THE HUMAN U5 SNRNP-SPECIFIC 100-KD PROTEIN IS AN RS DOMAIN-CONTAINING, PUTATIVE RNA HELICASE WITH SIGNIFICANT HOMOLOGY TO THE YEAST SPLICING FACTOR PRP28P

Through UV-crosslinking experiments, we previously provided evidence s uggesting that a U5 snRNP protein with a molecular weight in the 100-k Da range is an ATP-binding protein (Laggerbauer B, Lauber J, Luhrmann R, 1996, Nucleic Acid Res 24:868-875). Separation of HeLa U5 snRNP pro teins on 2D gels revealed multiple variants with apparent molecular ma sses of 100 kDa. Subsequent microsequencing of these variants led to t he isolation of a cDNA encoding a protein with an N-terminal RS domain and a C-terminal domain that contains all of the conserved motifs cha racteristic of members of the DEAD-box family of RNA-stimulated ATPase s and RNA helicases. Antibodies raised against cDNA-encoded 100-kDa pr otein specifically recognized native U5-100kD both on immunoblots and in purified HeLa U5 snRNPs or [U4/U6.U5] tri-snRNP complexes, confirmi ng that the bona fide 100-kDa cDNA had been isolated. In vitro phospho rylation studies demonstrated that U5-100kD can serve as a substrate f or both Clk/Sty and the U1 snRNP-associated kinase, and further sugges ted that the multiple U5-100kD variants observed on 2D gels represent differentially phosphorylated forms of the protein. A database homolog y search revealed a significant degree of homology (60% similarity, 37 % identity) between the Saccharomyces cerevisiae splicing factor, Prp2 8p, which lacks an N-terminal RS domain, and the C-terminal domain of U5-100kD. Consistent with their designation as structural homologues, anti-Prp28 antibodies recognized specifically the human U5-100kD prote in on immunoblots. Together with the DEXH-box U5-200kD protein (Lauber J et al., 1996, EMBO J 15:4001-4015), U5-100kD is the second example of a putative RNA helicase that is tightly associated with the U5 snRN P. Given the recent identification of the U5-116kD protein as a homolo gue of the ribosomal translocase EF-2 (Fabrizio P, Laggerbauer B, Laub er J, Lane WS, Luhrmann R, 1997, EMBO J 16:4092-4106), at least three integral U5 snRNP proteins thus potentially facilitate conformational changes in the spliceosome during nuclear pre-mRNA splicing.