Cloning of mDEAH 9 , a putative RNA helicase and mammalian homologue of Saccharomyces cerevisiae splicing factor Prp 43

Yeast splicing factor Prp43, a DEAH box protein of the putative RNA helicaseyRNA-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 '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 temperaturesensitive 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. Pre-mRNA splicing in yeast and mammalian systems is a multistep process involving recognition and assembly of premRNA splice sites into spliceosomal complexes, intron excision and exon ligation mediated by two transesterification reactions, and release of splice products concomitant with spliceosome disassembly (1–3). In contrast to self-splicing group I introns, pre-mRNA splicing requires ATP and numerous protein factors at multiple steps in the pathway. Among these factors are a group of genetically defined Saccharomyces cerevisiae genes (PRP2, -5, -16, -22, -28, -43) that are members of a family of ‘‘DEADyDEAH box’’ proteins that contain conserved motifs characteristic of RNA-dependent NTPases and RNA helicases (4–6). More recently, an additional candidate pre-mRNA splicing factor (Slt22) with RNA-dependent ATPase activity has been identified in association with the U4yU6.U5 tri-snRNP in yeast (7, 8). The DEADyDEAH class of splicing factor proteins may help catalyze the intricate series of highly ordered small nuclear RNA (snRNA)–pre-mRNA and snRNA–snRNA interactions required for successful splicing. Alternatively, such proteins may serve a kinetic proofreading function to enhance fidelity of splicing (9). Extensive genetic and biochemical analysis has allowed temporal correlation of specific PRP gene functions with specific steps in spliceosome assembly, catalysis, and spliceosome disassembly. PRP2 and Slt22 are required early in assembly before the first catalytic step (8, 10–12); PRP16 activity is essential for the second catalytic step (13, 14); and PRP22 functions during spliceosome disassembly (15). Results in the accompanying paper (16) support the hypothesis that PRP43 acts at a late stage of the disassembly process: alteration of a conserved helicase motif in mutant prp43 allele yielded a phenotype characterized by excess accumulation of spliceosomal complexes retaining excised intron lariats. However, many mechanistic details of the reactions catalyzed by this intriguing class of enzymes, including the precise RNA substrate and the secondary structural change presumably effected by each, remain to be fully elucidated. Thus far, few counterparts of these yeast DEADyDEAH box splicing factors have been identified in Drosophila or mammalian systems. Evidence that such factors are expressed in mammalian cells was provided by the recent identification and characterization of HRH1, a mammalian homologue of yeast Prp22. Wild-type HRH1 protein exhibits extensive sequence homology with Prp22, functionally complements prp22 mutations in yeast, and is localized to nuclear splicing domains in cultured cells (17, 18). Moreover, dominant-negative mutants of HRH1 inhibit pre-mRNA splicing in vivo and inhibit release of spliced products from spliceosomal complexes in vitro (18). These results strongly support the hypothesis that HRH1 and Prp22 are functional homologues. In this paper we identify a mammalian homologue of a second yeast DEAH box splicing factor, Prp43. The mouse homologue, designated mDEAH9, was cloned initially by reverse transcriptase (RT)yPCR using degenerate oligonucleotides to conserved helicase motifs. By the criteria of amino acid sequence homology to Prp43, functional complementation of prp43 mutants, and immunofluorescence localization of mDEAH9 in the nucleus, mDEAH9 appears to represent a mammalian homologue of yeast splicing factor Prp43. MATERIALS AND METHODS Strategy for Cloning mDEAH9. Degenerate oligonucleotide primers were designed to hybridize with two conserved sequences encoding the ‘‘GKT’’ and ‘‘DEAH’’ amino acid motifs. The primers used were as follows. Sense strand primer 1, 59-CGGAATTCACIGGITCIGGIAA(GyA)AC-39; antisense strand primer 2, 59-CGGAATTC(TyC)(TyC)(TyGyC)(Ay G)T(CyG)IGC(TyC)TC(AyG)-39. Underlined bases represent EcoRI sites added to facilitate subcloning. Mouse erythThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y9411803-5$2.00y0 PNAS is available online at http:yywww.pnas. org. Abbreviations: snRNA, small nuclear RNA; snRNP, small nuclear ribonucleoprotein; MEL, mouse erythroleukemia; FITC, fluorescein isothiocyanate. Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AF017153). ‡Present address: Scriptgen Pharmaceuticals, Inc., 200 Boston Avenue, Suite 3000, Medford, MA 02155. §To whom reprint requests should be addressed.