HeLa 200 kDa U5 snRNP-specific protein and its homologue in Saccharomyces cerevisiae are members of the DEXH-box protein family of putative RNA helicases

• Published in: The EMBO journal 1996-08, Vol.15 (15), p.4001-4015
• Main Authors: Lauber, J, Fabrizio, P, Teigelkamp, S, Lane, W.S, Hartmann, E, Luhrmann, R
• Format: Article
• Language: English
• Subjects: adenosinetriphosphatase; alternative splicing; Amino Acid Sequence; amino acid sequences; Antibodies; Base Sequence; complementary DNA; genbank/u18922; genbank/z70200; Genes, Fungal; Genes, Homeobox; HeLa Cells; Homeodomain Proteins - chemistry; Humans; inhibition; messenger RNA; Molecular Sequence Data; Molecular Weight; nucleoproteins; nucleotide sequences; Plasmids - metabolism; Ribonucleoprotein, U5 Small Nuclear - chemistry; Ribonucleoprotein, U5 Small Nuclear - metabolism; rna dependent adenosinetriphosphatase; RNA Helicases; RNA Nucleotidyltransferases - chemistry; RNA Nucleotidyltransferases - metabolism; RNA Precursors - metabolism; RNA Splicing; Saccharomyces cerevisiae; Sequence Homology, Amino Acid; small nuclear ribonucleoproteins; snu246 gene; structural genes
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1002/j.1460-2075.1996.tb00774.x

The primary structure of the 200 kDa protein of purified HeLa U5 snRNPs (U5-200kD) was characterized by cloning and sequencing of its cDNA. In order to confirm that U5-200kD is distinct from U5-220kD we demonstrate by protein sequencing that the human U5-specific 220 kDa protein is homologous to the yeast U5-specific protein Prp8p. A 246 kDa protein (Snu246p) homologous to U5-200kD was identified in Saccharomyces cerevisiae. Both proteins contain two conserved domains characteristic of the DEXH-box protein family of putative RNA helicases and RNA-stimulated ATPases. Antibodies raised against fusion proteins produced from fragments of the cloned mammalian cDNA interact specifically with the HeLa U5-200kD protein on Western blots and co-immunoprecipitate U5 snRNA and to a lesser extent U4 and U6 snRNAs from HeLa snRNPs. Similarly, U4, U5 and U6 snRNAs can be co-immunoprecipitated from yeast splicing extracts containing an HA-tagged derivative of Snu246p with HA-tag specific antibodies. U5-200kD and Snu246p are thus the first putative RNA helicases shown to be intrinsic components of snRNPs. Disruption of the SNU246 gene in yeast is lethal and leads to a splicing defect in vivo, indicating that the protein is essential for splicing. Anti-U5-200kD antibodies specifically block the second step of mammalian splicing in vitro, demonstrating for the first time that a DEXH-box protein is involved in mammalian splicing. We propose that U5-200kD and Snu246p promote one or more conformational changes in the dynamic network of RNA-RNA interactions in the spliceosome.