EVIDENCE THAT SEQUENCE-INDEPENDENT BINDING OF HIGHLY CONSERVED U2 SNRNP PROTEINS UPSTREAM OF THE BRANCH SITE IS REQUIRED FOR ASSEMBLY OF SPLICEOSOMAL COMPLEX-A

A critical step in the pre-mRNA splicing reaction is the stable bindin g of U2 snRNP to the branchpoint sequence (BPS) to form the A complex. The multimeric U2 snRNP protein complexes SF3a and SF3b are required for A complex assembly, but their specific roles in this process are n ot known. Saccharomyces cerevisiae homologs of all of the SF3a, but no ne of the SF3b, subunits have been identified. Here we report the isol ation of a cDNA encoding the mammalian SF3b subunit SAP 145 and the id entification of its probable yeast homolog (29% identity). This first indication that the homology between yeast and metazoan A complex prot eins can be extended to SF3b adds strong new evidence that the mechani sm of A complex assembly is highly conserved. To investigate this mech anism in the mammalian system we analyzed proteins that cross-link to P-32-site-specifically labeled pre-mRNA in the A complex. This analysi s revealed that SAP 145, together with four other SF3a/SF3b subunits, UV cross-links to pre-mRNA in a 20-nucleotide region upstream of the B PS. Mutation of this region, which we have designated the anchoring si te, has no apparent effect on U2 snRNP binding. In contrast, when a 2' O methyl oligonucleotide complementary to the anchoring site is added to the spliceosome assembly reaction, A complex assembly and cross-lin king of the SF3a/SF3b subunits are blocked. These data indicate that s equence-independent binding of the highly conserved SF3a/SF3b subunits upstream of the branch site is essential for anchoring U2 snRNP to pr e-mRNA.