A dominant negative mutation in the conserved RNA helicase motif 'SAT' causes splicing factor PRP2 to stall in spliceosomes.
To characterize sequences in the RNA helicase-like PRP2 protein of Saccharomyces cerevisiae that are essential for its function in pre-mRNA splicing, a pool of random PRP2 mutants was generated. A dominant negative allele was isolated which, when overexpressed in a wild-type yeast strain, inhibited cell growth by causing a defect in pre-mRNA splicing. This defect was partially alleviated by simultaneous co-overexpression of wild-type PRP2. The dominant negative PRP2 protein inhibited splicing in vitro and caused the accumulation of stalled splicing complexes. Immunoprecipitation with anti-PRP2 antibodies confirmed that dominant negative PRP2 protein competed with its wild-type counterpart for interaction with spliceosomes, with which the mutant protein remained associated. The PRP2-dn1 mutation led to a single amino acid change within the conserved SAT motif that in the prototype helicase eIF-4A is required for RNA unwinding. Purified dominant negative PRP2 protein had approximately 40% of the wild-type level of RNA-stimulated ATPase activity. As ATPase activity was reduced only slightly, but splicing activity was abolished, we propose that the dominant negative phenotype is due primarily to a defect in the putative RNA helicase activity of PRP2 protein.
Plumpton
M
M
Institute of Cell and Molecular Biology, University of Edinburgh, UK.
McGarvey
M
M
Beggs
J D
JD
D001483:Base Sequence; D002846:Chromatography, Affinity; D017124:Conserved Sequence; D053487:DEAD-box RNA Helicases; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005799:Genes, Dominant; D007930:Leucine / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000235:genetics*; D012326:RNA Splicing; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012694:Serine / Q000235:genetics; D017381:Spliceosomes / Q000378:metabolism*
D005656:Fungal Proteins; D009838:Oligodeoxyribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D012694:Serine; D012316:RNA Nucleotidyltransferases; C066490:PRP2 protein, S cerevisiae; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases; D007930:Leucine