Cancer-relevant Splicing Factor CAPERα Engages the Essential Splicing Factor SF3b155 in a Specific Ternary Complex

U2AF homology motifs (UHMs) mediate protein-protein interactions with U2AF ligand motifs (ULMs) of pre-mRNA splicing factors. The UHM-containing alternative splicing factor CAPERα regulates splicing of tumor-promoting VEGF isoforms, yet the molecular target of the CAPERα UHM is unknown. Here we present structures of the CAPERα UHM bound to a representative SF3b155 ULM at 1.7 Å resolution and, for comparison, in the absence of ligand at 2.2 Å resolution. The prototypical UHM/ULM interactions authenticate CAPERα as a bona fide member of the UHM family of proteins. We identify SF3b155 as the relevant ULM-containing partner of full-length CAPERα in human cell extracts. Isothermal titration calorimetry comparisons of the purified CAPERα UHM binding known ULM-containing proteins demonstrate that high affinity interactions depend on the presence of an intact, intrinsically unstructured SF3b155 domain containing seven ULM-like motifs. The interplay among bound CAPERα molecules gives rise to the appearance of two high affinity sites in the SF3b155 ULM-containing domain. In conjunction with the previously identified, UHM/ULM-mediated complexes of U2AF⁶⁵ and SPF45 with SF3b155, this work demonstrates the capacity of SF3b155 to offer a platform for coordinated recruitment of UHM-containing splicing factors.     

Backbone assignment of the UHM domain of Puf60 free and bound to five ligands

U2AF homology motifs (UHM) are protein domains that bind peptidic UHM ligand motifs (ULM) and thus form an intricate network of interactions involved in splicing regulation. Here, we report the backbone assignment of the UHM domain of the splicing factor Puf60 as well as ¹H, ¹⁵N chemical shifts upon binding of the ULM peptides U2AF⁶⁵ (85–112), SF1 (1–25), SF3b155 (194–229), SF3b155 (317–357), and Prp16 (201–238).     

Major phosphorylation of SF1 on adjacent Ser-Pro motifs enhances interaction with U2AF65

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.     

Multiple U2AF65 binding sites within SF3b155: thermodynamic and spectroscopic characterization of protein-protein interactions among pre-mRNA splicing factors

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.     

Dimerization and protein binding specificity of the U2AF homology motif of the splicing factor Puf60

PUF60 is an essential splicing factor functionally related and homologous to U2AF(65). Its C-terminal domain belongs to the family of U2AF (U2 auxiliary factor) homology motifs (UHM), a subgroup of RNA recognition motifs that bind to tryptophan-containing linear peptide motifs (UHM ligand motifs, ULMs) in several nuclear proteins. Here, we show that the Puf60 UHM is mainly monomeric in physiological buffer, whereas its dimerization is induced upon the addition of SDS. The crystal structure of PUF60-UHM at 2.2 angstroms resolution, NMR data, and mutational analysis reveal that the dimer interface is mediated by electrostatic interactions involving a flexible loop. Using glutathione S-transferase pulldown experiments, isothermal titration calorimetry, and NMR titrations, we find that Puf60-UHM binds to ULM sequences in the splicing factors SF1, U2AF65, and SF3b155. Compared with U2AF65-UHM, Puf60-UHM has distinct binding preferences to ULMs in the N terminus of SF3b155. Our data suggest that the functional cooperativity between U2AF65 and Puf60 may involve simultaneous interactions of the two proteins with SF3b155.     

U2AF-homology motif interactions are required for alternative splicing regulation by SPF45

The U2AF-homology motif (UHM) mediates protein-protein interactions between factors involved in constitutive RNA splicing. Here we report that the splicing factor SPF45 regulates alternative splicing of the apoptosis regulatory gene FAS (also called CD95). The SPF45 UHM is necessary for this activity and binds UHM-ligand motifs (ULMs) present in the 3' splice site-recognizing factors U2AF65, SF1 and SF3b155. We describe a 2.1-A crystal structure of SPF45-UHM in complex with a ULM peptide from SF3b155. Features distinct from those of previously described UHM-ULM structures allowed the design of mutations in the SPF45 UHM that selectively impair binding to individual ULMs. Splicing assays using the ULM-selective SPF45 variants demonstrate that individual UHM-ULM interactions are required for FAS splicing regulation by SPF45 in vivo. Our data suggest that networks of UHM-ULM interactions are involved in regulating alternative splicing.     

Structure,phosphorylation and U2AF65 binding of the N-terminal domain of splicing factor 1 during 3′-splice site recognition

Recognition of the 3′-splice site is a key step in pre-mRNA splicing and accomplished by a dynamic complex comprising splicing factor 1 (SF1) and the U2 snRNP auxiliary factor 65-kDa subunit (U2AF65). Both proteins mediate protein–protein and protein–RNA interactions for cooperative RNA-binding during spliceosome assembly. Here, we report the solution structure of a novel helix-hairpin domain in the N-terminal region of SF1 (SF1^NTD). The nuclear magnetic resonance- and small-angle X-ray scattering-derived structure of a complex of the SF1^NTD with the C-terminal U2AF homology motif domain of U2AF65 (U2AF65^UHM) reveals that, in addition to the known U2AF65^UHM–SF1 interaction, the helix-hairpin domain forms a secondary, hydrophobic interface with U2AF65^UHM, which locks the orientation of the two subunits. Mutational analysis shows that the helix hairpin is essential for cooperative formation of the ternary SF1–U2AF65–RNA complex. We further show that tandem serine phosphorylation of a conserved Ser80-Pro81-Ser82-Pro83 motif rigidifies a long unstructured linker in the SF1 helix hairpin. Phosphorylation does not significantly alter the overall conformations of SF1, SF1–U2AF65 or the SF1–U2AF65–RNA complexes, but slightly enhances RNA binding. Our results indicate that the helix-hairpin domain of SF1 is required for cooperative 3′-splice site recognition presumably by stabilizing a unique quaternary arrangement of the SF1–U2AF65–RNA complex.     

The SF3b155 N-terminal domain is a scaffold important for splicing

Recruitment of U2 snRNP to the branch point sequence of introns is a necessary step in pre-mRNA splicing. In the current model, U2AF65, bound at the polypyrimidine tract of the intron, recruits U2 snRNP to the branch point sequence by interacting with the U2 snRNP protein SF3b155. We demonstrate that the N-terminal domain of SF3b155 contains multiple U2AF65 binding sites that are distinct from the binding site for the U2 snRNP protein p14, mapped to amino acids 396-424 of SF3b155. The N-terminal domain of SF3b155 appears to adopt a primarily unfolded structure but is functional to inhibit splicing in vitro. RNA binding studies show that the N-terminal domain of SF3b155 binds RNA nonspecifically and that the sites for U2AF65 binding and RNA binding are overlapping (or the same) within SF3b155. We propose that the N-terminal domain of SF3b155 adopts a primarily unfolded structure that functions as a scaffold to facilitate SF3b155's multiple protein-protein and protein-RNA interactions. The multiple U2AF65 binding sites on SF3b155 suggest a model in which multiple U2AF65 molecules bound to the intron could enhance U2 snRNP recruitment to the branch point sequence.     

RNA induces conformational changes in the SF1/U2AF65 splicing factor complex

Spliceosomes assemble on pre-mRNA splice sites through a series of dynamic ribonucleoprotein complexes, yet the nature of the conformational changes remains unclear. Splicing factor 1 (SF1) and U2 auxiliary factor (U2AF⁶⁵) cooperatively recognize the 3′ splice site during the initial stages of pre-mRNA splicing. Here, we used small-angle X-ray scattering to compare the molecular dimensions and ab initio shape restorations of SF1 and U2AF⁶⁵ splicing factors, as well as the SF1/U2AF⁶⁵ complex in the absence and presence of AdML (adenovirus major late) splice site RNAs. The molecular dimensions of the SF1/U2AF⁶⁵/RNA complex substantially contracted by 15 Å in the maximum dimension, relative to the SF1/U2AF⁶⁵ complex in the absence of RNA ligand. In contrast, no detectable changes were observed for the isolated SF1 and U2AF⁶⁵ splicing factors or their individual complexes with RNA, although slight differences in the shapes of their molecular envelopes were apparent. We propose that the conformational changes that are induced by assembly of the SF1/U2AF⁶⁵/RNA complex serve to position the pre-mRNA splice site optimally for subsequent stages of splicing.     

RNA Binding Activity of Heterodimeric Splicing Factor U2AF: at Least One RS Domain Is Required for High-Affinity Binding

The pre-mRNA splicing factor U2AF (U2 small nuclear ribonucleoprotein particle [snRNP] auxiliary factor) plays a critical role in 3′ splice site selection. U2AF binds site specifically to the intron pyrimidine tract between the branchpoint and the 3′ splice site and targets U2 snRNP to the branch site at an early step in spliceosome assembly. Human U2AF is a heterodimer composed of large (hU2AF⁶⁵) and small (hU2AF³⁵) subunits. hU2AF⁶⁵ contains an arginine-serine-rich (RS) domain and three RNA recognition motifs (RRMs). hU2AF³⁵ has a degenerate RRM and a carboxyl-terminal RS domain. Genetic studies have recently shown that the RS domains on the Drosophila U2AF subunit homologs are each inessential and might have redundant functions in vivo. The site-specific pyrimidine tract binding activity of the U2AF heterodimer has previously been assigned to hU2AF⁶⁵. While the requirement for the three RRMs on hU2AF⁶⁵ is firmly established, a role for the large-subunit RS domain in RNA binding remains unresolved. We have analyzed the RNA binding activity of the U2AF heterodimer in vitro. When the Drosophila small-subunit homolog (dU2AF³⁸) was complexed with the large-subunit (dU2AF⁵⁰) pyrimidine tract, RNA binding activity increased 20-fold over that of free dU2AF⁵⁰. We detected a similar increase in RNA binding activity when we compared the human U2AF heterodimer and hU2AF⁶⁵. Surprisingly, the RS domain on dU2AF³⁸ was necessary for the increased binding activity of the dU2AF heterodimer. In addition, removal of the RS domain from the Drosophila large-subunit monomer (dU2AF⁵⁰ΔRS) severely impaired its binding activity. However, if the dU2AF³⁸ RS domain was supplied in a complex with dU2AF⁵⁰ΔRS, high-affinity binding was restored. These results suggest that the presence of one RS domain of U2AF, on either the large or small subunit, promotes high-affinity pyrimidine tract RNA binding activity, consistent with redundant roles for the U2AF RS domains in vivo.     

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 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/pre-mRNA. p14 comprises a canonical RNA recognition motif (RRM) with an additional C-terminal helix (alphaC) and a beta hairpin insertion. SF3b155 binds to the beta-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 alpha-helices, which are partially preformed in isolation. Helices alpha2 and alpha3 (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 beta hairpin insertion of the p14 RRM, consistent with the beta-sheet surface being occupied by the helical SF3b155 peptide and p14 helix alphaC. 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.     

