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.     

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.     

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.     

Different requirements of the kinase and UHM domains of KIS for its nuclear localization and binding to splicing factors

The protein kinase KIS is made by the juxtaposition of a unique kinase domain and a C-terminal domain with a U2AF homology motif (UHM), a sequence motif for protein interaction initially identified in the heterodimeric pre-mRNA splicing factor U2AF. This domain of KIS is closely related to the C-terminal UHM domain of the U2AF large subunit, U2AF⁶⁵. KIS phosphorylates the splicing factor SF1, which in turn enhances SF1 binding to U2AF⁶⁵ and the 3′ splice site, an event known to take place at an early step of spliceosome assembly. Here, the analysis of the subcellular localization of mutated forms of KIS indicates that the kinase domain of KIS is the necessary domain for its nuclear localization. As in the case of U2AF⁶⁵, the UHM-containing C-terminal domain of KIS is required for binding to the splicing factors SF1 and SF3b155. The efficiency of KIS binding to SF1 and SF3b155 is similar to that of U2AF⁶⁵ in pull-down assays. These results further support the functional link of KIS with splicing factors. Interestingly, when compared to other UHM-containing proteins, KIS presents a different specificity for the UHM docking sites that are present in the N-terminal region of SF3b155, thus providing a new insight into the variety of interactions mediated by UHM domains.     

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.     

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.     

Mammalian splicing factor SF1 interacts with SURP domains of U2 snRNP-associated proteins

Splicing factor 1 (SF1) recognizes the branch point sequence (BPS) at the 3′ splice site during the formation of early complex E, thereby pre-bulging the BPS adenosine, thought to facilitate subsequent base-pairing of the U2 snRNA with the BPS. The 65-kDa subunit of U2 snRNP auxiliary factor (U2AF65) interacts with SF1 and was shown to recruit the U2 snRNP to the spliceosome. Co-immunoprecipitation experiments of SF1-interacting proteins from HeLa cell extracts shown here are consistent with the presence of SF1 in early splicing complexes. Surprisingly almost all U2 snRNP proteins were found associated with SF1. Yeast two-hybrid screens identified two SURP domain-containing U2 snRNP proteins as partners of SF1. A short, evolutionarily conserved region of SF1 interacts with the SURP domains, stressing their role in protein–protein interactions. A reduction of A complex formation in SF1-depleted extracts could be rescued with recombinant SF1 containing the SURP-interaction domain, but only partial rescue was observed with SF1 lacking this sequence. Thus, SF1 can initially recruit the U2 snRNP to the spliceosome during E complex formation, whereas U2AF65 may stabilize the association of the U2 snRNP with the spliceosome at later times. In addition, these findings may have implications for alternative splicing decisions.     

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).     

Identification and characterization of srp1, a gene of fission yeast encoding a RNA binding domain and a RS domain typical of SR splicing factors.

The SR protein family is involved in constitutive and regulated pre-mRNA splicing and has been found to be evolutionarily conserved in metazoan organisms. In contrast, the genome of the unicellular yeast Saccharomyces cerevisiae does not contain genes encoding typical SR proteins. The mammalian SR proteins consist of one or two characteristic RNA binding domains (RBD), containing the signature sequences RDAEDA and SWQDLKD respectively, and a RS (arginine/serine-rich) domain which gave the family its name. We have now cloned from the fission yeast Schizosaccharomyces pombe the gene srp1. This gene is the first yeast gene encoding a protein with typical features of mammalian SR protein family members. The gene is not essential for growth. We show that overexpression of the RNA binding domain inhibits pre-mRNA splicing and that the highly conserved sequence RDAEDA in the RBD is involved. Overexpression of Srp1 containing mutations in the RS domain also inhibits pre-mRNA splicing activity. Furthermore, we show that overexpression of Srp1 and overexpression of the mammalian SR splicing factor ASF/SF2 suppress the pre-mRNA splicing defect of the temperature-sensitive prp4-73 allele. prp4 encodes a protein kinase involved in pre-mRNA splicing. These findings are consistent with the notion that Srp1 plays a role in the splicing process.     

Structural basis for the molecular recognition between human splicing factors U2AF65 and SF1/mBBP

The essential splicing factors SF1 and U2AF play an important role in the recognition of the pre-mRNA 3' splice site during early spliceosome assembly. The structure of the C-terminal RRM (RRM3) of human U2AF(65) complexed to an N-terminal peptide of SF1 reveals an extended negatively charged helix A and an additional helix C. Helix C shields the potential RNA binding surface. SF1 binds to the opposite, helical face of RRM3. It inserts a conserved tryptophan into a hydrophobic pocket between helices A and B in a way that strikingly resembles part of the molecular interface in the U2AF heterodimer. This molecular recognition establishes a paradigm for protein binding by a subfamily of noncanonical RRMs.     

剪接因子U2AF65相关蛋白的研究进展

U2核糖核蛋白小体辅助因子(U2AF)65是参与前体mRNA剪接的重要辅助因子,前体RNA生成之初,U1核糖核蛋白小体(snRNP)结合到内含子的5'剪接位点,U2AF65和U2AF35分别结合到多聚嘧啶序列和3'剪接位点,剪接因子1(SF1)结合到分支位点是剪接体形成的第一步。U2AF的存在又辅助U2snRNP代替SF1结合到分支位点,使剪接反应顺利进行。最近几年,发现基因组中存在一些U2AF65的旁系同源基因序列。这些旁系同源基因由祖先基因经连续复制而横向形成,复制出的基因副本经历了各自的进化途径,最终它们在结构和功能上有相似之处,又各有独特之处。我们简要讨论了U2AF65、PUF60、CAPERα和CAPERβ这4种同源蛋白的发现过程、结构特征、自身的多样性、基因的进化和生物学功能。     

茄子SmU2AF65基因的可变剪接

摘要: U2AF(U2 snRNP auxiliary factor)是参与前体mRNA剪接的重要辅助因子, 在进化上具有较高保守性。文章根据茄子BAC 77N19(GenBank登录号: EF517791)的基因组序列信息和烟草NpU2AF⁶⁵a和NpU2AF⁶⁵b基因的全长cDNA序列, 设计特异引物, 经cDNA末端快速扩增法(RACE)获得了1 986 bp的茄子同源基因(SmU2AF⁶⁵)全长cDNA, GenBank登录号为EU543263。序列分析表明该序列包含1 665 bp的可阅读框, 编码554个氨基酸, 在氨基酸序列的C末端有3个保守的RNA识别结构域RRM。RT-PCR分析表明, SmU2AF⁶⁵基因在不同组织中均有表达,但是该基因通过可变剪接至少能够产生两个转录本, 在根中产生与其他组织中不同的剪切子     

Alternative conformations at the RNA-binding surface of the N-terminal U2AF(65) RNA recognition motif

The essential pre-mRNA splicing factor, U2 auxiliary factor 65KD (U2AF(65)) recognizes the polypyrimidine tract (Py-tract) consensus sequence of the pre-mRNA using two RNA recognition motifs (RRMs), the most prevalent class of eukaryotic RNA-binding domain. The Py-tracts of higher eukaryotic pre-mRNAs are often interrupted with purines, yet U2AF(65) must identify these degenerate Py-tracts for accurate pre-mRNA splicing. Previously, the structure of a U2AF(65) variant in complex with poly(U) RNA suggested that rearrangement of flexible side-chains or bound water molecules may contribute to degenerate Py-tract recognition by U2AF(65). Here, the X-ray structure of the N-terminal RRM domain of U2AF(65) (RRM1) is described at 1.47 A resolution in the absence of RNA. Notably, RNA-binding by U2AF(65) selectively stabilizes pre-existing alternative conformations of three side-chains located at the RNA interface (Arg150, Lys225, and Arg227). Additionally, a flexible loop connecting the beta2/beta3 strands undergoes a conformational change to interact with the RNA. These pre-existing alternative conformations may contribute to the ability of U2AF(65) to recognize a variety of Py-tract sequences. This rare, high-resolution view of an important member of the RRM class of RNA-binding domains highlights the role of alternative side-chain conformations in RNA recognition.     

Mammalian splicing factor SF3a120 represents a new member of the SURP family of proteins and is homologous to the essential splicing factor PRP21p of Saccharomyces cerevisiae.

Mammalian splicing factor SF3a consists of three subunits of 60, 66, and 120 kDa and functions early during pre-mRNA splicing by converting the U2 snRNP into its active form. A cDNA encoding the 120-kDa subunit of SF3a has been cloned. The SF3a120 gene was localized to human chromosome 22, and three mRNAs of 3.2, 3.8, and 5.7 kb are ubiquitously expressed. The N-terminal half of the deduced SF3a120 amino acid sequence contains a tandemly repeated motif (the SURP module) that has recently been identified in the essential splicing factor PRP21p of Saccharomyces cerevisiae, the Drosophila alternative splicing regulator suppressor-of-white-apricot, and four proteins from nematodes and mammals; the C-terminal half is organized into a proline-rich region and a ubiquitin-like domain. The spacing between the SURP modules and the protein's essential function in constitutive splicing identify SF3a120 as the mammalian homologue of yeast PRP21p. Binding studies with truncated derivatives of SF3a120 revealed that the SURP domains function in binding to SF3a60, whereas a region of 130 amino acids C-terminal to these domains is essential for contacts with SF3a66.     

PUF60: a novel U2AF65-related splicing activity

We have identified a new pyrimidine-tract binding factor, PUF, that is required, together with U2AF, for efficient reconstitution of RNA splicing in vitro. The activity has been purified and consists of two proteins, PUF60 and the previously described splicing factor p54. p54 and PUF60 form a stable complex in vitro when cotranslated in a reaction mixture. PUF activity, in conjunction with U2AF, facilitates the association of U2 snRNP with the pre-mRNA. This reaction is dependent upon the presence of the large subunit of U2AF, U2AF65, but not the small subunit U2AF35. PUF60 is homologous to both U2AF65 and the yeast splicing factor Mud2p. The C-terminal domain of PUF60, the PUMP domain, is distantly related to the RNA-recognition motif domain, and is probably important in protein-protein interactions.     

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.     

Phosphorylation of splicing factor SF1 on Ser20 by cGMP-dependent protein kinase regulates spliceosome assembly.

Splicing factor 1 (SF1) functions at early stages of pre-mRNA splicing and contributes to splice site recognition by interacting with the essential splicing factor U2AF65 and binding to the intron branch site. We have identified an 80 kDa substrate of cGMP-dependent protein kinase-I (PKG-I) isolated from rat brain, which is identical to SF1. PKG phosphorylates SF1 at Ser20, which inhibits the SF1-U2AF65 interaction leading to a block of pre-spliceosome assembly. Mutation of Ser20 to Ala or Thr also inhibits the interaction with U2AF65, indicating that Ser20 is essential for binding. SF1 is phosphorylated in vitro by PKG, but not by cAMP-dependent protein kinase A (PKA). Phosphorylation of SF1 also occurs in cultured neuronal cells and is increased on Ser20 in response to a cGMP analogue. These results suggest a new role for PKG in mammalian pre-mRNA splicing by regulating in a phosphorylation-dependent manner the association of SF1 with U2AF65 and spliceosome assembly.