The mammalian spliceosome-associated protein, SAP 49, is associated specifically with U2 snRNP and is the most efficiently UV cross-linked protein in the spliceosomal complexes A, B, and C. We show here that SAP 49 cross-links to a region in the pre-mRNA immediately upstream of the branchpoint sequence in the prespliceosomal complex A. In addition to the RNA-binding activity of SAP 49, we show that this protein interacts directly and highly specifically with another U2 snRNP-associated spliceosomal protein, SAP 145. We have isolated a cDNA-encoding SAP 49 and find that it contains two amino-terminal RNA-recognition motifs (RRMs), consistent with the observation that SAP 49 binds directly to pre-mRNA. The remainder of the protein is highly proline-glycine rich (39% proline and 17% glycine). Unexpectedly, the SAP 49-SAP 145 protein-protein interaction requires the amino-terminus of SAP 49 that contains the two RRMs. The observation that SAP 49 and SAP 145 interact directly with both U2 snRNP and the pre-mRNA suggests that this protein complex plays a role in tethering U2 snRNP to the branch site.