snRNA U1 governs the dependence of splicing on Prp4 kinase activity. Proper transient interaction between the branch sequence and snRNA U2 was also important. Therefore, the Prp4 kinase is required for recognition and efficient splicing of introns displaying weak exon1/5’ splice sites and weak branch sequences.
Highlights
Introns are removed from pre-mRNAs by spliceosomes, which are highly dynamic macromolecular complexes consisting of five small nuclear RNAs associated with specific proteins in subcomplexes called snRNPs
Using a conditional mutant of Prp4, we showed that a subset of genes, including several cell cycle– regulatory genes, are dependent on Prp4 for splicing
This work shows that Prp4 activity is required for splicing surveillance in a subset of mRNAs
Summary
Introns are removed from pre-mRNAs by spliceosomes, which are highly dynamic macromolecular complexes consisting of five small nuclear RNAs (snRNAs; U1, U2, U5, and U4/U6) associated with specific proteins in subcomplexes called snRNPs. In vitro, spliceosomal subcomplexes assemble on pre-mRNAs in a time-dependent manner. The intron to be removed is defined during the formation of spliceosomal complex B, which contains the base-paired U4/ U6 snRNP as well as the other three snRNPs. ATP-dependent helicases control the specific rearrangement of the B complex so that catalysis of the transesterification reactions can occur in spliceosomal complex C, formed by the departure of snRNPs U1 and U4 and rearrangement of snRNPs U2, U6, and U5, culminating in the splicing reaction [1,2,3]. Several lines of evidence suggest that the 5’ splice site (SS) is defined by direct interactions with snRNA U1, and that definition of the 3’ SS involves the interaction of the branch sequence (bs) with snRNA U2 [4,5]
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