Motifs IV and V in the DEAH box splicing factor Prp22 are important for RNA unwinding, and helicase-defective Prp22 mutants are suppressed by Prp8.
The yeast pre-mRNA splicing factor Prp22 is a member of the DEAH box family of nucleic acid-stimulated ATPases and RNA helicases. Here we report a mutational analysis of 16 conserved residues in motifs Ia ((534)TQPRRVAA(541)), IV ((695)LVFLTG(700)), and V ((757)TNIAETSIT(765)). Mutants T757A, I764A, and T765A were lethal, and F697A cells did not grow at < or =30 degrees C. The mutant proteins failed to catalyze mRNA release from the spliceosome in vitro, and they were deficient for RNA unwinding. The F697A, I764A, and T765A proteins were active for ATP hydrolysis in the presence of RNA cofactor. The T757A mutant retained basal ATPase activity but was not stimulated by RNA, whereas ATP hydrolysis by T765A was strictly dependent on the RNA cofactor. Thus Thr-757 and Thr-765 in motif V link ATP hydrolysis to the RNA cofactor. To illuminate the mechanism of Prp22-catalyzed mRNA release, we performed a genetic screen to identify extragenic suppressors of the cold-sensitive growth defect of a helicase/release-defective Prp22 mutant. We identified one of the suppressors as a missense mutation of PRP8 (R1753K), a protein component of the U5 small nuclear ribonucleoprotein. We show that PRP8-R1753K suppressed multiple helicase-deficient prp22 mutations, including the lethal I764A mutation. Replacing Arg-1753 of Prp8 by either Lys, Ala, Gln, or Glu resulted in suppression of helicase-defective Prp22 mutants. Prp8-Arg1753 mutations by themselves caused temperature-sensitive growth defects in a PRP22 strain. These findings suggest a model whereby Prp22 disrupts an RNA/protein or RNA/RNA interaction in the spliceosome that is normally stabilized by Prp8.
Schneider
Susanne
S
Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021, USA.
Campodonico
Eva
E
Schwer
Beate
B
D000595:Amino Acid Sequence; D053487:DEAD-box RNA Helicases; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D009154:Mutation; D020365:RNA Helicases / Q000235:genetics*; D012322:RNA Precursors / Q000235:genetics; D012326:RNA Splicing; D000072260:RNA Splicing Factors; D012331:RNA, Fungal / Q000235:genetics; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*
C077018:PRP8 protein, S cerevisiae; D012322:RNA Precursors; D000072260:RNA Splicing Factors; D012331:RNA, Fungal; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; C067135:PRP22 protein, S cerevisiae; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases