Recognition of RNA branch point sequences by the KH domain of splicing factor 1 (mammalian branch point binding protein) in a splicing factor complex

Mammalian splicing factor 1 (SF1; also mammalian branch point binding prote in [mBBP]; hereafter SF1/mBBP) specifically recognizes the seven-nucleotide branch point sequence (BPS) located at 3 ' splice sites and participates i n the assembly of early spliceosomal complexes. SF1/mBBP utilizes a "maxi-K homology" (maxi-KH) domain for recognition of the single-stranded BPS and requires a cooperative interaction with splicing factor U2AF65 bound to an adjacent polypyrimidine tract (PPT) for high-affinity binding. To investiga te how the KH domain of SF1/mBBP recognizes the BPS in conjunction with U2A F and possibly other proteins, me constructed a transcriptional reporter sy stem utilizing human immunodeficiency virus type P Tat fusion proteins and examined the RNA-binding specificity of the complex using KH domain and RNA -binding site mutants. We first established that SF1/mBBP and U2AF cooperat ively assemble in our reporter system at RNA sites composed of the BPS, PPT , and AG dinucleotide found at 3 ' splice sites, with endogenous proteins a ssembled along With the Tat fusions, We next found that the activities of t he Tat fusion proteins on different BPS variants correlated well with the k nown splicing efficiencies of the variants, supporting a model in which the SF1/mBBP-BPS interaction helps determine splicing efficiency prior to the U2 snRNP-BPS interaction. Finally, the likely RNA-binding surface of the ma xi-KH domain was identified by mutagenesis and appears similar to that used by "simple" KH domains, involving residues from two putative cn helices, a highly conserved loop, and parts of a P sheet. Using a homology model cons tructed from the cocrystal structure of a Nova KH: domain-RNA complex (Lewi s et al,, Cell 100:323-332, 2000), we propose a plausible arrangement for S F1/mBBP-U2AF complexes assembled at 3 ' splice sites.