Abstract
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.
Highlights
Genetic and biochemical analyses implicate at least one member of the DEX(H/D) box protein family in each of the ATP-dependent steps of pre-mRNA splicing [1, 2]
We found that the prp22 alleles T757A, I764A, and T765A failed to complement the prp22⌬ mutation at all temperatures tested between 15 and 37 °C (Fig. 2)
The Thr-757 to Ala change had no impact on basal RNA-independent ATP hydrolysis but abated the stimulation by RNA. These findings suggest that residues Thr-757 and Thr-765 in motif V play a role in linking ATP hydrolysis to the nucleic acid cofactor
Summary
Genetic and biochemical analyses implicate at least one member of the DEX(H/D) box protein family in each of the ATP-dependent steps of pre-mRNA splicing [1, 2]. Prp plays two distinct roles during pre-mRNA splicing It is important for the second catalytic step that results in formation of mRNA, and it is essential for releasing mature mRNA from the spliceosome [13]. The interaction of Prp with the precursor RNA during the splicing reaction has been probed by crosslinking using chemically modified RNAs; Prp can be crosslinked to the last 8 nucleotides at the 3Ј splice site of the intron in a sequence-independent fashion. This suggests that Prp might bind to the precursor RNA prior to step 2 [35, 36].
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