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.

The expression of the genome requires the precise and controlled removal of intervening sequences within premessenger RNAs (pre-mRNA splicing). Assembly of the active spliceosome entails successive rearrangements, including the entry and exit of molecular partners (reviewed in [1]). Phosphorylation events are probably molecular switches to control these conformational changes. Indeed, experiments with phosphatase inhibitors, purified phosphatases and nonhydrolysable ATP analogues have shown that multiple phosphorylation and dephosphorylation events are required for spliceosome assembly and splicing [2][3][4]. Among the best-characterized of the phosphorylated splicing factors are the serine-arginine rich (SR) proteins (reviewed in [5]), whose intranuclear distribution and activity are influenced by phosphorylation by specific kinases including SRPK1, SRPK2 [6,7], and Clk ⁄ Sty [8].SF3b155 ⁄ SAP155, an integral spliceosome component and substrate of cyclin E ⁄ CDK2 [9], is a non-SR protein whose phosphorylation state is also regulated during the splicing process [10]. In addition, other factors that regulate splicing in a phosphorylation-dependent manner have been identified (reviewed in [11,12]).Splicing factor 1 (SF1) was identified as necessary for spliceosome assembly by in vitro reconstitution assays with protein fractions from HeLa cell nuclear extracts [13], and in a synthetic lethality screen with Mud2p, the yeast homologue of the splicing factor U2 auxiliary factor large subunit ( U2AF 65 ) 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 U2AF 65 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 U2AF 65 , and enhances formation of the ternary SF1-U2AF 65 -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.Abbreviations BPS, branch point sequence; CIP, calf intestinal phosphatase; DTT, dithiothreitol; GST, glutathione-S-transferase; SF1, splicing factor 1; siRNA, small interfering RNA; snRNP, small nuclear ribonucleoprotein particle; RRM, RNA recognition motif; U2AF, U2 auxiliary factor; UHM, U2AF homology motif.

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. KeywordsRRM; RNA binding domain; Pre-mRNA splicing; mRNA translation; TIA-1; TIAR Post-transcriptional mechanisms for regulating gene expression depend on numerous coordinated interactions among RNA sequences and trans-acting protein factors. The RNA binding protein TIA-1 (T-cell-restricted intracellular antigen-1) functions as a posttranscriptional regulator of gene expression by recognizing uridine-rich RNA sites. In the nucleus, TIA-1 regulates alternative splicing of pre-mRNAs, including those encoding fibroblast growth factor receptor-2 (fgfr-2), type II procollagen (col2a1), the cystic fibrosis transmembrane conductance regulator (cftr), and the pro-apoptotic Fas receptor (fas), among others [1;2;3;4;5;6]. In the cytoplasm, TIA-1 regulates mRNA translation [7;8;9;10], and suppresses mRNA translation during environmental stress (reviewed in [11]). The functional importance of TIA-1 is shown by its requirement for viability of DT40 cells [12], and the high rates of embryonic lethality among mice lacking TIA-1 [8;13].*Correspondence E-mail: clara_kielkopf@urmc.rochester.edu; Fax: 585-275-6007. ‡ These authors contributed equally to this work.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Materials and Methods Protein purificationThe human TIA-1-RRM2 fragment (residues 94-175) was expressed using the pGEX-6p vector (GE Healthcare). Glutathione-S-transferase (GST) fusion proteins were purified by glutathione affinity, and then the TIA-1 fragment was further purified by cation exchange chromatography following removal of the GST tag....