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 suggesting that a U5 snRNP protein with a molecular weight in the 100-kDa range is an ATP-binding protein (Laggerbauer B, Lauber J, Lührmann R, 1996, Nucleic Acid Res 24:868-875). Separation of HeLa U5 snRNP proteins on 2D gels revealed multiple variants with apparent molecular masses of 100 kDa. Subsequent microsequencing of these variants led to the 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 characteristic of members of the DEAD-box family of RNA-stimulated ATPases and RNA helicases. Antibodies raised against cDNA-encoded 100-kDa protein specifically recognized native U5-100kD both on immunoblots and in purified HeLa U5 snRNPs or [U4/U6.U5] tri-snRNP complexes, confirming that the bona fide 100-kDa cDNA had been isolated. In vitro phosphorylation studies demonstrated that U5-100kD can serve as a substrate for both Clk/Sty and the U1 snRNP-associated kinase, and further suggested that the multiple U5-100kD variants observed on 2D gels represent differentially phosphorylated forms of the protein. A database homology search revealed a significant degree of homology (60% similarity, 37% identity) between the Saccharomyces cerevisiae splicing factor, Prp28p, 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 protein 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 snRNP. Given the recent identification of the U5-116kD protein as a homologue of the ribosomal translocase EF-2 (Fabrizio P, Laggerbauer B, Lauber J, Lane WS, Lührmann 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.
Teigelkamp
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Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, Germany.
Mundt
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Achsel
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Will
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CL
Lührmann
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R
D000595:Amino Acid Sequence; D053487:DEAD-box RNA Helicases; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D011485:Protein Binding; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D017415:Ribonucleoprotein, U5 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; C498290:DDX23 protein, human; D012316:RNA Nucleotidyltransferases; C088939:PRP28 protein, S cerevisiae; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases