Prp45 affects Prp22 partition in spliceosomal complexes and splicing efficiency of non-consensus substrates.
Human transcription co-regulator SNW1/SKIP is implicated in the regulation of both transcription elongation and alternative splicing. Prp45, the SNW/SKIP ortholog in yeast, is assumed to be essential for pre-mRNA processing. Here, we characterize prp45(1-169), a temperature sensitive allele of PRP45, which at permissive temperature elicits cell division defects and hypersensitivity to microtubule inhibitors. Using a synthetic lethality screen, we found that prp45(1-169) genetically interacts with alleles of NTC members SYF1, CLF1/SYF3, NTC20, and CEF1, and 2nd step splicing factors SLU7, PRP17, PRP18, and PRP22. Cwc2-associated spliceosomal complexes purified from prp45(1-169) cells showed decreased stoichiometry of Prp22, suggesting its deranged interaction with the spliceosome. In vivo splicing assays in prp45(1-169) cells revealed that branch point mutants accumulated more pre-mRNA whereas 5' and 3' splice site mutants showed elevated levels of lariat-exon intermediate as compared to wild-type cells. Splicing of canonical intron was unimpeded. Notably, the expression of Prp45(119-379) in prp45(1-169) cells restored Prp22 partition in the Cwc2-pulldowns and rescued temperature sensitivity and splicing phenotype of prp45(1-169) strain. Our data suggest that Prp45 contributes, in part through its interaction with the 2nd step-proofreading helicase Prp22, to splicing efficiency of substrates non-conforming to the consensus.
Gahura
Ondrej
O
Faculty of Science, Department of Cell Biology, Charles University in Prague, Prague 128 00, Czech Republic.
Abrhámová
Katerina
K
Skruzný
Michal
M
Valentová
Anna
A
Munzarová
Vanda
V
Folk
Petr
P
Půta
Frantisek
F
D000483:Alleles; D000595:Amino Acid Sequence; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D007438:Introns; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing; D000072260:RNA Splicing Factors; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism*
C488197:PRP45 protein, S cerevisiae; D012322:RNA Precursors; D000072260:RNA Splicing Factors; D029701:Saccharomyces cerevisiae Proteins; C067135:PRP22 protein, S cerevisiae; D053487:DEAD-box RNA Helicases