Biochemical and NMR analyses of an SF3b155-p14-U2AF-RNA interaction network involved in branch point definition during pre-mRNA splicing

The p14 subunit of the essential splicing factor 3b (SF3b) can be cross-linked to the branch-point adenosine of pre-mRNA introns within the spliceosome. p14 stably interacts with the SF3b subunit SF3b1 55, which also binds the 65-kDa subunit of U2 auxiliary splicing factor (U2AF65). We combined biochemical and NMR techniques to study the conformation of p14 either alone or complexed with SF3b155 fragments, as well as an interaction network involving p14, SF3b155, U2AF65, and U2 snRNA/premRNA. p14 comprises a canonical RNA recognition motif (RRM) with an additional C-terminal helix (alpha C) and a beta hairpin insertion. SF3b155 binds to the beta-sheet surface of p14, thereby occupying the canonical RNA-binding site of the pl 4 RRM. The minimal region of SF3b155 interacting with p14 (i.e., residues 381-424) consists of four et-helices, which are partially preformed in isolation. Helices alpha 2 and alpha 3 (residues 401-415) constitute the core pl 4-binding epitope. Regions of SF3b1 55 binding to pl 4 and U2AF65 are nonoverlapping. This allows for a simultaneous interaction of SF3b1 55 with both proteins, which may support the stable association of U2 snRNP with the pre-mRNA. pl 4-RNA interactions are modulated by SF3b1 55 and the RNA-binding site of the p14-SF3b155 complex involves the noncanonical 0 hairpin insertion of the p14 RRM, consistent with the P-sheet surface being occupied by the helical SF3b155 peptide and p14 helix alpha C. Our data suggest that p14 lacks inherent specificity for recognizing the branch point, but that some specificity may be achieved by scaffolding interactions involving other components of SF3b.