Abstract
The fission yeast genome, which contains numerous short introns, is an apt model for studies on fungal splicing mechanisms and splicing by intron definition. Here we perform a domain analysis of the evolutionarily conserved Schizosaccharomyces pombe pre-mRNA-processing factor, SpPrp18. Our mutational and biophysical analyses of the C-terminal α-helical bundle reveal critical roles for the conserved region as well as helix five. We generate a novel conditional missense mutant, spprp18-5 To assess the role of SpPrp18, we performed global splicing analyses on cells depleted of prp18+ and the conditional spprp18-5 mutant, which show widespread but intron-specific defects. In the absence of functional SpPrp18, primer extension analyses on a tfIId+ intron 1-containing minitranscript show accumulated pre-mRNA, whereas the lariat intron-exon 2 splicing intermediate was undetectable. These phenotypes also occurred in cells lacking both SpPrp18 and SpDbr1 (lariat debranching enzyme), a genetic background suitable for detection of lariat RNAs. These data indicate a major precatalytic splicing arrest that is corroborated by the genetic interaction between spprp18-5 and spprp2-1, a mutant in the early acting U2AF59 protein. Interestingly, SpPrp18 depletion caused cell cycle arrest before S phase. The compromised splicing of transcripts coding for G1-S regulators, such as Res2, a transcription factor, and Skp1, a regulated proteolysis factor, are shown. The cumulative effects of SpPrp18-dependent intron splicing partly explain the G1 arrest upon the loss of SpPrp18. Our study using conditional depletion of spprp18+ and the spprp18-5 mutant uncovers an intron-specific splicing function and early spliceosomal interactions and suggests links with cell cycle progression.
Highlights
The fission yeast genome, which contains numerous short introns, is an apt model for studies on fungal splicing mechanisms and splicing by intron definition
Prp18 proteins from budding yeast, fission yeast, and humans share a high degree of similarity in their C-terminal halves, which adopt the five-helical bundle with a conserved region (CR) loop between helices 4 and 5 (Fig. 1A)
A comparison of the domain architecture across these three species revealed the splicing factor motif in the N-terminal region of SpPrp18 and hPrp18 that is absent in ScPrp18 (Fig. 1A)
Summary
The fission yeast genome, which contains numerous short introns, is an apt model for studies on fungal splicing mechanisms and splicing by intron definition. Prp has been analyzed extensively in budding yeast and human cell extracts for splicing of a specific model intron containing pre-mRNAs, but the Schizosaccharomyces pombe prp18ϩ has not been studied in detail Such studies have the potential to allow mechanistic insights into a genetic model system that better recapitulates splicing of short introns prevalent in several higher eukaryotes. Genome-wide splicing analyses of transcripts in a missense mutant of spslu7ϩ revealed its requirement before the first step of the splicing reaction, its widespread but intron- specific functions, and its novel interaction with another precatalytic splicing factor [22]. Genome-wide splicing studies and genetic interaction analyses using a missense mutant show that widespread SpPrp functions are in precatalytic spliceosomes, and its essential functions for early steps in splicing are intron-specific. Using a missense mutant in prp18ϩ, we uncover an important role in promoting G1-S cell cycle transition through intron-specific splicing effects of transcripts encoding some key regulators of this transition
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