Prp43 is an essential RNA-dependent ATPase required for release of lariat-intron from the spliceosome.
The essential Saccharomyces cerevisiae PRP43 gene encodes a 767-amino acid protein of the DEXH-box family. Prp43 has been implicated in spliceosome disassembly (Arenas, J. E., and Abelson, J. N. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11798-11802). Here we show that purified recombinant Prp43 is an RNA-dependent ATPase. Alanine mutations at conserved residues within motifs I ((119)GSGKT(123)), II ((215)DEAH(218)) and VI ((423)QRAGRAGR(430)) that diminished ATPase activity in vitro were lethal in vivo, indicating that ATP hydrolysis is necessary for the biological function of Prp43. Overexpression of lethal, ATPase-defective mutants in a wild-type strain resulted in dominant-negative growth inhibition. The ATPase-defective mutant T123A interfered in trans with the in vitro splicing function of wild-type Prp43. T123A did not affect the chemical steps of splicing or the release of mature mRNA from the spliceosome, but it blocked the release of the excised lariat-intron from the spliceosome. We show that the lariat-intron is not accessible to debranching by purified Dbr1 when it is held in the T123A-arrested splicing complex. Our results define a new ATP-dependent step of splicing that is catalyzed by Prp43.
Martin
Arnold
A
Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021, USA.
Schneider
Susanne
S
Schwer
Beate
B
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D053487:DEAD-box RNA Helicases; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D007438:Introns; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D020365:RNA Helicases / Q000302:isolation & purification / Q000378:metabolism*; D012316:RNA Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D012331:RNA, Fungal / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D017381:Spliceosomes
D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D012316:RNA Nucleotidyltransferases; C048135:lariat debranching enzyme; D000251:Adenosine Triphosphatases; C109034:PRP43 protein, S cerevisiae; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases