THE HUMAN U5-220KD PROTEIN (HPRP8) FORMS A STABLE RNA-FREE COMPLEX WITH SEVERAL US-SPECIFIC PROTEINS, INCLUDING AN RNA UNWINDASE, A HOMOLOGOF 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 co re proteins but also 10 particle-specific proteins. Several of these p roteins have sequence motifs which suggest that they participate in co nformational changes of RNA and protein. Together, the specific protei ns comprise 85% of the mass of the U5 snRNP particle. Therefore, prote in-protein interactions should be highly important for both the archit ecture and the function of this particle. We investigated protein-prot ein interactions using both native and recombinant US-specific protein s. Native U5 proteins were obtained by dissociation of U5 snRNP partic les with the chaotropic salt sodium thiocyanate. A stable, RNA-free co mplex containing the 116-kDa EF-2 homologue (116kD), the 200kD RNA unw indase, the 220kD protein, which is the orthologue of the yeast Prp8p protein, and the U5-40kD protein was detected by sedimentation analysi s 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 analyse s demonstrated that the 220kD protein binds simultaneously to the 40- and the 116kD proteins and probably also to the 200kD protein. Since t he 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 splic eosome to the other proteins in the complex and thus modulate their ac tivity.