Functional domains of the yeast splicing factor Prp22p.
The essential Saccharomyces cerevisiae PRP22 gene encodes a 1145-amino acid DEXH box RNA helicase. Prp22p plays two roles during pre-mRNA splicing as follows: it is required for the second transesterification step and for the release of mature mRNA from the spliceosome. Whereas the step 2 function of Prp22p does not require ATP hydrolysis, spliceosome disassembly is dependent on the ATPase and helicase activities. Here we delineate a minimal functional domain, Prp22(262-1145), that suffices for the activity of Prp22p in vivo when expressed under the natural PRP22 promoter and for pre-mRNA splicing activity in vitro. The biologically active domain lacks an S1 motif (residues 177-256) that had been proposed to play a role in RNA binding by Prp22p. The deletion mutant Prp22(351-1145) can function in vivo when provided at a high gene dosage. We suggest that the segment from residues 262 to 350 enhances Prp22p function in vivo, presumably by targeting Prp22p to the spliceosome. We characterize an even smaller catalytic domain, Prp22(466-1145) that suffices for ATP hydrolysis, RNA binding, and RNA unwinding in vitro and for nuclear localization in vivo but cannot by itself support cell growth. However, the ATPase/helicase domain can function in vivo if the N-terminal region Prp22(1-480) is co-expressed in trans.
Department of Microbiology, Weill Medical College of Cornell University, New York, New York 10021, USA.
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D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D017124:Conserved Sequence; D053487:DEAD-box RNA Helicases; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011131:Polyribonucleotides / Q000737:chemistry / Q000378:metabolism; D020365:RNA Helicases / Q000378:metabolism*; D012326:RNA Splicing; D000072260:RNA Splicing Factors; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D017381:Spliceosomes / Q000378:metabolism; D013379:Substrate Specificity
D005656:Fungal Proteins; D011131:Polyribonucleotides; D000072260:RNA Splicing Factors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases; C067135:PRP22 protein, S cerevisiae; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases