The adenovirus E4-ORF4 splicing enhancer protein interacts with a subset of phosphorylated SR proteins

SR proteins purified from uninfected HeLa cells inhibit adenovirus IIIa pre-mRNA splicing by binding to the intronic IIIa repressor element (3RE). In contrast, SR proteins purified from late adenovirus-infected cells are functionally inactivated as splicing repressor proteins by a virus-induced dephosphorylation. We have shown that the adenovirus E4-ORF4 protein, which binds the cellular protein phos phatase 2A (PP2A) and activates IIIa splicing in vitro and in vivo, induces SR protein dephosphorylation. Here we show that E4-ORF4 interacts with only a subset of SR proteins present in HeLa cells. Thus, E4-ORF4 interacts efficiently with SF2/ASF and SRp30c, but not with other SR proteins. Interestingly, E4-ORF4 interacts with SF2/ASF through the latter's RNA recognition motifs. Furthermore, E4-ORF4 interacts preferentially with the hyperphosphorylated form of SR proteins found in uninfected HeLa cells. E4-ORF4 mutant proteins that fail to bind strongly to PP2A or SF2/ASF do not relieve the repressive effect of HeLa SR proteins on IIIa pre-mRNA splicing in transient transfection experiments, suggesting that an interaction between all three proteins is required for E4-ORF4-induced SR protein dephosphorylation.     

Regulation of SRp20 exon 4 splicing

SR proteins are essential splicing factors involved in the use of both constitutive and alternative exons. We previously showed that the SR proteins SRp20 and ASF/SF2 have antagonistic activities on SRp20 pre-mRNA splicing. SRp20 activates exon 4 recognition in its pre-mRNA, whereas ASF/SF2 inhibits this recognition. In experiments aimed at testing the specificity of SRp20 and ASF/SF2 for exon 4 splicing regulation, we show here that this specificity lies in the RNA binding domains of SRp20 and ASF/SF2 and not in the RS domains. Surprisingly, a deletion of 14 amino acids at the end of ASF/SF2-RBD2 converts ASF/SF2 from an inhibitor to an activator of exon 4 splicing. We found that ASF3 also inhibits exon 4 recognition, thus acting similarly to ASF/SF2, while SC35 activates a cryptic 5' splice site downstream of exon 3 and, in doing so, represses exon 4 use. In contrast, Tra2 and the SR proteins 9G8 and SRp40 do not appear to affect exon 4 splicing.     

Functional analysis of pre-mRNA splicing factor SF2/ASF structural domains.

Human pre-mRNA splicing factor SF2/ASF has an activity required for general splicing in vitro and promotes utilization of proximal alternative 5' splice sites in a concentration-dependent manner by opposing hnRNP A1. We introduced selected mutations in the N-terminal RNA recognition motif (RRM) and the C-terminal Arg/Ser (RS) domain of SF2/ASF, and assayed the resulting recombinant proteins for constitutive and alternative splicing in vitro and for binding to pre-mRNA and mRNA. Mutants inactive in constitutive splicing can affect alternative splice site selection, demonstrating that these activities involve distinct molecular interactions. Specific protein-RNA contact mediated by Phe56 and Phe58 in the RNP-1 submotif of the SF2/ASF RRM are essential for constitutive splicing, although they are not required for RRM-mediated binding to pre-mRNA. The RS domain is also required for constitutive splicing activity and both Arg and Ser residues are important. Analysis of domain deletion mutants demonstrated strong synergy between the RRM and a central degenerate RRM repeat in binding to RNA. These two domains are sufficient for alternative splicing activity in the absence of an RS domain.     

The splicing factor-associated protein, p32, regulates RNA splicing by inhibiting ASF/SF2 RNA binding and phosphorylation

The cellular protein p32 was isolated originally as a protein tightly associated with the essential splicing factor ASF/SF2 during its purification from HeLa cells. ASF/SF2 is a member of the SR family of splicing factors, which stimulate constitutive splicing and regulate alternative RNA splicing in a positive or negative fashion, depending on where on the pre-mRNA they bind. Here we present evidence that p32 interacts with ASF/SF2 and SRp30c, another member of the SR protein family. We further show that p32 inhibits ASF/SF2 function as both a splicing enhancer and splicing repressor protein by preventing stable ASF/SF2 interaction with RNA, but p32 does not block SRp30c function. ASF/SF2 is highly phosphorylated in vivo, a modification required for stable RNA binding and protein-protein interaction during spliceosome formation, and this phosphorylation, either through HeLa nuclear extracts or through specific SR protein kinases, is inhibited by p32. Our results suggest that p32 functions as an ASF/SF2 inhibitory factor, regulating ASF/SF2 RNA binding and phosphorylation. These findings place p32 into a new group of proteins that control RNA splicing by sequestering an essential RNA splicing factor into an inhibitory complex.     

Deletion of the N-terminus of SF2/ASF permits RS-domain-independent pre-mRNA splicing

Serine/arginine-rich (SR) proteins are essential splicing factors with one or two RNA-recognition motifs (RRMs) and a C-terminal arginine- and serine-rich (RS) domain. SR proteins bind to exonic splicing enhancers via their RRM(s), and from this position are thought to promote splicing by antagonizing splicing silencers, recruiting other components of the splicing machinery through RS-RS domain interactions, and/or promoting RNA base-pairing through their RS domains. An RS domain tethered at an exonic splicing enhancer can function as a splicing activator, and RS domains play prominent roles in current models of SR protein functions. However, we previously reported that the RS domain of the SR protein SF2/ASF is dispensable for in vitro splicing of some pre-mRNAs. We have now extended these findings via the identification of a short inhibitory domain at the SF2/ASF N-terminus; deletion of this segment permits splicing in the absence of this SR protein's RS domain of an IgM pre-mRNA substrate previously classified as RS-domain-dependent. Deletion of the N-terminal inhibitory domain increases the splicing activity of SF2/ASF lacking its RS domain, and enhances its ability to bind pre-mRNA. Splicing of the IgM pre-mRNA in S100 complementation with SF2/ASF lacking its RS domain still requires an exonic splicing enhancer, suggesting that an SR protein RS domain is not always required for ESE-dependent splicing activation. Our data provide additional evidence that the SF2/ASF RS domain is not strictly required for constitutive splicing in vitro, contrary to prevailing models for how the domains of SR proteins function to promote splicing.     

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.     

The splicing factors 9G8 and SRp20 transactivate splicing through different and specific enhancers

The activity of the SR protein family of splicing factors in constitutive or alternative splicing requires direct interactions with the pre-mRNA substrate. Thus it is important to define the high affinity targets of the various SR species and to evaluate their ability to discriminate between defined RNA targets. We have analyzed the binding specificity of the 30-kDa SR protein 9G8, which contains a zinc knuckle in addition to the RNA binding domain (RBD). Using a SELEX approach, we demonstrate that 9G8 selects RNA sequences formed by GAC triplets, whereas a mutated zinc knuckle variant selects different RNA sequences, centered around a (A/U)C(A/U)(A/U)C motif, indicating that the zinc knuckle is involved in the RNA recognition specificity of 9G8. In contrast, SC35 selects sequences composed of pyrimidine or purine-rich motifs. Analyses of RNA-protein interactions with purified recombinant 30-kDa SR proteins or in nuclear extracts, by means of UV crosslinking and immunoprecipitation, demonstrate that 9G8, SC35, and ASF/SF2 recognize their specific RNA targets with high specificity. Interestingly, the RNA sequences selected by the mutated zinc knuckle 9G8 variant are efficiently recognized by SRp20, in agreement with the fact that the RBD of 9G8 and SRp20 are similar. Finally, we demonstrate the ability of 9G8 and of its zinc knuckle variant, or SRp20, to act as efficient splicing transactivators through their specific RNA targets. Our results provide the first evidence for cooperation between an RBD and a zinc knuckle in defining the specificity of an RNA binding domain.     

Multiple properties of the splicing repressor SRp38 distinguish it from typical SR proteins

The SR protein SRp38 is a general splicing repressor that is activated by dephosphorylation during mitosis and in response to heat shock. Here we describe experiments that provide insights into the mechanism by which SRp38 functions in splicing repression. We first show that SRp38 redistributes and colocalizes with snRNPs, but not with a typical SR protein, SC35, during mitosis and following heat shock. Supporting the functional significance of this association, a micrococcal nuclease-sensitive component, i.e., an snRNP(s), completely rescued heat shock-induced splicing repression in vitro, and purified U1 snRNP did so partially. SRp38 contains an N-terminal RNA binding domain (RBD) and a C-terminal RS domain composed of two subdomains (RS1 and RS2 domains). Unexpectedly, an RS1 deletion mutant derivative specifically inhibited the second step of splicing, while an RS2 deletion mutant retained significant dephosphorylation-dependent repression activity. Using chimeric SRp38/SC35 proteins, we show that SC35-RBD/SRp38-RS can function as a general splicing activator and that the dephosphorylated version can act as a strong splicing repressor. SRp38-RBD/SC35-RS, however, was essentially inactive in these assays. Together, our results help to define the unusual features of SRp38 that distinguish it from other SR proteins.     

Roles for SR proteins and hnRNP A1 in the regulation of c-src exon N1

The splicing of the c-src exon N1 is controlled by an intricate combination of positive and negative RNA elements. Most previous work on these sequences focused on intronic elements found upstream and downstream of exon N1. However, it was demonstrated that the 5' half of the N1 exon itself acts as a splicing enhancer in vivo. Here we examine the function of this regulatory element in vitro. We show that a mutation in this sequence decreases splicing of the N1 exon in vitro. Proteins binding to this element were identified as hnRNP A1, hnRNP H, hnRNP F, and SF2/ASF by site-specific cross-linking and immunoprecipitation. The binding of these proteins to the RNA was eliminated by a mutation in the exonic element. The activities of hnRNP A1 and SF2/ASF on N1 splicing were examined by adding purified protein to in vitro splicing reactions. SF2/ASF and another SR protein, SC35, are both able to stimulate splicing of c-src pre-mRNA. However, splicing activation by SF2/ASF is dependent on the N1 exon enhancer element whereas activation by SC35 is not. In contrast to SF2/ASF and in agreement with other systems, hnRNP A1 repressed c-src splicing in vitro. The negative activity of hnRNP A1 on splicing was compared with that of PTB, a protein previously demonstrated to repress splicing in this system. Both proteins repress exon N1 splicing, and both counteract the enhancing activity of the SR proteins. Removal of the PTB binding sites upstream of N1 prevents PTB-mediated repression but does not affect A1-mediated repression. Thus, hnRNP A1 and PTB use different mechanisms to repress c-src splicing. Our results link the activity of these well-known exonic splicing regulators, SF2/ASF and hnRNP A1, to the splicing of an exon primarily controlled by intronic factors.     

The human splicing factors ASF/SF2 and SC35 possess distinct, functionally significant RNA binding specificities.

