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J. Biol. Chem., Vol. 277, Issue 18, 15452-15458, May 3, 2002
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From the Department of Microbiology and Immunology, Weill Medical
College of Cornell University, New York, New York 10021
Saccharomyces cerevisiae Prp22 and
Prp16 are RNA-dependent ATPases required for pre-mRNA
splicing. Both proteins are members of the DEXH-box family
of nucleic acid-dependent NTPases. Prior mutational
analysis of Prp22 and Prp16 identified residues within conserved motifs
I (GXGKT), II (DEAH), and VI
(QRXGRXGR) that are required for their
biological activity. Nonfunctional Prp22 and Prp16 mutants exerted a
dominant negative effect on cell growth. Here we show that
overexpression of lethal Prp22 mutants leads to accumulation of
unspliced pre-mRNAs and excised introns in vivo. The
biochemical basis for the lethality and inhibition of splicing in
vivo was determined by purifying and characterizing recombinant
mutant proteins. The lethal Prp22 mutants D603A and E604A in motif II
and Q804A and R808A in motif VI were defective for ATP hydrolysis and
mRNA release from the spliceosome, but were active in promoting
step 2 transesterification. Lethal Prp16 mutants G378A and K379A in
motif I; D473A and E474A in motif II; and Q685A, G688A, R689A, and
R692A in motif VI were defective for ATP hydrolysis and step 2 transesterification chemistry. The ATPase-defective mutants of Prp16
and Prp22 bound to spliceosomes in vitro and blocked the
function of the respective wild-type proteins in trans.
Comparing the mutational effects in Prp16 and Prp22 highlights
common as well as distinct structural requirements for the
ATP-dependent steps in pre-mRNA splicing.
Characterization of Dominant-negative Mutants of the DEAH-box
Splicing Factors Prp22 and Prp16*
, and
*
This work was supported by National Institutes of Health
Grant GM50288.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Present address: Wellcome Trust Sanger Inst., Hinxton, Cambridge
CB10 1SA, United Kingdom.
§
To whom correspondence should be addressed. Tel.: 212-746-6518;
Fax: 212-746-8587; E-mail:
bschwer@mail.med.cornell.edu.
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