THE [U4 U6-CENTER-DOT-U5] TRI-SNRNP-SPECIFIC 27K PROTEIN IS A NOVEL SR PROTEIN THAT CAN BE PHOSPHORYLATED BY THE SNRNP-ASSOCIATED PROTEIN-KINASE/

SR proteins play important roles in the recognition and selection of t he 3' and 5' splice site of a given intron and contribute to the phosp horylation/dephosphorylation-mediated regulation of pre-mRNA splicing. Recent studies have demonstrated that the U1 snRNP is recruited to th e 5' splice site by protein/protein interactions involving the SR doma ins of the U1-70K protein and SF2/ASF. Recently, it was suggested that SR proteins might also contribute to the binding of the [U4/U6 . U5] tri-snRNP to the pre-spliceosome (Roscigno RF, Garcia-Blanco MA, 1995, RNA 1:692-706), although it remains unclear whether these SR proteins interact with proteins of the tri-snRNP complex. As a first step towa rd the identification of proteins that could potentially mediate the i ntegration of the [U4/U6 . U5] tri-snRNP complex into the spliceosome, we investigated whether purified [U4/U6 . U5] tri-snRNP complexes con tain SR proteins. Three proteins in the tri-snRNP complex with approxi mate molecular weights of 27, 60, and 100 kDa were phosphorylated by p urified snRNP-associated protein kinase, which has been shown previous ly to phosphorylate the serine/arginine-rich domains of U1-70K and SF2 /ASF (Woppmann A et al., 1993, Nucleic Acids Res 21:2815-2822). These proteins are thus prime candidates for novel tri-snRNP SR proteins. He re, we describe the biochemical and molecular characterization of the 27K protein. Analysis of a cDNA encoding the 27K protein revealed an N -terminal SR domain strongly homologous (54% identity) to the SR domai n of the U1 snRNP-specific 70K protein. In contrast to many other SR p roteins, the 27K protein does not contain an RNA-binding domain. The 2 7K protein can be phosphorylated in vitro by the snRNP-associated prot ein kinase and exhibits several isoelectric variants upon 2D gel elect rophoresis. Thus, the tri-snRNP-specific 27K protein could potentially be involved in SR protein-mediated protein/protein interactions and, additionally, its phosphorylation state could modulate pre-mRNA splici ng.