DExD/H-box Prp5 protein is in the spliceosome during most of the splicing cycle.
The DExD/H-box Prp5 protein (Prp5p) is an essential, RNA-dependent ATPase required for pre-spliceosome formation during nuclear pre-mRNA splicing. In order to understand how this protein functions, we used in vitro, biochemical assays to examine its association with the spliceosome from Saccharomyces cerevisiae. GST-Prp5p in splicing assays pulls down radiolabeled pre-mRNA ... as well as splicing intermediates and lariat product, but reduced amounts of spliced mRNA. It cosediments with active spliceosomes isolated by glycerol gradient centrifugation. In ATP-depleted extracts, GST-Prp5p associates with pre-mRNA even in the absence of spliceosomal snRNAs. Maximal selection in either the presence or absence of ATP requires a pre-mRNA with a functional intron. Prp5p is present in the commitment complex and functions in subsequent pre-spliceosome formation. Reduced Prp5p levels decrease levels of commitment, pre-spliceosomal and spliceosomal complexes. Thus Prp5p is most likely an integral component of the spliceosome, being among the first splicing factors associating with pre-mRNA and remaining until spliceosome disassembly. The results suggest a model in which Prp5p recruits the U2 snRNP to pre-mRNA in the commitment complex and then hydrolyzes ATP to promote stable association of U2 in the pre-spliceosome. They also suggest that Prp5p could have multiple ATP-independent and ATP-dependent functions at several stages of the splicing cycle.
Mesh Terms:
Adenosine Triphosphate, Blotting, Western, DEAD-box RNA Helicases, RNA Precursors, RNA Splicing, RNA, Small Nuclear, Recombinant Fusion Proteins, Ribonucleoprotein, U2 Small Nuclear, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Spliceosomes, Temperature, Transcription, Genetic
Adenosine Triphosphate, Blotting, Western, DEAD-box RNA Helicases, RNA Precursors, RNA Splicing, RNA, Small Nuclear, Recombinant Fusion Proteins, Ribonucleoprotein, U2 Small Nuclear, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Spliceosomes, Temperature, Transcription, Genetic
RNA
Date: Jul. 01, 2009
PubMed ID: 19451545
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