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.