The DEAH-box protein PRP22 is an ATPase that mediates ATP-dependent mRNA release from the spliceosome and unwinds RNA duplexes.
Of the proteins required for pre-mRNA splicing, at least four, the DEAH-box proteins, are closely related due to the presence of a central 'RNA helicase-like' region, and extended homology through a large portion of the protein. A major unresolved question is the function of these proteins. Indirect evidence suggests that several of these proteins are catalysts for important structural rearrangements in the spliceosome. However, the mechanism for the proposed alterations is presently unknown. We present evidence that PRP22, a DEAH-box protein required for mRNA release from the spliceosome, unwinds RNA duplexes in a concentration- and ATP-dependent manner. This demonstrates that PRP22 can modify RNA structure directly. We also show that the PRP22-dependent release of mRNA from the spliceosome is an ATP-dependent process and that recombinant PRP22 is an ATPase. Non-hydrolyzable ATP analogs did not substitute for ATP in the RNA-unwinding reaction, suggesting that ATP hydrolysis is required for this reaction. Specific mutation of a putative ATP phosphate-binding motif in the recombinant protein eliminated the ATPase and RNA-unwinding capacity. Significantly, these data suggest that the DEAH-box proteins act directly on RNA substrates within the spliceosome.
Wagner
J D
JD
Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
Jankowsky
E
E
Company
M
M
Pyle
A M
AM
Abelson
J N
JN
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D053487:DEAD-box RNA Helicases; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D006868:Hydrolysis; D009154:Mutation; D010641:Phenotype; D020365:RNA Helicases; D012326:RNA Splicing; D000072260:RNA Splicing Factors; D012330:RNA, Double-Stranded; D012333:RNA, Messenger / Q000378:metabolism; D012342:RNA, Small Nuclear; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017381:Spliceosomes
D005656:Fungal Proteins; D000072260:RNA Splicing Factors; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D011993:Recombinant Fusion Proteins; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C067135:PRP22 protein, S cerevisiae; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases