Cloning of mDEAH9, a putative RNA helicase and mammalian homologue of Saccharomyces cerevisiae splicing factor Prp43.
Yeast splicing factor Prp43, a DEAH box protein of the putative RNA helicase/RNA-dependent NTPase family, is a splicing factor that functions late in the pre-mRNA splicing pathway to facilitate spliceosome disassembly. In this paper we report cDNA cloning and characterization of mDEAH9, an apparent mammalian homologue of Prp43. Amino acid sequence comparison revealed that the two proteins are approximately 65% identical over a 500-aa region spanning the central helicase domain and the C-terminal region. Expression of mDEAH9 in S. cerevisiae bearing a temperature-sensitive mutation in prp43 was sufficient to restore growth at the nonpermissive temperature. This functional complementation was specific, as mouse mDEAH9 failed to complement mutations in related splicing factor genes prp16 or prp22. Finally, double label immunofluorescence experiments performed with mammalian cells revealed colocalization of mDEAH9 and splicing factor SC35 in punctate nuclear speckles. Thus, the hypothesis that mDEAH9 represents the mammalian homologue of yeast Prp43 is supported by its high sequence homology, functional complementation, and colocalization with a known splicing factor in the nucleus. Our results provide additional support for the hypothesis that the spliceosomal machinery that mediates regulated, dynamic changes in conformation of pre-mRNA and snRNP RNAs has been highly conserved through evolution.
Gee
S
S
Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720, USA.
Krauss
S W
SW
Miller
E
E
Aoyagi
K
K
Arenas
J
J
Conboy
J G
JG
D000595:Amino Acid Sequence; D000818:Animals; D002451:Cell Compartmentation; D003001:Cloning, Molecular; D017124:Conserved Sequence; D053487:DEAD-box RNA Helicases; D005816:Genetic Complementation Test; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics*; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000235:genetics*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000302:isolation & purification; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
D009687:Nuclear Proteins; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D012316:RNA Nucleotidyltransferases; C109034:PRP43 protein, S cerevisiae; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases