The 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.
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.
Lauber
J
J
Institut fur Molekularbiologie und Tumorforschung, Philipps-Universitat Marburg, Emil Mannkopff-Strasse 2, D35037 Marburg, Germany.
Fabrizio
P
P
Teigelkamp
S
S
Lane
W S
WS
Hartmann
E
E
Luhrmann
R
R
D000595:Amino Acid Sequence; D000906:Antibodies; D001483:Base Sequence; D005800:Genes, Fungal; D005801:Genes, Homeobox; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids / Q000378:metabolism; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid
D000906:Antibodies; D018398:Homeodomain Proteins; D012322:RNA Precursors; D017415:Ribonucleoprotein, U5 Small Nuclear; D012316:RNA Nucleotidyltransferases; D020365:RNA Helicases