ASF/SF2 and SC35 belong to a highly conserved family of nuclear proteins that are both essential for splicing of pre-mRNA in vitro and are able to influence selection of alternative splice sites. An important question is whether these proteins display distinct RNA binding specificities and, if so, whether this influences their functional interactions with pre-mRNA. To address these issues, we first performed selection/amplification from pools of random RNA sequences (SELEX) with portions of the two proteins comprising the RNA binding domains (RBDs). Although both molecules selected mainly purine-rich sequences, comparison of individual sequences indicated that the motifs recognized are different. Binding assays performed with the full-length proteins confirmed that ASF/SF2 and SC35 indeed have distinct specificities, and at the same time provided evidence that the highly charged arginine-serine region of each protein is not a major determinant of specificity. In the case of ASF/SF2, evidence is presented that binding specificity involves cooperation between the protein's two RBDs. Finally, we demonstrate that an element containing three copies of a high-affinity ASF/SF2 binding site constitutes a powerful splicing enhancer. In contrast, a similar element consisting of three SC35 sites was inactive. The ASF/SF2 enhancer can be activated specifically in splicing-deficient S100 extracts by recombinant ASF/SF2 in conjunction with one or more additional protein factors. These and other results suggest a central role for ASF/SF2 in the function of purine-rich splicing enhancers.     

Dephosphorylation-dependent sorting of SR splicing factors during mRNP maturation

SR proteins are a family of sequence-specific RNA binding proteins originally discovered as essential factors for pre-mRNA splicing and recently implicated in mRNA transport, stability, and translation. Here, we used a genetic complementation system derived from conditional knockout mice to address the function and regulation of SR proteins in vivo. We demonstrate that ASF/SF2 and SC35 are each required for cell viability, but, surprisingly, the effector RS domain of ASF/SF2 is dispensable for cell survival in MEFs. Although shuttling SR proteins have been implicated in mRNA export, prevention of ASF/SF2 from shuttling had little impact on mRNA export. We found that shuttling and nonshuttling SR proteins are segregated in an orderly fashion during mRNP maturation, indicating distinct recycling pathways for different SR proteins. We further showed that this process is regulated by differential dephosphorylation of the RS domain, thus revealing a sorting mechanism for mRNP transition from splicing to export.     

Functional domains of the human splicing factor ASF/SF2.

The human splicing factor ASF/SF2 displays two predominant activities in in vitro splicing assays: (i) it is an essential factor apparently required for all splices and (ii) it is able to switch utilization of alternative 5' splice sites in a concentration-dependent manner. ASF/SF2 is the prototype of a family of proteins typified by the presence of one or two RNP-type RNA binding domains (RBDs) and a region highly enriched in repeating arginine-serine dipeptides (RS regions). Here we describe a functional analysis of ASF/SF2, which defines several regions essential for one, or both, of its two principal activities, and provides insights into how this type of protein functions in splicing. Two isoforms of the protein, which arise from alternative splicing, are by themselves inactive, but each can block the activity of ASF/SF2, thereby functioning as splicing repressors. Some, but not all, mutations in the RS region prevent ASF/SF2 from functioning as an essential splicing factor. However, the entire RS region can be deleted without reducing splice site switching activity, indicating that it is not absolutely required for interaction with other splicing factors. Experiments with deletion and substitution mutants reveal that the protein contains two related, but highly diverged, RBDs, and that both are essential for activity. Each RBD by itself retains the ability to bind RNA, although optimal binding requires both domains.     

SRp30c is a repressor of 3' splice site utilization

Several intron elements influence exon 7B skipping in the mammalian hnRNP A1 pre-mRNA. We have shown previously that the 38-nucleotide CE9 element located in the intron separating alternative exon 7B from exon 8 can repress the use of a downstream 3' splice site. The ability of CE9 to act on heterologous substrates, combined with the results of competition and gel shift assays, indicates that the activity of CE9 is mediated by a trans-acting factor. UV cross-linking analysis revealed the specific association of a 25-kDa nuclear protein with CE9. Using RNA affinity chromatography, we isolated a 25-kDa protein that binds to CE9 RNA. This protein corresponds to SRp30c. Consistent with a role for SRp30c in the activity of CE9, recombinant SRp30c interacts specifically with CE9 and can promote splicing repression in vitro in a CE9-dependent manner. The closest homologue of SRp30c, ASF/SF2, does not bind to CE9 and does not repress splicing even when the intronic SRp30c binding sites are replaced with high-affinity ASF/SF2 binding sites. Only the first 7 nucleotides of CE9 are sufficient for binding to SRp30c, and mutations that abolish binding also prevent repression. Our results indicate that SRp30c can function as a repressor of 3' splice site utilization and suggest that the SRp30c-CE9 interaction may contribute to the control of hnRNP A1 alternative splicing.     