The conserved RNA recognition motif 3 of U2 snRNA auxiliary factor (U2AF 65) is essential in vivo but dispensable for activity in vitro

The general splicing factor U2AF(65) recognizes the polypyrimidine tract (Py tract) that precedes 3' splice sites and has three RNA recognition motifs (RRMs). The C-terminal RRM (RRM3), which is highly conserved, has been proposed to contribute to Py-tract binding and establish protein-protein contacts with splicing factors mBBP/SF1 and SAP155. Unexpectedly, we find that the human RRM3 domain is dispensable for U2AF(65) activity in vitro. However, it has an essential function in Schizosaccharomyces pombe distinct from binding to the Py tract or to mBBP/SF1 and SAP155. First, deletion of RRM3 from the human protein has no effect on Py-tract binding. Second, RRM123 and RRM12 select similar sequences from a random pool of RNA. Third, deletion of RRM3 has no effect on the splicing activity of U2AF(65) in vitro. However, deletion of the RRM3 domain of S. pombe U2AF(59) abolishes U2AF function in vivo. In addition, certain amino acid substitutions on the four-stranded beta-sheet surface of RRM3 compromise U2AF function in vivo without affecting binding to mBBP/SF1 or SAP155 in vitro. We propose that RRM3 has an unrecognized function that is possibly relevant for the splicing of only a subset of cellular introns. We discuss the implications of these observations on previous models of U2AF function.     

Interactome for auxiliary splicing factor U2AF65 suggests diverse roles

U2 small nuclear ribonucleoprotein auxiliary factor (U2AF) is an essential component of the splicing machinery that is composed of two protein subunits, the 35 kDa U2AF³⁵ (U2AF1) and the 65 kDa U2AF⁶⁵ (U2AF2). U2AF interacts with various splicing factors within this machinery. Here we expand the list of mammalian splicing factors that are known to interact with U2AF⁶⁵ as well as the list of nuclear proteins not known to participate in splicing that interact with U2AF⁶⁵. Using a yeast two-hybrid system, we found fourteen U2AF⁶⁵-interacting proteins. The validity of the screen was confirmed by identification of five known U2AF⁶⁵-interacting proteins, including its heterodimeric partner, U2AF³⁵. In addition to binding these known partners, we found previously unrecognized U2AF⁶⁵ interactions with four splicing-related proteins (DDX39, SFRS3, SFRS18, SNRPA), two zinc finger proteins (ZFP809 and ZC3H11A), a U2AF⁶⁵ homolog (RBM39), and two other regulatory proteins (DAXX and SERBP1). We report which regions of U2AF⁶⁵ each of these proteins interacts with and we discuss their potential roles in regulation of pre-mRNA splicing, 3′-end mRNA processing, and U2AF⁶⁵ sub-nuclear localization. These findings suggest expanded roles for U2AF⁶⁵ in both splicing and non-splicing functions.     

Interaction between Subunits of Heterodimeric Splicing Factor U2AF Is Essential In Vivo

The heterodimeric pre-mRNA splicing factor, U2AF (U2 snRNP auxiliary factor), plays a critical role in 3′ splice site selection. Although the U2AF subunits associate in a tight complex, biochemical experiments designed to address the requirement for both subunits in splicing have yielded conflicting results. We have taken a genetic approach to assess the requirement for the Drosophila U2AF heterodimer in vivo. We developed a novel Escherichia coli copurification assay to map the domain on the Drosophila U2AF large subunit (dU2AF⁵⁰) that interacts with the Drosophila small subunit (dU2AF³⁸). A 28-amino-acid fragment on dU2AF⁵⁰ that is both necessary and sufficient for interaction with dU2AF³⁸ was identified. Using the copurification assay, we scanned this 28-amino-acid interaction domain for mutations that abrogate heterodimer formation. A collection of these dU2AF⁵⁰ point mutants was then tested in vivo for genetic complementation of a recessive lethal dU2AF⁵⁰ allele. A mutation that completely abolished interaction with dU2AF³⁸ was incapable of complementation, whereas dU2AF⁵⁰ mutations that did not effect heterodimer formation rescued the recessive lethal dU2AF⁵⁰ allele. Analysis of heterodimer formation in embryo extracts derived from these interaction mutant lines revealed a perfect correlation between the efficiency of subunit association and the ability to complement the dU2AF⁵⁰ recessive lethal allele. These data indicate that Drosophila U2AF heterodimer formation is essential for viability in vivo, consistent with a requirement for both subunits in splicing in vitro.