Role of SR protein modular domains in alternative splicing specificity in vivo.

The SR proteins constitute a family of nuclear phosphoproteins which are required for constitutive splicing and also influence alternative splicing regulation. They have a modular structure consisting of one or two RNA recognition motifs (RRMs) and a C-terminal domain, rich in arginine and serine residues. The functional role of the different domains of SR proteins in constitutive splicing activity has been extensively studied in vitro; however, their contribution to alternative splicing specificity in vivo has not been clearly established. We sought to address how the modular domains of SR proteins contribute to alternative splicing specificity. The activity of a series of chimeric proteins consisting of domain swaps between different SR proteins showed that splice site selection is determined by the nature of the RRMs and that RRM2 of SF2/ASF has a dominant role and can confer specificity to a heterologous protein. In contrast, the identity of the RS domain is not important, as the RS domains are functionally interchangeable. The contribution of the RRMs to alternative splicing specificity in vivo suggests that sequence-specific RNA binding by SR proteins is required for this activity.

Related papers:

title: Sorting out the complexity of SR protein functions.
abstract: been clear whether all of these activities occur during the removal of each intron+ Recent studies now suggest that all of the proposed SR protein functions are carried out during each round of splicing, and at least some of these functions are performed by independent SR protein molecules+ This review discusses recent advances in understanding the diverse functions of SR proteins in metazoan pre-mRNA splicing and presents a model that takes these new findings into account+ Although the reader should keep in mind that the activity of SR proteins in vivo can be influenced by modulating their subcellular localization, this review focuses on biochemical experiments that specifically address the mechanisms by which SR proteins directly function in the splicing reaction+ (The reader may also refer to related reviews recently published elsewhere (Tacke & Manley, 1999; Blencowe, 2000)+)

title: A coactivator of pre-mRNA splicing.
abstract: The nuclear matrix antigen recognized by the monoclonal antibody (mAb) B1C8 is a novel serine (S) and arginine (R)-rich protein associated with splicing complexes and is named here SRm160 (SR-related matrix protein of 160 kD). SRm160 contains multiple SR repeats, but unlike proteins of the SR family of splicing factors, lacks an RNA recognition motif. SRm160 and a related protein SRm300 (the 300-kD nuclear matrix antigen recognized by mAb B4A11) form a complex that is required for the splicing of specific pre-mRNAs. The SRm160/300 complex associates with splicing complexes and promotes splicing through interactions with SR family proteins. Binding of SRm160/300 to pre-mRNA is normally also dependent on U1 snRNP and is stabilized by U2 snRNP. Thus, SRm160/300 forms multiple interactions with components bound directly to important sites within pre-mRNA. The results suggest that a complex of the nuclear matrix proteins SRm160 and SRm300 functions as a coactivator of pre-mRNA splicing.

title: A specific subset of SR proteins shuttles continuously between the nucleus and the cytoplasm.
abstract: The SR proteins constitute a large family of nuclear phosphoproteins required for constitutive pre-mRNA splicing. These factors also have global, concentration-dependent effects on alternative splicing regulation and this activity is antagonized by members of the hnRNP A/B family of proteins. We show here that whereas some human SR proteins are confined to the nucleus, three of them-SF2/ASF, SRp20, and 9G8-shuttle rapidly and continuously between the nucleus and the cytoplasm. By swapping the corresponding domains between shuttling and nonshuttling SR proteins, we show that the carboxy-terminal arginine/serine-rich (RS) domain is required for shuttling. This domain, however, is not sufficient to promote shuttling of an unrelated protein reporter, suggesting that stable RNA binding mediated by the RNA-recognition motifs may be required for shuttling. Consistent with such a requirement, a double point-mutation in RRM1 of SF2/ASF that impairs RNA binding prevents the protein from shuttling. In addition, we show that phosphorylation of the RS domain affects the shuttling properties of SR proteins. These findings show that different SR proteins have unique intracellular transport properties and suggest that the family members that shuttle may have roles not only in nuclear pre-mRNA splicing but also in mRNA transport, cytoplasmic events, and/or processes that involve communication between the nucleus and the cytoplasm.

title: Regulated tissue-specific expression of antagonistic pre-mRNA splicing factors.
abstract: The SR proteins are essential metazoan pre-mRNA splicing factors that can also influence the selection of alternative 5' splice sites in a concentration-dependent manner. Their activity in alternative splicing in vitro is antagonized by members of the hnRNP A/B family of proteins. The opposite effects of members of these two families of antagonistic splicing factors in vitro and upon overexpression in vivo suggest that changes in their relative levels may be a natural mechanism for the regulation of alternative splicing in vivo. One prediction of this model is that the ratios of these antagonists should vary in different cell types and in other situations in which cellular or viral transcripts are differentially spliced. We raised monoclonal antibodies specific for SF2/ASF and used them to measure the abundance of SF2/ASF protein and its isoforms, its phosphorylation state in vivo and during splicing in vitro, and its association with the spliceosome. SF2/ASF exists predominantly or exclusively in a highly phosphorylated state in vivo in all cell types examined, and unphosphorylated protein was not detectable. Unphosphorylated recombinant SF2/ASF becomes rapidly phosphorylated under splicing conditions in HeLa cell extracts and associates stably with one or more exons of beta-globin pre-mRNA. This interaction appears to persist through the splicing reaction and SF2/ASF remains bound to spliced mRNA. We compared the distribution of SF2/ASF to that of its antagonist, hnRNP A1, in different rat tissues and in immortal and transformed cell lines. We found that the protein levels of these antagonistic splicing factors vary naturally over a very wide range, supporting the notion that changes in the ratio of these proteins can affect alternative splicing of a variety of pre-mRNAs in vivo.

title: Antagonism between RSF1 and SR proteins for both splice-site recognition in vitro and Drosophila development.
abstract: Specific recognition of splice sites within metazoan mRNA precursors (pre-mRNAs) is a potential stage for gene regulation by alternative splicing. Splicing factors of the SR protein family play a major role in this regulation, as they are required for early recognition of splice sites during spliceosome assembly. Here, we describe the characterization of RSF1, a splicing repressor isolated from Drosophila, that functionally antagonizes SR proteins. Like the latter, RSF1 comprises an amino-terminal RRM-type RNA-binding domain, whereas its carboxy-terminal part is enriched in glycine (G), arginine (R), and serine (S) residues (GRS domain). RSF1 induces a dose-sensitive inhibition of splicing for several reporter pre-mRNAs, an inhibition that occurs at the level of early splicing complexes formation. RSF1 interacts, through its GRS domain, with the RS domain of the SR protein SF2/ASF and prevents the latter from cooperating with the U1 small nuclear ribonucleoprotein particle (U1 snRNP) in binding pre-mRNA. Furthermore, overproduction of RSF 1 in the fly rescues several developmental defects caused by overexpression of the splicing activator SR protein B52/ SRp55. Therefore, RSF1 may correspond to the prototypical member of a novel family of general splicing repressors that selectively antagonize the effect of SR proteins on 5' splice-site recognition.

title: The SR splicing factors ASF/SF2 and SC35 have antagonistic effects on intronic enhancer‐dependent splicing of the β‐tropomyosin alternative exon 6A
abstract: Exons 6A and 6B of the chicken β‐tropomyosin gene are mutually exclusive and selected in a tissue‐specific manner. Exon 6A is present in non‐muscle and smooth muscle cells, while exon 6B is present in skeletal muscle cells. In this study we have investigated the mechanism underlying exon 6A recognition in non‐muscle cells. Previous reports have identified a pyrimidine‐rich intronic enhancer sequence (S4) downstream of exon 6A as essential for exon 6A 5′‐splice site recognition. We show here that preincubation of HeLa cell extracts with an excess of RNA containing this sequence specifically inhibits exon 6A recognition by the splicing machinery. Splicing inhibition by an excess of this RNA can be rescued by addition of the SR protein ASF/SF2, but not by the SR proteins SC35 or 9G8. ASF/SF2 stimulates exon 6A splicing through specific interaction with the enhancer sequence. Surprisingly, SC35 behaves as an inhibitor of exon 6A splicing, since addition to HeLa nuclear extracts of increasing amounts of the SC35 protein completely abolish the stimulatory effect of ASF/SF2 on exon 6A splicing. We conclude that exon 6A recognition in vitro depends on the ratio of the ASF/SF2 to SC35 SR proteins. Taken together our results suggest that variations in the level or activity of these proteins could contribute to the tissue‐specific choice of β‐tropomyosin exon 6A. In support of this we show that SR proteins isolated from skeletal muscle tissues are less efficient for exon 6A stimulation than SR proteins isolated from HeLa cells.

title: The splicing factor SRp20 modifies splicing of its own mRNA and ASF/SF2 antagonizes this regulation
abstract: SRp20 is a member of the highly conserved SR family of splicing regulators. Using a variety of reporter gene constructs, we show that SRp20 regulates alternative splicing of its own mRNA. Overexpression of SRp20 results in a reduction in the level of exon 4‐skipped SRp20 transcripts and activates the production of transcripts containing exon 4. These exon 4‐included transcripts encode a truncated protein lacking the C‐terminal RS domain. We provide evidence that SRp20 probably enhances the recognition of the otherwise unused, weak splice acceptor of exon 4. The recognition of exons with weak splice acceptor sites may be a general activity of SRp20. Unexpectedly, ASF/SF2, another member of the SR family, antagonizes the effect of SRp20 on SRp20 pre‐mRNA splicing and suppresses the production of the exon 4‐included form. Our results indicate that ASF/SF2 suppresses the use of the alternative exon 4, most likely by inhibiting the recognition of the splice donor of exon 4. These results demonstrate, for the first time, an auto‐regulatory activity of an SR protein which is antagonized by a second SR protein.

title: SR-related proteins and the processing of messenger RNA precursors.
abstract: The processing of messenger RNA precursors (pre-mRNA) to mRNA in metazoans requires a large number of proteins that contain domains rich in alternating arginine and serine residues (RS domains). These include members of the SR family of splicing factors and proteins that are structurally and functionally distinct from the SR family, collectively referred to below as SR-related proteins. Both groups of RS domain proteins function in constitutive and regulated pre-mRNA splicing. Recently, several SR-related proteins have been identified that are associated with the transcriptional machinery. Other SR-related proteins are associated with mRNA 3' end formation and have been implicated in export. We review these findings and evidence that proteins containing RS domains may play a fundamental role in coordinating different steps in the synthesis and processing of pre-mRNA.

title: Determinants of SR protein specificity.
abstract: The SR (Ser-Arg) proteins are a family of nuclear factors that play multiple important roles in splicing of mRNA precursors in metazoan organisms, functioning in both constitutive and regulated splicing. Certain of these functions are redundant, such that any single SR proteins will suffice, but other functions are unique and are specific to a given family member. A number of studies during the past year have investigated the basis for SR protein specificity.

title: Distinct functions of SR proteins in alternative pre-mRNA splicing.
abstract: Alternative splicing of precursor messenger RNAs (pre-mRNAs) is a common mechanism of regulating gene expression. SR proteins are a family of pre-mRNA splicing factors that are structurally related and evolutionarily conserved. Any member of the SR family can complement a splicing-deficient extract that lacks the entire family of SR proteins. Here it is demonstrated that particular SR proteins have distinct functions in alternative pre-mRNA splicing in vitro. In addition, SR proteins are differentially expressed in a variety of tissues. These results suggest a fundamental role for SR proteins in the regulation of alternative splicing.

title: Identification and characterization of three members of the human SR family of pre‐mRNA splicing factors.
abstract: SR proteins have a characteristic C‐terminal Ser/Arg‐rich repeat (RS domain) of variable length and constitute a family of highly conserved nuclear phosphoproteins that can function as both essential and alternative pre‐mRNA splicing factors. We have cloned a cDNA encoding a novel human SR protein designated SRp30c, which has an unusually short RS domain. We also cloned cDNAs encoding the human homologues of Drosophila SRp55/B52 and rat SRp40/HRS. Recombinant proteins expressed from these cDNAs are active in constitutive splicing, as shown by their ability to complement a HeLa cell S100 extract deficient in SR proteins. Additional cDNA clones reflect extensive alternative splicing of SRp40 and SRp55 pre‐mRNAs. The predicted protein isoforms lack the C‐terminal RS domain and might be involved in feedback regulatory loops. The ability of human SRp30c, SRp40 and SRp55 to modulate alternative splicing in vivo was compared with that of other SR proteins using a transient contransfection assay. The overexpression of individual SR proteins in HeLa cells affected the choice of alternative 5′ splice sites of adenovirus E1A and/or human beta‐thalassemia reporters. The resulting splicing patterns were characteristic for each SR protein. Consistent with the postulated importance of SR proteins in alternative splicing in vivo, we demonstrate complex changes in the levels of mRNAs encoding the above SR proteins upon T cell activation, concomitant with changes in the expression of alternatively spliced isoforms of CD44 and CD45.

title: Overexpression of the SR proteins ASF/SF2 and SC35 influences alternative splicing in vivo in diverse ways.
abstract: The SR proteins are a family of essential splicing factors highly conserved throughout metazoa. Here we examine the effects of two prototypical SR proteins, ASF/SF2 and SC35, when overexpressed by transfection in cultured cells together with plasmids encoding alternatively spliced model transcripts. As expected from past work, both proteins were found to affect alternative splicing, but differences as well as similarities in their behavior were observed. With adenovirus E1a pre-mRNA, ASF/SF2 caused shifts in alternative splicing similar to those observed previously, and the effects of mutations, in the protein and the pre-mRNA, were largely consistent with in vitro results. For example, the C-terminal RS domain was not required to alter splice site selection. SC35 overexpression also altered E1a splicing, but the pattern was distinct from that detected with ASF/SF2, indicating that the two proteins can function differently in vivo. Unexpectedly, with SV40 early pre-mRNA, overexpression of either protein resulted in a marked inhibition of splicing, with the downstream small t 5' splice site more sensitive than the upstream large T 5' splice site. This is essentially the opposite of what has been observed when the concentration of these proteins is increased in vitro. The RS domain was necessary but not sufficient for this effect. Finally, overexpression of SC35, but not ASF/SF2, resulted in substantial accumulation of the unspliced SV40 pre-mRNA, which was efficiently transported to the cytoplasm. This finding suggests that SC35 may play an unanticipated role in mRNA stability and/or transport.

title: RNA splicing specificity determined by the coordinated action of RNA recognition motifs in SR proteins.
abstract: Pre-mRNA splicing requires a large number of RNA-binding proteins that have one or more RNA-recognition motifs (RRMs). Among these is the SR protein family, whose members are essential for splicing and are able to commit pre-mRNAs to the splicing pathway with overlapping but distinct substrate specificity. Some SR proteins, such as SC35, contain an N-terminal RRM and a C-terminal arginine/serine-rich (RS) domain, whereas others, such as SF2/ASF, also contain a second, atypical RRM. Although both the RRMs and the RS domain of SR proteins are required for constitutive splicing, it is unclear which domain(s) defines their substrate specificity, and whether two RRMs in a given SR protein function independently or act coordinately. Using domain swaps between SC35 and SF2/ASF and a functional commitment assay, we demonstrate that individual domains are functional modules, RS domains are interchangeable, and substrate specificity is defined by the RRMs. The atypical RRM of SF2/ASF does not appear to function alone in splicing, but can either activate or suppress the splicing specificity of an N-terminal RRM. Therefore, multiple RRMs in SR proteins act coordinately to achieve a unique spectrum of pre-mRNA substrate specificity.

title: Targeted disruption of an essential vertebrate gene: ASF/SF2 is required for cell viability.
abstract: Alternative splicing factor/splicing factor 2 (ASF/SF2) is the prototype of a family of nuclear proteins highly conserved throughout metazoa, the SR (serine/arginine) proteins. Based largely on in vitro studies, SR proteins have been suggested to play important roles in constitutive and alternative splicing of pre-mRNAs. Here we describe the development of a genetic system employing the chicken B-cell line DT40 to study the function of ASF/SF2 in vivo. The high level of homologous recombination and rapid growth rate of these cells allowed us to show first that ASF/SF2 is an essential gene, and then to perform targeted disruption of both ASF/SF2 alleles, by creating a cell line in which the only source of ASF/SF2 is a human cDNA controlled by a tetracycline (tet)-repressible promoter. We show that addition of tet to these cells results in rapid depletion of ASF/SF2, concomitant accumulation of incompletely processed pre-mRNA, and subsequent cell death. The tet-induced lethality could be rescued by plasmids expressing wild-type ASF/SF2, but not several mutant derivatives, or other SR proteins. Heterozygous cell lines overexpressing human ASF/SF2 displayed significant reductions of endogenous ASF/SF2 mRNA, suggesting that ASF/SF2 mRNA levels are controlled by an autoregulatory loop. This system provides a novel method for genetic analysis of factors that function in basic processes in vertebrate cells.

title: The SR protein B52/SRp55 is essential for Drosophila development.
abstract: B52, also called SRp55, is a 52-kDa member of the Drosophila SR protein family of general splicing factors. Escherichia coli-produced B52 is capable of both activating splicing and affecting the alternative splice site choice in human in vitro splicing reactions. Here we report the isolation of a B52 null mutant generated by remobilizing a P element residing near the B52 gene. The resulting deletion, B52(28), is confined to the B52 gene and its neighbor the Hrb87F gene. Second-instar larvae homozygous for the deletion are deficient in both B52 mRNA and protein. The B52 null mutant is lethal at the first- and second-instar larval stages. Germ line transformation of Drosophila flies with B52 genomic DNA rescues this lethality. Thus, B52 is an essential gene and has a critical role in Drosophila development. Larvae deficient in B52 are still capable of splicing the five endogenous pre-mRNAs tested here, including both constitutively and alternatively spliced genes. Therefore, B52 is not required for all splicing in vivo. This is the first in vivo deficiency analysis of a member of the SR protein family.

title: Blastocyst formation is blocked in mouse embryos lacking the splicing factor SRp20
abstract: SRp20 is a splicing factor belonging to the highly conserved family of SR proteins [1]   , which have multiple roles in the regulation of constitutive and alternative splicing in vivo. It has been suggested that SR proteins are involved in bringing together the splice sites during spliceosome assembly . SR proteins show partial redundancy, as each single SR protein can restore splicing activity to a splicing-deficient cytoplasmic extract (termed S-100 extract). Nevertheless, several studies demonstrate that individual SR proteins have different effects on the selection of specific alternative splice sites, and they recognize distinct RNA sequences       . Also, inactivation of two SR proteins, B52/SRp55 in Drosophila [13] or ASF/SF2 in the chicken cell line DT40 [14], is lethal, indicating the existence of nonredundant functions. Here, using Cre-loxP-mediated recombination in mice to inactivate the SRp20 gene, we found that it is essential for mouse development. Mutant preimplantation embryos failed to form blastocysts and died at the morula stage. Immunofluorescent staining showed that SRp20 is present in oocytes and early stages of embryonic development. This is the first report of mice deficient for a member of the SR protein family. Our experiments confirm that, although similar in structure, the SR proteins are not functionally redundant.

title: Functional characterization of SR and SR‐related genes in Caenorhabditis elegans
abstract: The SR proteins constitute a family of nuclear phosphoproteins, which are required for constitutive splicing and also influence alternative splicing regulation. Initially, it was suggested that SR proteins were functionally redundant in constitutive splicing. However, differences have been observed in alternative splicing regulation, suggesting unique functions for individual SR proteins. Homology searches of the Caenorhabditis elegans genome identified seven genes encoding putative orthologues of the human factors SF2/ASF, SRp20, SC35, SRp40, SRp75 and p54, and also several SR‐related genes. To address the issue of functional redundancy, we used dsRNA interference (RNAi) to inhibit specific SR protein function during C.elegans development. RNAi with CeSF2/ASF caused late embryonic lethality, suggesting that this gene has an essential function during C.elegans development. RNAi with other SR genes resulted in no obvious phenotype, which is indicative of gene redundancy. Simultaneous interference of two or more SR proteins in certain combinations caused lethality or other developmental defects. RNAi with CeSRPK, an SR protein kinase, resulted in early embryonic lethality, suggesting an essential role for SR protein phosphorylation during development.

title: Primary structure of the human splicing factor asf reveals similarities with drosophila regulators
abstract: We described previously the purification of a human protein, called alternative splicing factor (ASF), that can switch utilization of alternative 5' splice sites in an SV40 early pre-mRNA. We now report the isolation of a cDNA, designated ASF-1, that encodes this protein. ASF-1 consists of 248 amino acid residues, including an 80 residue RNA-binding domain at its N-terminus and a 50 residue C-terminal region that is 80% serine plus arginine. ASF-1 produced in E. coli can activate splicing in vitro and switch 5' splice-site utilization, establishing that the recombinant protein is sufficient to supply these activities. Analysis of additional cDNAs revealed that ASF pre-mRNA can itself be alternatively spliced, surprisingly, by utilization of a shared 5' splice site and two closely spaced 3' splice sites. Use of the upstream site results in a second mRNA (ASF-2) in which translation of the downstream exon occurs extensively in an alternative reading frame distinct from ASF-1.

title: Functional expression of cloned human splicing factor SF2: homology to rna-binding proteins, U1 70K, and drosophila splicing regulators
abstract: SF2 is a protein factor essential for constitutive pre-mRNA splicing in HeLa cell extracts and also activates proximal alternative 5' splice sites in a concentration-dependent manner. This latter property suggests a role for SF2 in preventing exon skipping, ensuring the accuracy of splicing, and regulating alternative splicing. Human SF2 cDNAs have been isolated and overexpressed in bacteria. Recombinant SF2 is active in splicing and stimulates proximal 5' splice sites. SF2 has a C-terminal region rich in arginine-serine dipeptides, similar to the RS domains of the U1 snRNP 70K polypeptide and the Drosophila alternative splicing regulators transformer, transformer-2, and suppressor-of-white-apricot. Like transformer-2 and 70K, SF2 contains an RNP-type RNA recognition motif.

title: Functional analysis of pre‐mRNA splicing factor SF2/ASF structural domains.
abstract: 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.

title: Functional domains of the human splicing factor ASF/SF2.
abstract: 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.

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

title: The Drosophila SR protein RBP1 contributes to the regulation of doublesex alternative splicing by recognizing RBP1 RNA target sequences.
abstract: The SR proteins represent a family of splicing factors several of which have been implicated in the regulation of sex‐specific alternative splicing of doublesex (dsx) pre‐mRNA in Drosophila. The dsx gene is involved in Drosophila sex determination. We have identified two RNA target sequence motifs recognized by the SR protein RBP1 from Drosophila using an in vitro selection approach. Several copies of these RBP1 target sequences were found within two regions of the dsx pre‐mRNA which are important for the regulation of dsx alternative splicing, the repeat region and the purine‐rich polypyrimidine tract of the regulated female‐specific 3′ splice site. We show that RBP1 target sequences within the dsx repeat region are required for the efficient splicing of dsx pre‐mRNA. Moreover, our studies reveal that RBP1 contributes to the activation of female‐specific dsx splicing in vivo by recognizing the RBP1 target sequences within the purine‐rich polypyrimidine tract of the female‐specific 3′ splice site.

title: Sequence-specific RNA binding by an SR protein requires RS domain phosphorylation: creation of an SRp40-specific splicing enhancer.
abstract: We showed previously that ASF/SF2, a member of the SR protein family of splicing factors, can activate a splicing enhancer element composed of high-affinity ASF/SF2 binding sites. To determine whether other SR proteins can behave similarly, we selected a high-affinity RNA-binding site (B1) for the SR protein SRp40. Strikingly, the success of this selection was completely dependent on phosphorylation of the RS domain, as unphosphorylated SRp40 failed to select specific sequences. We show that three copies of B1 function as a strong splicing enhancer, activating an intron with suboptimal splicing signals in nuclear extracts. Enhancer activity in S100 extracts (which lack SR proteins) required SRp40 and a nuclear fraction previously found to be required for ASF/SF2-dependent splicing. Importantly, enhancer activity was lost when SRp40 was replaced by ASF/SF2 or SC35, and SRp40 was the only classical SR protein found to be associated with the enhancer. Together, our results indicate that phosphorylation-dependent, sequence-specific RNA binding can impart unique activities to individual SR proteins.

title: A specific RNA hairpin loop structure binds the RNA recognition motifs of the Drosophila SR protein B52
abstract: B52, also known as SRp55, is a member of the Drosophila melanogaster SR protein family, a group of nuclear proteins that are both essential splicing factors and specific splicing regulators. Like most SR proteins, B52 contains two RNA recognition motifs in the N terminus and a C-terminal domain rich in serine-arginine dipeptide repeats. Since B52 is an essential protein and is expected to play a role in splicing a subset of Drosophila pre-mRNAs, its function is likely to be mediated by specific interactions with RNA. To investigate the RNA-binding specificity of B52, we isolated B52-binding RNAs by selection and amplification from a pool of random RNA sequences by using full-length B52 protein as the target. These RNAs contained a conserved consensus motif that constitutes the core of a secondary structural element predicted by energy minimization. Deletion and substitution mutations defined the B52-binding site on these RNAs as a hairpin loop structure covering about 20 nucleotides, which was confirmed by structure-specific enzymatic probing. Finally, we demonstrated that both RNA recognition motifs of B52 are required for RNA binding, while the RS domain is not involved in this interaction.

title: Identification of functional exonic splicing enhancer motifs recognized by individual SR proteins.
abstract: Using an in vitro randomization and functional selection procedure, we have identified three novel classes of exonic splicing enhancers (ESEs) recognized by human SF2/ASF, SRp40, and SRp55, respectively. These ESEs are functional in splicing and are highly specific. For SF2/ASF and SRp55, in most cases, only the cognate SR protein can efficiently recognize an ESE and activate splicing. In contrast, the SRp40-selected ESEs can function with either SRp40 or SRp55, but not with SF2/ASF. UV cross-linking/competition and immunoprecipitation experiments showed that SR proteins recognize their cognate ESEs in nuclear extract by direct and specific binding. A motif search algorithm was used to derive consensus sequences for ESEs recognized by these SR proteins. Each SR protein yielded a distinct 5- to 7-nucleotide degenerate consensus. These three consensus sequences occur at higher frequencies in exons than in introns and may thus help define exon-intron boundaries. They occur in clusters within regions corresponding to naturally occurring, mapped ESEs. We conclude that a remarkably diverse set of sequences can function as ESEs. The degeneracy of these motifs is consistent with the fact that exonic enhancers evolved within extremely diverse protein coding sequences and are recognized by a small number of SR proteins that bind RNA with limited sequence specificity.

title: The splicing factors 9G8 and SRp20 transactivate splicing through different and specific enhancers.
abstract: 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.

title: Exonic splicing enhancers: mechanism of action, diversity and role in human genetic diseases.
abstract: Exonic splicing enhancers (ESEs) are discrete sequences within exons that promote both constitutive and regulated splicing. The precise mechanism by which ESEs facilitate the assembly of splicing complexes has been controversial. However, recent studies have provided insights into this question and have led to a new model for ESE function. Other recent work has suggested that ESEs are comprised of diverse sequences and occur frequently within exons. Ominously, these latter studies predict that many human genetic diseases linked to mutations within exons might be caused by the inactivation of ESEs.

title: Inhibition by SR proteins of splicing of a regulated adenovirus pre-mRNA
abstract: THE adenovirus L1 unit1 represents an example of an alternatively spliced precursor messenger (pre-mRNA) where one 5' splice can be joined to one of two alternative 3' splice sites, producing the 52,55K or the IIIa mRNAs . Efficient usage of the distal IIIa 3' splice site requires late viral protein synthesis and is therefore confined to the late phase of virus infection2–4. Here we show that, in extracts from uninfected cells, the classical SR proteins5, which are essential splicing factors5–7, inhibit IIIa pre-mRNA splicing by binding to an intronic repressor element and preventing recruitment of the U2 small nuclear ribonucleoprotein particle to the spliceosome. We further show that the viral repressor element has splicing-enhancer activity when appropriately placed in the pre-mRNA. Together, our results demonstrate that SR proteins function as activators or repressors of splicing depending on where on the pre-mRNA they bind.

title: Specific interactions between proteins implicated in splice site selection and regulated alternative splicing
abstract: Specific recognition and pairing of the 5' and 3' splice sites are critical steps in pre-mRNA splicing. We report that the splicing factors SC35 and SF2/ASF specifically interact with both the integral U1 small nuclear ribonucleoprotein (snRNP U1-70K) and with the 35 kd subunit of the splicing factor U2AF (U2AF35). Previous studies indicated that the U1 snRNP binds specifically to the 5' splice site, while U2AF35-U2AF65 heterodimer binds to the 3' splice site. Together, these observations suggest that SC35 and other members of the SR family of splicing factors may function in splice site selection by acting as a bridge between components bound to the 5' and 3' splice sites. Interestingly, SC35, SF2/ASF, and U2AF35 also interact with the Drosophila splicing regulators Transformer (Tra) and Transformer-2 (Tra2), suggesting that protein-protein interactions mediated by SR proteins may also play an important role in regulating alternative splicing.

