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

Abstract

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 SF3b155, 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 (aC) and a b hairpin insertion. SF3b155 binds to the b-sheet surface of p14, thereby occupying the canonical RNA-binding site of the p14 RRM. The minimal region of SF3b155 interacting with p14 (i.e., residues 381-424) consists of four a-helices, which are partially preformed in isolation. Helices a2 and a3 (residues 401-415) constitute the core p14-binding epitope. Regions of SF3b155 binding to p14 and U2AF65 are nonoverlapping. This allows for a simultaneous interaction of SF3b155 with both proteins, which may support the stable association of U2 snRNP with the pre-mRNA. p14-RNA interactions are modulated by SF3b155 and the RNA-binding site of the p14-SF3b155 complex involves the noncanonical b hairpin insertion of the p14 RRM, consistent with the b-sheet surface being occupied by the helical SF3b155 peptide and p14 helix aC. 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.

Dimerization and Protein Binding Specificity of the U2AF Homology Motif of the Splicing Factor Puf60

“…S4B and Table 2). As reported previously, U2AF 65 -UHM preferentially binds the ULMs in SF1 (8,15) and SF3b155(317-357) (13,18,24 (Fig. 5A).…”

Unmasking the U2AF homology motif family: a bona fide protein–protein interaction motif in disguise

“…The ULM-containing region of SF3b155 adjoins its binding site for p14, an RRM-like U2 snRNP subunit that ultimately contacts the branch point sequence of the pre-mRNA Cass and Berglund 2006;Schellenberg et al 2006;Spadaccini et al 2006). Considering the above data, multiple UHM-containing proteins are likely to simultaneously associate with the SF3b155 IDR alongside the p14 subunit, possibly with positive cooperativity (Fig.…”

Multivalent Binding of Formin-binding Protein 21 (FBP21)-Tandem-WW Domains Fosters Protein Recognition in the Pre-spliceosome

“…The SF3 complex plays a pivotal role in maturation of the early spliceosome with the SF3B subunit enabling branch point recognition (26), whereas its dissociation commits the spliceosome to its first catalytic step (27). Several components of SF3 contain multiple PRS motifs and the SF3B subunit directly recognize the branch point region of the intron (26). Similar to FBP21, small molecule interference with SF3B leads to the modulation of VEGF receptor expression (28,29).…”