Binding of DAZAP1 and hnRNPA1/A2 to an exonic splicing silencer in a natural BRCA1 exon 18 mutant

A disease-causing G-to-T transversion at position +6 of BRCA1 exon 18 induces exclusion of the exon from the mRNA and, as has been suggested by in silico analysis, disrupts an ASF/SF2-dependent splicing enhancer. We show here using a pulldown assay with an internal standard that wild-type (WT) and mutant T6 sequences displayed similar ASF/SF2 binding efficiencies, which were significantly lower than that of a typical exonic splicing enhancer derived from the extra domain A exon of fibronectin. Overexpression or small interfering RNA (siRNA)-mediated depletion of ASF/SF2 did not affect the splicing of a WT BRCA1 minigene but resulted in an increase and decrease of T6 exon 18 inclusion, respectively. Furthermore, extensive mutation analysis using hybrid minigenes indicated that the T6 mutant creates a sequence with a prevalently inhibitory function. Indeed, RNA-protein interaction and siRNA experiments showed that the skipping of T6 BRCA1 exon 18 is due to the creation of a splicing factor-dependent silencer. This sequence specifically binds to the known repressor protein hnRNPA1/A2 and to DAZAP1, the involvement of which in splicing inhibition we have demonstrated. Our results indicate that the binding of the splicing factors hnRNPA1/A2 and DAZAP1 is the primary determinant of T6 BRCA1 exon 18 exclusion.     

A splicing repressor domain in polypyrimidine tract-binding protein

Polypyrimidine tract-binding protein (PTB) is an hnRNP with four RRM type domains. It plays roles as a repressive alternative splicing regulator of multilple target genes, as well as being involved in pre-mRNA 3' end processing, mRNA localization, stability, and internal ribosome entry site-mediated translation. Here we have used a tethered function assay, in which a fusion protein of PTB and the bacteriophage MS2 coat protein is recruited to a splicing regulatory site by binding to an artificially inserted MS2 binding site. Deletion mutations of PTB in this system allowed us to identify RRM2 and the following inter-RRM linker region as the minimal region of PTB that can act as splicing repressor domain when recruited to RNA. Splicing repression by the minimal repressor domain remained cell type-specific and dependent upon other defined regulatory elements in the alpha-tropomyosin test minigene. Our results highlight the fact that splicing repression by PTB can be uncoupled from the mode by which it binds to RNA.     

Activation and repression functions of an SR splicing regulator depend on exonic versus intronic-binding position

SR proteins and related factors play widespread roles in alternative pre-mRNA splicing and are known to promote splice site recognition through their Arg-Ser-rich effector domains. However, binding of SR regulators to some targets results in repression of splice sites through a distinct mechanism. Here, we investigate how activated and repressed targets of the Drosophila SR regulator Transformer2 elicit its differing effects on splicing. We find that, like activation, repression affects early steps in the recognition of splice sites and spliceosome assembly. Repositioning of regulatory elements reveals that Tra2 complexes that normally repress splicing from intronic positions activate splicing when located in an exon. Protein tethering experiments demonstrate that this position dependence is an intrinsic property of Tra2 and further show that repression and activation are mediated by separate effector domains of this protein. When other Drosophila SR factors (SF2 and Rbp1) that activate splicing from exonic positions were tethered intronically they failed to either activate or repress splicing. Interestingly, both activities of Tra2 favor the exonic identity of the RNA sequences that encompass its binding sites. This suggests a model in which these two opposite functions act in concert to define both the position and extent of alternatively spliced exons.