The human U5-220kD protein (hPrp8) forms a stable RNA-free complex with several U5-specific proteins, including an RNA unwindase, a homologue of ribosomal elongation factor EF-2, and a novel WD-40 protein.
The human small nuclear ribonucleoprotein (snRNP) U5 is biochemically the most complex of the snRNP particles, containing not only the Sm core proteins but also 10 particle-specific proteins. Several of these proteins have sequence motifs which suggest that they participate in conformational changes of RNA and protein. Together, the specific proteins comprise 85% of the mass of the U5 snRNP particle. Therefore, protein-protein interactions should be highly important for both the architecture and the function of this particle. We investigated protein-protein interactions using both native and recombinant U5-specific proteins. Native U5 proteins were obtained by dissociation of U5 snRNP particles with the chaotropic salt sodium thiocyanate. A stable, RNA-free complex containing the 116-kDa EF-2 homologue (116kD), the 200kD RNA unwindase, the 220kD protein, which is the orthologue of the yeast Prp8p protein, and the U5-40kD protein was detected by sedimentation analysis of the dissociated proteins. By cDNA cloning, we show that the 40kD protein is a novel WD-40 repeat protein and is thus likely to mediate regulated protein-protein interactions. Additional biochemical analyses demonstrated that the 220kD protein binds simultaneously to the 40- and the 116kD proteins and probably also to the 200kD protein. Since the 220kD protein is also known to contact both the pre-mRNA and the U5 snRNA, it is in a position to relay the functional state of the spliceosome to the other proteins in the complex and thus modulate their activity.
Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, 35037 Marburg, Germany.
T
T
Achsel
K
K
Ahrens
H
H
Brahms
S
S
Teigelkamp
R
R
Lührmann
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry*; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000737:chemistry*; D011485:Protein Binding; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017421:Sequence Analysis; D013861:Thiocyanates / Q000494:pharmacology
D002352:Carrier Proteins; D005656:Fungal Proteins; C077018:PRP8 protein, S cerevisiae; C115374:PRPF8 protein, human; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; D013861:Thiocyanates; C024553:sodium thiocyanate