title: Arginine/serine-rich domains of the su(wa) and tra RNA processing regulators target proteins to a subnuclear compartment implicated in splicing
abstract: Two unrelated pre-mRNA splicing regulators-suppressor-of-white-apricot (su(wa)) and transformer (tra)-contain distinctive, approximately 120 amino acid arginine/serine (RS)-rich domains. Deletion of the su(wa) RS domain eliminates function. Replacement with the tra RS domain restores su(wa) function to nearly wild-type levels. Replacement with a 10 amino acid simple nuclear entry signal allows partial, inefficient function. Thus, the su(wa) RS domain apparently serves a generic function(s) subsuming nuclear entry. Moreover, immunocytochemical studies demonstrate that both RS domains specifically direct localization of a fused reporter protein to a punctate subnuclear compartment shown previously to be enriched in several constitutive splicing components. We propose that RS domains are a new class of targeting signals directing concentration of proteins in a subnuclear compartment implicated in splicing metabolism.

title: An amino acid sequence motif sufficient for subnuclear localization of an arginine/serine-rich splicing factor.
abstract: We have identified an amino acid sequence in the Drosophila Transformer (Tra) protein that is capable of directing a heterologous protein to nuclear speckles, regions of the nucleus previously shown to contain high concentrations of spliceosomal small nuclear RNAs and splicing factors. This sequence contains a nucleoplasmin-like bipartite nuclear localization signal (NLS) and a repeating arginine/serine (RS) dipeptide sequence adjacent to a short stretch of basic amino acids. Sequence comparisons from a number of other splicing factors that colocalize to nuclear speckles reveal the presence of one or more copies of this motif. We propose a two-step subnuclear localization mechanism for splicing factors. The first step is transport across the nuclear envelope via the nucleoplasmin-like NLS, while the second step is association with components in the speckled domain via the RS dipeptide sequence.

title: Role of the Modular Domains of SR Proteins in Subnuclear Localization and Alternative Splicing Specificity
abstract: SR proteins are required for constitutive pre-mRNA splicing and also regulate alternative splice site selection in a concentration-dependent manner. They have a modular structure that consists of one or two RNA-recognition motifs (RRMs) and a COOH-terminal arginine/serine-rich domain (RS domain). We have analyzed the role of the individual domains of these closely related proteins in cellular distribution, subnuclear localization, and regulation of alternative splicing in vivo. We observed striking differences in the localization signals present in several human SR proteins. In contrast to earlier studies of RS domains in the Drosophila suppressor-of-white-apricot (SWAP) and Transformer (Tra) alternative splicing factors, we found that the RS domain of SF2/ASF is neither necessary nor sufficient for targeting to the nuclear speckles. Although this RS domain is a nuclear localization signal, subnuclear targeting to the speckles requires at least two of the three constituent domains of SF2/ASF, which contain additive and redundant signals. In contrast, in two SR proteins that have a single RRM (SC35 and SRp20), the RS domain is both necessary and sufficient as a targeting signal to the speckles. We also show that RRM2 of SF2/ASF plays an important role in alternative splicing specificity: deletion of this domain results in a protein that, although active in alternative splicing, has altered specificity in 5′ splice site selection. These results demonstrate the modularity of SR proteins and the importance of individual domains for their cellular localization and alternative splicing function in vivo.

title: Arginine/serine-rich domains of SR proteins can function as activators of pre-mRNA splicing.
abstract: Serine/arginine (SR)-rich splicing factors contain an RNA binding domain and an arginine/serine (RS)-rich domain required for protein-protein interactions. In addition to their roles in the basic splicing reaction, SR proteins function as components of splicing enhancer complexes. Here, we investigate the role of RS domains in splicing enhancer function. Hybrid proteins containing RS domains fused to the MS2 RNA binding protein were tested in vitro with RNA substrates bearing an MS2 recognition sequence. These hybrid proteins activated splicing in nuclear extracts, but not in S100 extracts lacking SR proteins. However, intact recombinant SR proteins could complement the activity of the hybrid proteins in S100 extracts. These data demonstrate that RS domains function as splicing activators and suggest that the general and enhancer-dependent functions of SR proteins can be uncoupled.

title: The HCF repeat is an unusual proteolytic cleavage signal.
abstract: The herpes simplex virus VP16-associated protein HCF is a nuclear host-cell factor that exists as a family of polypeptides encoded by a single gene. The mature HCF polypeptides are amino- and carboxy-terminal fragments of a large approximately 300-kD precursor protein that arise through cleavage at one or more centrally located sites. The sites of cleavage are the HCF repeats, highly conserved 26-amino-acid sequences repeated six times in the HCF precursor protein. The HCF repeat alone is sufficient to induce cleavage of a heterologous protein, and cleavage occurs at a defined site--PPCE/THET--within the HCF repeat. Alanine-scan mutagenesis was used to identify a large 18-amino-acid segment of the HCF repeat that is important to induce cleavage of a heterologous protein. Even though HCF is cleaved, the majority of amino- and carboxy-terminal cleavage products remain tightly, albeit noncovalently, associated. Modulation of this noncovalent association may provide a mechanism for regulating HCF activity. For example, the cleaved products of an alternative mRNA splicing variant of HCF do not remain associated.

title: Regulation of alternative splicing in vivo by overexpression of antagonistic splicing factors.
abstract: The opposing effects of SF2/ASF and heterogeneous nuclear ribonucleoprotein (hnRNP) A1 influence alternative splicing in vitro. SF2/ASF or hnRNP A1 complementary DNAs were transiently overexpressed in HeLa cells, and the effect on alternative splicing of several cotransfected reporter genes was measured. Increased expression of SF2/ASF activated proximal 5' splice sites, promoted inclusion of a neuron-specific exon, and prevented abnormal exon skipping. Increased expression of hnRNP A1 activated distal 5' splice sites. Therefore, variations in the intracellular levels of antagonistic splicing factors influence different modes of alternative splicing in vivo and may be a natural mechanism for tissue-specific or developmental regulation of gene expression.

title: The Mkk3/6-p38–Signaling Cascade Alters the Subcellular Distribution of Hnrnp A1 and Modulates Alternative Splicing Regulation
abstract: Individual members of the serine-arginine (SR) and heterogeneous nuclear ribonucleoprotein (hnRNP) A/B families of proteins have antagonistic effects in regulating alternative splicing. Although hnRNP A1 accumulates predominantly in the nucleus, it shuttles continuously between the nucleus and the cytoplasm. Some but not all SR proteins also undergo nucleo-cytoplasmic shuttling, which is affected by phosphorylation of their serine/arginine (RS)–rich domain. The signaling mechanisms that control the subcellular localization of these proteins are unknown. We show that exposure of NIH-3T3 and SV-40 transformed green monkey kidney (COS) cells to stress stimuli such as osmotic shock or UVC irradiation, but not to mitogenic activators such as PDGF or EGF, results in a marked cytoplasmic accumulation of hnRNP A1, concomitant with an increase in its phosphorylation. These effects are mediated by the MKK3/6-p38 pathway, and moreover, p38 activation is necessary and sufficient for the induction of hnRNP A1 cytoplasmic accumulation. The stress-induced increase in the cytoplasmic levels of hnRNP A/B proteins and the concomitant decrease in their nuclear abundance are paralleled by changes in the alternative splicing pattern of an adenovirus E1A pre-mRNA splicing reporter. These results suggest the intriguing possibility that signaling mechanisms regulate pre-mRNA splicing in vivo by influencing the subcellular distribution of splicing factors.

