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Essential, protein-protein complexes between the large subunit of the U2 small nuclear RNA Auxiliary Factor (U2AF65) with the Splicing Factor 1 (SF1) or the spliceosomal component SF3b155 are exchanged during a critical, ATP-dependent step of pre-mRNA splicing. Both SF1 and the N-terminal domain of SF3b155 interact with a U2AF homology motif (UHM) of U2AF65. SF3b155 contains seven tryptophan-containing sites with sequence similarity to the previously characterized U2AF65-binding domain of SF1. We show that the SF3b155 domain lacks detectable secondary structure using circular dichroism spectroscopy, and demonstrate that five of the tryptophan-containing SF3b155 sites are recognized by the U2AF65-UHM using intrinsic tryptophan fluorescence experiments with SF3b155 variants. When compared with SF1, similar spectral shifts and sequence requirements indicate that U2AF65 interactions with each of the SF3b155 sites are similar to the minimal SF1 site. However, thermodynamic comparison of SF1 or SF3b155 proteins with minimal peptides demonstrates that formation the SF1/U2AF65 complex is likely to affect regions of SF1 beyond the previously identified, linear interaction site, in a remarkably distinct manner from the local U2AF65 binding mode of SF3b155. Furthermore, the complex of the SF1/U2AF65 interacting domains is stabilized by 3.3 kcal mol−1 relative to the complex of the SF3b155/U2AF65 interacting domains, consistent with the need for ATP hydrolysis to drive exchange of these partners during pre-mRNA splicing. We propose that the multiple U2AF65 binding sites within SF3b155 regulate conformational rearrangements during spliceosome assembly. Comparison of the SF3b155 sites defines an (R/K)nXRW(DE) consensus sequence for predicting U2AF65-UHM ligands from genomic sequences, where parentheses denote residues that contribute to, but are not required for binding.

T-cell-restricted intracellular antigen-1 (TIA-1) regulates alternative pre-mRNA splicing in the nucleus, and mRNA translation in the cytoplasm, by recognizing uridine-rich sequences of RNAs. As a step towards understanding of RNA recognition by this regulatory factor, the X-ray structure of the central RNA recognition motif (RRM2) of human TIA-1 is presented at 1.95Å resolution. Comparison with structurally homologous RRM-RNA complexes identifies residues at the RNA interfaces that are conserved in TIA-1–RRM2. The versatile capability of RNP motifs to interact with either proteins or RNA is reinforced by symmetry-related protein-protein interactions mediated by the RNP motifs of TIA-1–RRM2. Importantly, the TIA-1–RRM2 structure reveals the locations of mutations responsible for inhibiting nuclear import. In contrast with previous assumptions, the mutated residues are buried within the hydrophobic interior of the domain, where they would be likely to destabilize the RRM fold rather than directly inhibit RNA binding.

Protein phosphorylation ensures the accurate and controlled expression of the genome, for instance by regulating the activities of pre-mRNA splicing factors. Here we report that splicing factor 1 (SF1), which is involved in an early step of intronic sequence recognition, is highly phosphorylated in mammalian cells on two serines within an SPSP motif at the junction between its U2AF65 and RNA binding domains. We show that SF1 interacts in vitro with the protein kinase KIS, which possesses a ‘U2AF homology motif’ (UHM) domain. The UHM domain of KIS is required for KIS and SF1 to interact, and for KIS to efficiently phosphorylate SF1 on the SPSP motif. Importantly, SPSP phosphorylation by KIS increases binding of SF1 to U2AF65, and enhances formation of the ternary SF1–U2AF65–RNA complex. These results further suggest that this phosphorylation event has an important role for the function of SF1, and possibly for the structural rearrangements associated with spliceosome assembly and function.