The Fission Yeast Pre-mRNA-processing Factor 18 (prp18+) Has Intron-specific Splicing Functions with Links to G1-S Cell Cycle Progression

Abstract

Edited by Linda SpremulliThe 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 ␣-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 G 1 -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 G 1 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.Pre-mRNA splicing, a fundamental step in the processing of nascent eukaryotic RNA polymerase II transcripts, achieves precise excision of introns coupled with exon ligation to generate functional mRNAs. The spliceosome, which is composed of five U snRNPs 7 and Ͼ100 auxiliary proteins, assembles onto cis splicing signals, namely the 5Ј splice site (5Јss), the branch point nucleotide, the 3Ј splice site (3Јss), and polypyrimidine (Pyn) tracts. The spliceosomal catalytic core consists of the U2, U5, and U6 snRNPs and accessory proteins. This complex mediates the two trans-esterification reactions required for splicing to occur. First, the 5Јss is cleaved to yield the branched lariat intron-exon 2 and exon 1 intermediates, followed by the second reaction, where the 3Јss is cleaved, the exons are joined, and lariat intron is excised (1, 2).Genetic and biochemical analyses in budding yeast and biochemical studies with mammalian cell extracts have established a network of interactions among factors that act at the second step of splicing (i.e. Prp8, Prp16, Prp17, Prp18, Slu7, and Prp22) (3-8). PRP18, a non-essential budding yeast gene, encodes a U5 snRNP-associated factor. Prp18 has been analyzed extensively in budding yeast and human cell...

Sde2 is an intron‐specific pre‐ mRNA splicing regulator activated by ubiquitin‐like processing

“…Splicing defects of a subset of pre‐mRNAs were reported in the deletion strain of tls1 (Wang et al , ). Specific pre‐mRNA splicing defects have also been reported for mutants of U1 snRNA gene (Alvarez et al , ), U2AF, prp18 and survival of motor neuron (SMN) gene (Webb et al , ; Campion et al , ; Vijaykrishna et al , ). We have previously reported an intron‐specific pre‐mRNA splicing defect of cdc2 intron‐3 in a hub1 mutant of S. pombe (Mishra et al , ).…”

Functions for fission yeast splicing factors SpSlu7 and SpPrp18 in alternative splice-site choice and stress-specific regulated splicing

“…The fission yeast strain slu7+ O/E (with the wild-type allele under nmt81 control and the normal complement of the slu7+ gene); slu7 + (with the wild-type allele under nmt81 control and deletion of endogenous slu7+ gene), slu7-2 strain (with missense mutant allele under nmt81 control and deletion of the endogenous slu7+ gene) are described in ( Fig 1 , Table 1 ). The strains WT (with the wild-type allele under nmt81 control and deletion of endogenous prp18+ gene) and prp18-5 (with missense mutant allele under nmt81 control deletion of endogenous prp18+ gene) are detailed in [ 37 ]. The weak, yet thiamine-repressible, nmt81 promoter drives the wild-type and variant prp18+ and slu7 + alleles.…”

“…104 exon skipping events had a total read count greater than three that was used as cutoff. The raw intensity values for these probes from splicing microarray data (two replicates) of wild-type slu7+ O/E strain reported in [ 36 ] and in WT ( prp18+ ) [ 37 ] ( Fig 1C ) were extracted. A high confidence set of top ranking ten exon skipping events was curated based on their higher microarray raw intensity values in slu7 + O/E cells.…”

“…To assess SpSlu7 protein levels before and after thiamine supplementation (28 hours) western blotting of lysates from slu7-2 cells was done. Crude whole cell extracts were prepared as reported previously [ 37 ]. 30 μl of protein lysate corresponding to 20 μg protein was electrophoresed on SDS-PAGE gels (15% SDS-PAGE for SpAts1 HA; 10% SDS-PAGE for SpSlu7).…”

“…Further, both human Slu7 and hPrp18 function during second step in in vitro splicing assays [ 34 , 35 ]. Genome-wide splicing studies from our laboratory utilised the fission yeast missense mutants spslu7-2 and spprp18-5 to demonstrate intron-specific functions for these essential factors in the splicing of constitutive exons [ 36 , 37 ]. In this study, we integrate publicly available fission yeast transcriptome sequences with data from splicing-sensitive microarrays to identify consistent and robust alternative splice events which we then experimentally assessed in fission yeast cells.…”