title: Adenovirus E1A coding sequences that enable ras and pmt oncogenes to transform cultured primary cells.
abstract: Plasmids expressing partial adenovirus early region 1A (E1A) coding sequences were tested for activities which facilitate in vitro establishment (immortalization) of primary baby rat kidney cells and which enable the T24 Harvey ras-related oncogene and the polyomavirus middle T antigen (pmt) gene to transform primary baby rat kidney cells. E1A cDNAs expressing the 289- and 243-amino acid proteins expressed both E1A transforming functions. Mutant hrA, which encodes a 140-amino acid protein derived from the amino-terminal domain shared by the 289- and 243-amino acid proteins, enabled ras (but not pmt) to transform and facilitated in vitro establishment to a low, but detectable, extent. These studies suggest that E1A functions which collaborate with ras oncogenes and those which facilitate establishment are linked. Furthermore, E1A transforming functions are not associated with activities of the 289-amino acid E1A proteins required for efficient transcriptional activation of viral early region promoters.

title: Expression of a human alpha‐globin/fibronectin gene hybrid generates two mRNAs by alternative splicing.
abstract: We have isolated genomic clones for human fibronectin (FN), by screening a human gene library with previously isolated FN cDNA clones. We have recently reported two different FN mRNAs, one of them containing an additional 270 nucleotide insert coding for a structural domain ED. Restriction mapping and DNA sequencing of the genomic clones show that the ED type III unit corresponds to exactly one exon in the gene, whilst the two flanking type III units are split in two exons at variable positions. When an alpha‐globin/FN gene hybrid construct, containing the ED exon, flanking introns and neighbouring FN exons, is transfected into HeLa cells, two hybrid mRNAs differing by the ED exon are synthesized. These experiments confirmed that the two FN mRNAs observed in vivo arise from the same gene by alternative splicing.

title: A novel bipartite splicing enhancer modulates the differential processing of the human fibronectin EDA exon.
abstract: EDA is a facultative type III homology of human fibronectin encoded by an alternative spliced exon. The EDA+ and EDA- mRNA forms show a cell type specific distribution with their relative proportion varying during development, aging and oncogenic transformation. We have previously demonstrated that an 81 bp nucleotide sequence within the exon itself is essential for differential RNA processing. Fine mapping of cis acting elements within this region has been carried out to identify possible target sites for the modulation of alternative splicing. There are at least two short nucleotide sequences involved. Element A (GAAGAAGA) is a positive modulator for the recognition of the exon, its deletion results in constitutive exclusion of the EDA exon. Element B (CAAGG) is a negative modulator for exon recognition, its deletion results in constitutive inclusion of the EDA exon. This bipartite structure of the splicing enhancer is a novel feature of the mammalian exons.

title: Functional association between promoter structure and transcript alternative splicing.
abstract: It has been assumed that constitutive and regulated splicing of RNA polymerase II transcripts depends exclusively on signals present in the RNA molecule. Here we show that changes in promoter structure strongly affect splice site selection. We investigated the splicing of the ED I exon, which encodes a facultative type III repeat of fibronectin, whose inclusion is regulated during development and in proliferative processes. We used an alternative splicing assay combined with promoter swapping to demonstrate that the extent of ED I splicing is dependent on the promoter structure from which the transcript originated and that this regulation is independent of the promoter strength. Thus, these results provide the first evidence for coupling between alternative splicing and promoter-specific transcription, which agrees with recent cytological and biochemical evidence of coordination between splicing and transcription.

title: Human fibronectin: molecular cloning evidence for two mRNA species differing by an internal segment coding for a structural domain.
abstract: Two different fibronectin (FN) mRNA species were detected in the human cell line Hs578T. One species, mRNA I, contains an additional 270 nucleotide long insert (ED) that encodes exactly one of the internally repeated structural domains of the protein. The 90 amino acid extra domain belongs to the so‐called type III homology and it is located in the carboxy‐terminal half of FN, in between the cell attachment and the heparin binding sites of the protein. The evidence of two mRNAs is provided by the isolation and characterisation of four independent cDNA clones from a library prepared with a synthetic oligonucleotide primer, and it was confirmed by S1 nuclease analysis of cDNA/mRNA hybrids. This kind of analysis also showed that in the human cell line, mRNA I is present at a lower level than mRNA II (the mRNA species without the ED), whilst in human liver, mRNA I is virtually undetectable. Since liver tissue has recently been reported to be the source of plasma FN, our results indicate that the presence of the ED insert could be a particular feature of cellular FN.

title: A splicing enhancer in the human fibronectin alternate ED1 exon interacts with SR proteins and stimulates U2 snRNP binding.
abstract: The inclusion of the 270-nucleotide human fibronectin ED1 exon in HeLa cells requires the presence of a centrally located 81-nucleotide exon sequence. We have conducted a series of in vitro experiments aimed at understanding the structural and functional features associated with this splicing enhancer (SE). Using hybrid model pre-mRNA substrates, we show that the SE element markedly stimulates the use of the 3' splice site of ED1. Deletion and replacement analysis identifies the stimulating sequences as a purine-rich stretch of 9 nucleotides (GAAGAAGAC). The SE element stimulates splicing to the ED1 3' splice site from various positions within the exon except when placed beyond 293 nucleotides downstream from that 3' splice site. The action of the enhancer is not limited to the ED1 acceptor site because the SE element stimulates human beta-globin splicing and also induces the use of a 3' splice site in a prokaryotic sequence in vitro. We have explored the mechanism of action of the fibronectin splicing enhancer and found that the SE element is required for efficient assembly of early splicing complexes, allowing a more efficient interaction of the U2 snRNP with branch site sequences. In competition experiments, an RNA containing mainly SE sequences specifically abolished the action of the SE element, suggesting that factors bind the enhancer element to mediate stimulation of splicing. Using RNA mobility shift assays we show that SR proteins interact specifically with the SE element. Our results demonstrate that exon sequences lying in the SE element play a crucial role in specifying splice site recognition through interactions with factors binding to the 3' splice site.

title: A role for exon sequences in alternative splicing of the human fibronectin gene.
abstract: Exon EDIIIA of the fibronectin (Fn) gene is alternatively spliced via pathways which either skip or include the whole exon in the messenger RNA (mRNA). We have investigated the role of EDIIIA exon sequences in the human Fn gene in determining alternative splicing of this exon during transient expression of alpha globin/Fn minigene hybrids in HeLa cells. We demonstrate that a DNA sequence of 81bp within the central region of exon EDIIIA is required for alternative splicing during processing of the primary transcript to generate both EDIIIA+ and EDIIIA- mRNA's. Furthermore, alternative splicing of EDIIIA only occurs when this sequence is present in the correct orientation since when it is in antisense orientation splicing always occurs via exon-skipping generating EDIIIA- mRNA.

title: Identification of a Bidirectional Splicing Enhancer: Differential Involvement of SR Proteins in 5′ or 3′ Splice Site Activation
abstract: The adenovirus E1A pre-mRNA undergoes alternative splicing whose modulation occurs during infection, through the use of three different 5′ splice sites and of one major or one minor 3′ splice site. Although this pre-mRNA has been extensively used as a model to compare the transactivation properties of SR proteins, no cis-acting element has been identified in the transcript sequence. Here we describe the identification and the characterization of a purine-rich splicing enhancer, located just upstream of the 12S 5′ splice site, which is formed from two contiguous 9-nucleotide (nt) purine motifs (Pu1 and Pu2). We demonstrate that this sequence is a bidirectional splicing enhancer (BSE) in vivo and in vitro, because it activates both the downstream 12S 5′ splice site through the Pu1 motif and the upstream 216-nt intervening sequence (IVS) 3′ splice site through both motifs. UV cross-linking and immunoprecipitation experiments indicate that the BSE interacts with several SR proteins specifically, among them 9G8 and ASF/SF2, which bind preferentially to the Pu1 and Pu2 motifs, respectively. Interestingly, we show by in vitro complementation assays that SR proteins have distinct transactivatory properties. In particular, 9G8, but not ASF/SF2 or SC35, is able to strongly activate the recognition of the 12S 5′ splice site in a BSE-dependent manner in wild-type E1A or in a heterologous context, whereas ASF/SF2 or SC35, but not 9G8, activates the upstream 216-nt IVS splicing. Thus, our results identify a novel exonic BSE and the SR proteins which are involved in its differential activity.

title: U1 snRNP-ASF/SF2 interaction and 5' splice site recognition: characterization of required elements.
abstract: Members of the SR family of proteins, can collaborate with U1 snRNP in the recognition of 5' splice sites in pre-messenger RNAs. We have previously shown that purified U1 snRNP and ASF/SF2 form a ternary complex with pre-mRNA, which is dependent on a functional 5' splice site. In this manuscript we dissect the requirements for the formation of this complex. Sequences in the pre-mRNA, domains in ASF/SF2 and components of the U1 snRNP particle are shown to be required for complex formation. We had shown that sequences at the 5' splice site of PIP7. A are necessary and now we show these are sufficient for complex formation. Furthermore, we show that one functional RNA binding domain and the RS domain are both required for ASF/SF2 to participate in complex formation. The RNA binding domains were redundant in this assay, suggesting that either domain can interact with the pre-messenger RNA. Finally, our experiments show no function for the U1-specific A protein in complex formation, whereas a function for U1-specific C protein was strongly suggested. The study of the earliest interactions between pre-mRNA and splicing factors suggests a model for 5' splice site recognition.

title: Pathways for selection of 5′ splice sites by U1 snRNPs and SF2/ASF.
abstract: We have used protection against ribonuclease H to investigate the mechanisms by which U1 small nuclear ribonucleoprotein particles (snRNPs) determine the use of two alternative 5′ splice sites. The initial binding of U1 snRNPs to alternative consensus splice sites was indiscriminate, and on a high proportion of pre‐mRNA molecules both sites were occupied simultaneously. When the sites were close, this inhibited splicing. We propose that double occupancy leads to the use of the downstream site for splicing and that this is the cause of the proximity effect seen with strong alternative splice sites. This model predicts that splicing to an upstream site of any strength requires a low affinity of U1 snRNPs for the downstream site. This prediction was tested both by cleaving the 5′ end of U1 snRNA and by altering the sequence of the downstream site of an adenovirus E1A gene. The enhancement of downstream 5′ splice site use by splicing factor SF2/ASF appears to be mediated by an increase in the strength of U1 snRNP binding to all sites indiscriminately.

title: Selection of Alternative 5′ Splice Sites: Role of U1 snRNP and Models for the Antagonistic Effects of SF2/ASF and hnRNP A1
abstract: The first component known to recognize and discriminate among potential 5′ splice sites (5′SSs) in pre-mRNA is the U1 snRNP. However, the relative levels of U1 snRNP binding to alternative 5′SSs do not necessarily determine the splicing outcome. Strikingly, SF2/ASF, one of the essential SR protein-splicing factors, causes a dose-dependent shift in splicing to a downstream (intron-proximal) site, and yet it increases U1 snRNP binding at upstream and downstream sites simultaneously. We show here that hnRNP A1, which shifts splicing towards an upstream 5′SS, causes reduced U1 snRNP binding at both sites. Nonetheless, the importance of U1 snRNP binding is shown by proportionality between the level of U1 snRNP binding to the downstream site and its use in splicing. With purified components, hnRNP A1 reduces U1 snRNP binding to 5′SSs by binding cooperatively and indiscriminately to the pre-mRNA. Mutations in hnRNP A1 and SF2/ASF show that the opposite effects of the proteins on 5′SS choice are correlated with their effects on U1 snRNP binding. Cross-linking experiments show that SF2/ASF and hnRNP A1 compete to bind pre-mRNA, and we conclude that this competition is the basis of their functional antagonism; SF2/ASF enhances U1 snRNP binding at all 5′SSs, the rise in simultaneous occupancy causing a shift in splicing towards the downstream site, whereas hnRNP A1 interferes with U1 snRNP binding such that 5′SS occupancy is lower and the affinities of U1 snRNP for the individual sites determine the site of splicing.

title: Direct interaction of the U1 snRNP-A protein with the upstream efficiency element of the SV40 late polyadenylation signal.
abstract: An integral component of the splicing machinery, the U1 snRNP, is here implicated in the efficient polyadenylation of SV40 late mRNAs. This occurs as a result of an interaction between U1 snRNP-A protein and the upstream efficiency element (USE) of the polyadenylation signal. UV cross-linking and immunoprecipitation demonstrate that this interaction can occur while U1 snRNP-A protein is simultaneously bound to U1 RNA as part of the snRNP. The target RNA of the first RRM (RRM1) has been shown previously to be the second stem-loop of U1 RNA. We have found that a target for the second RRM (RRM2) is within the AUUUGURA motifs of the USE of the SV40 late polyadenylation signal. RNA substrates containing the wild-type USE efficiently bind to U1 snRNP-A protein, whereas substrates fail to bind when motifs of the USE were replaced by linker sequences. The addition of an oligoribonucleotide containing a USE motif to an in vitro polyadenylation reaction inhibits polyadenylation of a substrate representing the SV40 late polyadenylation signal, whereas a mutant oligoribonucleotide, a nonspecific oligoribonucleotide, and an oligoribonucleotide containing the U1 RNA-binding site had much reduced or no inhibitory effects. In addition, antibodies to bacterially produced, purified U1 snRNP-A protein specifically inhibit in vitro polyadenylation of the SV40 late substrate. These data suggest that the U1 snRNP-A protein performs an important role in polyadenylation through interaction with the USE. Because this interaction can occur when U1 snRNP-A protein is part of the U1 snRNP, our data provide evidence to support a link between the processes of splicing and polyadenylation, as suggested by the exon definition model.

title: Crystal structure of human UP1, the domain of hnRNP A1 that contains two RNA-recognition motifs.
abstract: Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is one of the most abundant core proteins of hnRNP complexes in metazoan nuclei. It behaves as a global regulator of alternative pre-mRNA splicing by antagonizing the activities of several serine/arginine-rich splicing factors (SR proteins), resulting in the activation of distal alternative 5' splice sites and skipping of optional exons. Purified hnRNP A1 has nucleic acid annealing activity. The protein also shuttles continuously between the nucleus and the cytoplasm, a process mediated by signals within its C-terminal glycine-rich domain. The N-terminal region of human hnRNP A1, termed unwinding protein 1 (UP1), contains two RNA-recognition motifs (RRMs), RRM1 and RRM2. Understanding the structural elements by which hnRNP A1 interacts with RNA will have broad implications for studies of RNA processing.



The crystal structure of UP1 has been determined to 1.9 A resolution. Each RRM independently adopts the characteristic RRM fold, consisting of a four-stranded antiparallel beta-pleated sheet and two alpha helices packed on one side of the beta sheet. The two RRMs are antiparallel and held in close contact, mainly by two Arg-Asp ion pairs. As a result, the two four-stranded beta sheets are brought together to form an extended RNA-binding surface. A segment of the linker connecting the two RRMs is flexible in the absence of bound RNA, but the general location of the linker suggests that it can make direct contacts with RNA. Comparison with other RRM structures indicates that a short 310 helix, found immediately N-terminal to the first beta strand in RRM1, may interact with RNA directly.



The RRM is one of the most common and best characterized RNA-binding motifs. In certain cases, one RRM is sufficient for sequence-specific and high affinity RNA binding; but in other cases, synergy between several RRMs within a single protein is required. This study shows how two RRMs are organized in a single polypeptide. The two independently folded RRMs in UP1 are held together in a fixed geometry, enabling the two RRMs to function as a single entity in binding RNA, and so explaining the synergy between the RRMs. The UP1 structure also suggests that residues which lie outside of the RRMs can make potentially important interactions with RNA.

title: Distinct functions of the closely related tandem RNA-recognition motifs of hnRNP A1.
abstract: hnRNP A1 regulates alternative splicing by antagonizing SR proteins. It consists of two closely related, tandem RNA-recognition motifs (RRMs), followed by a glycine-rich domain. Analysis of variant proteins with duplications, deletions, or swaps of the RRMs showed that although both RRMs are required for alternative splicing function, each RRM plays distinct roles, and their relative position is important. Surprisingly, RRM2 but not RRM1 could support this function when duplicated, despite their very similar structure. Specific RNA binding and annealing are not sufficient for hnRNP A1 alternative splicing function. These observations, together with phylogenetic and structural data, suggest that the two RRMs are quasi-symmetric but functionally nonequivalent modules that evolved as components of a single bipartite domain.

title: A mammalian germ cell-specific RNA-binding protein interacts with ubiquitously expressed proteins involved in splice site selection.
abstract: RNA-binding motif (RBM) genes are found on all mammalian Y chromosomes and are implicated in spermatogenesis. Within human germ cells, RBM protein shows a similar nuclear distribution to components of the pre-mRNA splicing machinery. To address the function of RBM, we have used protein-protein interaction assays to test for possible physical interactions between these proteins. We find that RBM protein directly interacts with members of the SR family of splicing factors and, in addition, strongly interacts with itself. We have mapped the protein domains responsible for mediating these interactions and expressed the mouse RBM interaction region as a bacterial fusion protein. This fusion protein can pull-down several functionally active SR protein species from cell extracts. Depletion and add-back experiments indicate that these SR proteins are the only splicing factors bound by RBM which are required for the splicing of a panel of pre-mRNAs. Our results suggest that RBM protein is an evolutionarily conserved mammalian splicing regulator which operates as a germ cell-specific cofactor for more ubiquitously expressed pre-mRNA splicing activators.

title: The role of specific protein-RNA and protein-protein interactions in positive and negative control of pre-mRNA splicing by Transformer 2

abstract: We have investigated the function of different structural domains of the Drosophila splicing regulator Transformer 2 (Tra2). We find that the ribonucleoprotein consensus sequence (RNP-CS) of Tra2 is required for male fertility and positive and negative control of alternative splicing in transgenic flies, as well as for in vitro binding of recombinant Tra2 to doublesex and tra2 pre-mRNAs. Thus, all of the known functions of Tra2 require specific protein-RNA interactions. We also show that one of the two arginine-serine (RS)-rich domains of Tra2 is dispensable, while the other is essential for all of the in vivo functions. Part of this domain is also required for RNA binding in vitro. Significantly, the essential RS domain is also required for specific protein-protein interactions. We find that Tra2 interacts with itself, with the splicing regulator Transformer, and with the general splicing factor SF2 in vitro and in the yeast two-hybrid system. These results demonstrate that both protein-RNA and protein-protein interactions are involved in tra2-dependent activation and repression of alternative splicing.

title: Analysis of the RNA-recognition motif and RS and RGG domains: conservation in metazoan pre-mRNA splicing factors.
abstract: We present a systematic analysis of sequence motifs found in metazoan protein factors involved in constitutive pre-mRNA splicing and in alternative splicing regulation. Using profile analysis we constructed a database enriched in protein sequences containing one or more presumptive copies of the RNA-recognition motif (RRM). We provide an accurate alignment of RRMs and structure-based criteria for identifying new RRMs, including many that lack the prototype RNP-1 submotif. We present a comprehensive table of 125 sequences containing 252 RRMs, including 22 previously unreported RRMs in 17 proteins. The presence of a putative RRM in these proteins, which are implicated in a variety of cellular processes, strongly suggests that their function involves binding to RNA. Unreported homologies in the RRM-enriched database to the metazoan SR family of splicing factors are described for an Arg-rich human nuclear protein and two yeast proteins (S. pombe mei2 and S. cerevisiae Npl3). We have rigorously tested the phylogenetic relationships of a large sample of RRMs. This analysis indicates that the RRM is an ancient conserved region (ACR) that has diversified by duplication of genes and intragenic domains. Statistical analyses and classification of repeated Arg-Ser (RS) and RGG domains in various protein splicing factors are presented.

title: Genetic analysis of the SR protein ASF/SF2: interchangeability of RS domains and negative control of splicing.
abstract: The SR proteins constitute a family of splicing factors, highly conserved in metazoans, that contain one or two amino-terminal RNA-binding domains (RBDs) and a region enriched in arginine/serine repeats (RS domain) at the carboxyl terminus. Previous studies have shown that SR proteins possess distinct RNA-binding specificities that likely contribute to their unique functions, but it is unclear whether RS domains have specific roles in vivo. Here, we used a genetic system developed in the chicken B cell line DT40 to address this question. Expression of chimeric proteins generated by fusion of the RS domains of heterologous SR proteins, or a human TRA-2 protein, with the RBDs of ASF/SF2 allowed cell growth following genetic inactivation of endogenous ASF/SF2, indicating that RS domains are interchangeable for all functions required to maintain cell viability. However, a chimera containing the RS domain from a related splicing factor, U2AF65, could not rescue viability and was inactive in in vitro splicing assays, suggesting that this domain performs a distinct function. We also used the DT40 system to show that depletion of ASF/SF2 affects splicing of specific transcripts in vivo. Although splicing of several simple constitutive introns was not significantly affected, the alternative splicing patterns of two model pre-mRNAs switched in a manner consistent with predictions from previous studies. Unexpectedly, ASF/SF2 depletion resulted in a substantial increase in splicing of an HIV-1 tat pre-mRNA substrate, indicating that ASF/SF2 can repress tat splicing in vivo. These results provide the first demonstration that an SR protein can influence splicing of specific pre-mRNAs in vivo.

title: Analysis of the functional specificity of RS domains in vivo
abstract: A number of splicing factors contain extensive regions that are rich in arginine and serine (RS domains). These domains are thought to facilitate protein–protein interactions that are critical in the regulation of alternative splicing. Using a domain swap strategy, we have tested the ability of RS domains from several proteins to substitute in vivo for an essential RS domain in the Drosophila splicing regulator TRA‐2. By several criteria, RS domains were found to vary significantly in their ability to support the splicing regulation functions of TRA‐2. The RS domain of dU2AF50 functioned efficiently, while that of the dSRp55 protein did not. Moreover, we find similar differences in the ability of RS domains to direct fusion proteins to discrete subnuclear sites at which TRA‐2 associates with spermatocyte chromosomes. These results indicate that RS domains are not all functionally equivalent in vivo.