Prp22, a DExH-box RNA helicase, plays two distinct roles in yeast pre-mRNA splicing.
In order to assess the role of Prp22 in yeast pre-mRNA splicing, we have purified the 130 kDa Prp22 protein and developed an in vitro depletion/reconstitution assay. We show that Prp22 is required for the second step of actin pre-mRNA splicing. Prp22 can act on pre-assembled spliceosomes that are arrested after step 1 in an ATP-independent fashion. The requirement for Prp22 during step 2 depends on the distance between the branchpoint and the 3' splice site, suggesting a previously unrecognized role for Prp22 in splice site selection. We characterize the biochemical activities of Prp22, a member of the DExH-box family of proteins, and we show that purified recombinant Prp22 protein is an RNA-dependent ATPase and an ATP-dependent RNA helicase. Prp22 uses the energy of ATP hydrolysis to effect the release of mRNA from the spliceosome. Thus, Prp22 has two distinct functions in yeast pre-mRNA splicing: an ATP-independent role during the second catalytic step and an ATP-requiring function in disassembly of the spliceosome.
Schwer
B
B
Department of Microbiology, Cornell University Medical College, New York, NY 10021, USA. bschwer@mail.med.cornell.edu
Gross
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CH
D000199:Actins / Q000235:genetics; D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology; D000255:Adenosine Triphosphate / Q000502:physiology; D053487:DEAD-box RNA Helicases; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing / Q000502:physiology*; D000072260:RNA Splicing Factors; D012331:RNA, Fungal / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D029701:Saccharomyces cerevisiae Proteins; D017381:Spliceosomes / Q000378:metabolism; D015003:Yeasts / Q000201:enzymology / Q000235:genetics*
D000199:Actins; D005656:Fungal Proteins; D012322:RNA Precursors; D000072260:RNA Splicing Factors; D012331:RNA, Fungal; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D012316:RNA Nucleotidyltransferases; D000251:Adenosine Triphosphatases; C067135:PRP22 protein, S cerevisiae; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases