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The SR protein family

The SR protein family Protein family review Peter J Shepard and Klemens J Hertel Address: Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697-4025, USA. Correspondence: Klemens J Hertel. Email: [email protected] More recent genome-wide studies have identified several Summary other RS-domain-containing proteins, most of which are The processing of pre-mRNAs is a fundamental step required conserved in higher eukaryotes and function in pre-mRNA for the expression of most metazoan genes. Members of the splicing or RNA metabolism [10]. Because of differences in family of serine/arginine (SR)-rich proteins are critical compo- domain structure, lack of mAb104 recognition, or lack of a nents of the machineries carrying out these essential processing events, highlighting their importance in maintaining efficient prototypical RRM, these proteins are referred to as SR-like gene expression. SR proteins are characterized by their ability or SR-related proteins. An extensive list of SR-related to interact simultaneously with RNA and other protein compo- proteins and their functional roles in RNA metabolism was nents via an RNA recognition motif (RRM) and through a recently discussed [11]. domain rich in arginine and serine residues, the RS domain. Their functional roles in gene expression are surprisingly diverse, ranging from their classical involvement in constitutive and While introns are common to all eukaryotes, the complex- alternative pre-mRNA splicing to various post-splicing activities, ity of alternative splicing varies among species. SR proteins including mRNA nuclear export, nonsense-mediated decay, and exist in all metazoan species [8] as well as in some lower mRNA translation. These activities point up the importance of SR eukaryotes, such as the fission yeast Schizosaccharomyces proteins during the regulation of mRNA metabolism. pombe [12,13]. However, classical SR proteins are not present in all eukaryotes and are apparently missing from Gene organization and evolutionary history the budding yeast Saccharomyces cerevisiae, which lacks The discovery of SR proteins goes back to studies in alternative splicing. Instead, three SR-like proteins have Drosophila where genetic screens identified SWAP been identified in S. cerevisiae, one of which has been (suppressor-of-white-apricot) [1], Tra (transformer) [2] shown to modulate the efficiency of pre-mRNA splicing and Tra-2 (transformer-2) [3] as splicing factors. Their [14]. In general, the species-specific presence of SR sequence characterization led to the identification of a proteins correlates with the presence of RS domains within protein domain rich in arginine and serine dipeptides, other components of the general splicing machinery. The termed the arginine/serine (RS) domain. Subsequent observation that the density of RS repeats correlates with identification of the splicing factors SF2/ASF and SC35 the conservation of the branch-point signal, a critical from human cell lines also revealed the presence of sequence element of the 3’ splice site, argues for an ancestral extended RS domains in addition to at least one RNA- origin of SR proteins [15]. As such, SR proteins appear to be binding domain of the RNA recognition motif (RRM)-type ancestral to eukaryotes and were subsequently lost [4-6]. The family of SR proteins was classified following independently in some lineages (Figure 2). Phylo genetic tree the identification of additional RS-domain-containing analyses further suggest that successive gene duplications proteins on the basis of the presence of a phosphoepitope played an important role in SR protein evolution [16]. These recognized by the monoclonal antibody mAb104 [7], their duplication events are coupled with high rates of conservation across vertebrates and invertebrates, and nonsynonymous substitutions that promoted positive their activity in splicing complementation assays [8]. In selection favoring the gain of new functions, supporting the humans, the SR protein family is encoded by nine genes, hypothesis that the expansion of RS repeats during designated splicing factor, arginine/serine-rich (SFRS) 1-7, evolution had a fundamental role in the relaxation of the 9, and 11 (Table 1). All nine members of the human SR splicing signals and in the evolution of regulated splicing. protein family - SF2/ASF, SC35, SRp20, SRp40, SRp55, SRp75, SRp30c, 9G8, and SRp54 - have a common Characteristic structural features structural organization (Figure 1), containing either one or All SR proteins share two main structural features: the RS two amino-terminal RNA-binding domains that provide domain and at least one RRM (Figure 1). For the majority RNA-binding specificity, and a variable-length RS domain of SR proteins with two RNA-binding domains, the second at their carboxyl terminus that functions as a protein is a poor match to the RRM consensus and is referred to as interaction domain [9]. an RRM homolog (RRMH). The only exception is 9G8, http://genomebiology.com/2009/10/10/242 Shepard and Hertel: Genome Biology 2009, 10:242 242.2 Table 1 SRp20 RRM RS Human genes encoding SR proteins SC35 RRM RS Gene name SR protein Chromosomal location UniProt SRp54 RRM RS SFRS1 SF2/ASF/SRp30a 17q21.3-q22 Q07955 SF2/ASF RRM RRMH RS SFRS2 SC35/SRp30b 17q25.1 Q01130 SRp30c RRM RRMH RS SFRS3 SRp20 6p21.31 P84103 SFRS4 SRp75 1p35.3 Q08170 SRp40 RRM RRMH RS SFRS5 SRp40 14q24.2 Q13243 SRp55 RRM RRMH RS SFRS6 SRp55 20q13.11 Q13247 SRp75 RRM RRMH RS SFRS7 9G8 2p22.1 Q16629 9G8 RRM Zn RS SFRS9 SRp30c 12q24.23 Q13242 SFRS11 SRp54 1p31.1 Q05519 Figure 1 The human SR protein family. The structural organization of the which contains an RRM and a zinc-knuckle domain that is nine human SR proteins is shown. RRM, RNA recognition motif; RRMH, RRM homology; RS, arginine/serine-rich domain; Zn, Zinc thought to contact the RNA [17]. In the cases where it has knuckle. been determined, SR proteins have specific, yet degenerate RNA-binding specificities [18,19]. The RS domains of SR proteins participate in protein interactions with a number of other RS-domain-containing splicing factors [20,21]. structural insights provided an explanation for the These include other SR proteins, SR-related proteins [22], seemingly low specificity of RNA binding exhibited by and components of the general splicing machinery [20,21, SRp20 [31,32]. 23-25]. Furthermore, the RS domain can function as a nuclear localization signal by mediating the interaction Localization and function with the SR protein nuclear import receptor, trans- Many proteins involved in pre-mRNA splicing, including portin-SR [26-28]. the SR proteins, are enriched in nuclear compartments termed speckles, which occur throughout the nucleus. Structural characterization of a complete SR protein has Speckles are of two distinct structural types [33]: inter- not yet been achieved. Consequently, only isolated RRMs chromatin granule clusters (IGCs) about 20-25 nm in of SR proteins have been analyzed structurally by nuclear diameter, which are storage/reassembly sites for pre- magnetic resonance spectroscopy. Unfortunately, no mRNA splicing factors; and perichromatin fibrils approxi- structural information detailing the RS domain is available mately 5 nm in diameter, which are sites of actively to date. This may be explained by the poor solubility of transcribing genes and co-transcriptional splicing [34]. these proteins in their free state and the unknown The SR proteins are a prominent component of nuclear phosphorylation state of the serines within the RS domain. speckles (Figure 4) [35,36], and biochemical analyses have In addition, the degenerate RNA-binding sequences indicated that RS domains are responsible for targeting the recognized by SR proteins may have prevented their study SR proteins to these structures [26,37]. The intranuclear in the bound form. To tackle the solubility issues, the organization of SR proteins is dynamic, and they are RRMs of SRp20 and 9G8 were fused to the immuno- recruited from the IGC storage clusters to the sites of globulin G-binding domain 1 of Streptococcal protein G co-transcriptional splicing, the perichromatin fibrils (GB1) solubility tag [29] or overexpressed RRMs were [38,39]. Interestingly, both the RNA-binding domains and suspended in a solution containing charged amino acids RS domains are required for recruitment of SR proteins [30]. Using these manipulations it was possible to obtain from the IGCs to the perichromatin fibrils, as is phos- solution structures of the free 9G8 and SRp20 RRMs and phory lation of the RS domain [40]. of the SRp20 RRM in complex with the RNA sequence 5’-CAUC-3’ (Figure 3). When examining the unbound Splicing activation RRMs of SRp20 and 9G8, one is struck by an unusually In classic cases of alternative splicing, it has been shown large exposed hydrophobic surface, which could explain that cis-acting RNA sequence elements, known as splicing why the solubility of SR proteins is so low. The SRp20 enhancers, increase exon inclusion by serving as sites for RRM complex with RNA shows that although all four recruitment of the splicing machinery - the spliceosome - nucleotides present are contacted by the RRM, only the which is a complex of ribonucleoprotein splicing factors, 5’ cytosine is recognized in a specific manner. These such as U1 and U2 small nuclear ribonucleoproteins http://genomebiology.com/2009/10/10/242 Shepard and Hertel: Genome Biology 2009, 10:242 242.3 (a) Human SR proteins (b) Main species tree 0.083 0.1335 0.13 Hs p54 Hs SF2-ASF Dm SRP54 0.165 Ce RSP7 0.19 Hs SC35 Hs SRp30c Dm SC35 Ce RSP4 0.05 0.17 Sp SRP Hs SRP75 Hs SRp20 0.04 Dm SFRS3 0.16 Hs 9G8 Hs SRP55 0.08 Dm xl6 0.04 Dm RBP 1 0.2 Ce RSP6 Hs SRP40 Hs SF2-ASF Hs SRp30c 0.2 Hs SRp20 Dm SF2 Ce RSP3 0.07 Sp SRP2 0.22 Hs 9G8 Hs SRP75 Hs SRP55 Hs SRP40 0.27 Hs SC35 Dm B52 Ce RSP1 Ce RSP2 0.42 Hs_p54 Ce RSP5 Dm CG4266 Sp RNPS1 At SC35 At SR33 At SCL28 At RSZ33 At RSZp22 At SR1 At SRP34a At SRp30 At RSP31 At RSP 40 At RSP40 Figure 2 Evolutionary relationship between members of the SR family. The phylogeny was inferred using the neighbor-joining method. ClustalW was used to align sequences and perform phylogenetic analysis. Trees were drawn by CTree. The horizontal lines in each panel indicate the similarity between SR proteins. (a) Phylogenetic tree based on the alignment of the human (Hs) SR protein family. The numbers above each bar indicate the degree of similarity. (b) Phylogenetic tree based on the alignment of Homo sapiens (Hs), Drosophila melanogaster (Dm), Caenorhabditis elegans (Ce), Arabidopsis thaliana (At), and Schizosaccharomyces pombe (Sp) SR protein sequences. Green and blue lines indicate different clusters. Cluster set selection is based on minimizing the subtype diversity ratio, a measure that groups related subclasses. http://genomebiology.com/2009/10/10/242 Shepard and Hertel: Genome Biology 2009, 10:242 242.4 [9,41,42]. SR proteins act at several steps during the splicing reaction [4,5,8,43-45] and require phosphorylation for efficient splice-site recognition and dephosphorylation for splicing catalysis [46,47]. A number of SR protein kinases have been identified that specifically phosphorylate serine residues within the RS domain of SR proteins. These include SR protein kinase 1 (SRPK1) [48], Clk/Sty kinase [49], cdc2p34 [50], and topoisomerase [51]. Surprisingly, binding sites for SR proteins are not only limited to alternatively spliced exons, but have also been verified for exons of constitutively spliced pre-mRNAs [52,53]. It is therefore likely that SR proteins bind to sequences found in most, if not all, exons. One model for the mechanism of splicing activation proposes that the RS domain of an enhancer-bound SR protein interacts directly with other splicing factors contain ing an RS domain, thus facilitating the recruitment of spliceosomal components such as the snRNP U1 to the 5’ splice site or U2AF65 (the large subunit of the splicing factor U2AF) to the 3’ splice site [9]. An alternative mode of spliceosomal recruitment was suggested by experiments showing that RS domains of SR proteins contact the pre- mRNA within the functional spliceosome [54,55]. Irres pec- tive of the RS domain activation mode, SR proteins facili- tate the recruitment of spliceosomal components to the regulated splice site [42,56] (Figure 5a). Thus, SR proteins bound to ESEs function as general activators of exon definition [57]. Kinetic analyses have shown that the relative activity of ESE-bound SR proteins determines the magnitude of splicing promotion. This activity depended on the number of SR proteins assembled on an ESE and the distance between the ESE and the intron. It was also shown that activation of splicing was proportional to the number of serine-arginine repeats within the RS domain of the bound SR protein. Thus, the quantity of serine-arginine repeats appears to dictate the activation potential of SR proteins [58]. In addition to their exon-dependent functions, SR proteins Figure 3 have activities that do not require interaction with exon sequences [59]. The role of the exon-independent function Solution structure of an SR protein RRM from human SRp20 (blue) in may be to promote the pairing of 5’ and 3’ splice sites complex with the RNA sequence 5’-CAUC-3’ (red). All four nucleotides present are contacted by the RRM, but only the 5’ cytosine is across the intron or to facilitate the incorporation of the recognized specifically. The structure was generated using the Visual tri-snRNP U4/U6•U5 into the spliceosome [44] (Figure 5b). Molecular Dynamics program [78] from coordinates deposited in the U4/U6•U5 is a complex of snRNPs that contains the Brookhaven National Laboratory Protein Data Bank [30]. splicing activity. Although the RRM of the SR protein is essential for its exon-independent activity [59], it is likely that SR proteins interact with the partially assembled (snRNPs), and their associated proteins, such as U2 spliceosome or the tri-snRNP through RS domain contacts. auxiliary factor (U2AF), that splices exons together and releases the intron RNA. Splicing enhancers are usually Splicing repression located within the regulated exon, and are thus known as One striking feature of SR proteins is their prevalent exonic splicing enhancers (ESEs) [41,42]. ESEs are usually location within the pre-mRNA. In nearly all cases SR recognized by at least one member of the SR protein family proteins have been found to interact with exonic sequences and recruit the splicing machinery to the adjacent intron of the pre-mRNA. This is a surprising finding considering http://genomebiology.com/2009/10/10/242 Shepard and Hertel: Genome Biology 2009, 10:242 242.5 10 µm 10 µm 10 µm GFP-SRp20 SC35 Merge Figure 4 Localization of SR proteins within the nucleus. Left panel: HeLa cells transfected with GFP-SRp20. The GFP fluorescence is visualized directly. Middle panel: cells are also stained with anti-SC35 hybridoma supernatant to highlight clusters of SR proteins in the nucleus (red), which are referred to as nuclear speckles. Speckles are believed to be storage compartments for SR proteins and other splicing factors. Right panel: merge of GFP-SRp20 and SC35 images. The bar in each panel indicates the scale. Images courtesy of Lin Li and Rozanne Sandri-Goldin. the fact that their relatively promiscuous binding mammalian cells and Xenopus oocytes [63] by binding to a specificity predicts that introns are littered with potential 22-nucleotide sequence within the H2A mRNA (Figure SR-protein-binding sites. The fact that SR proteins are 6a). In addition, the S. cerevisiae protein Npl3p, which is mainly observed to bind within exonic sequences suggests closely related to the SR proteins, assists in mRNA export that additional requirements need to be met for functional in yeast [64]. Once again, phosphorylation of specific SR protein binding to the pre-mRNA. There are, however, serine residues within the RS domain seems to control the some instances of SR proteins binding within the intron, efficiency of the mRNA-export function of Npl3p [65]. where they function as negative regulators of splicing. The Given the fact that SR proteins are essential for splicing best-characterized example occurs during adenovirus [9], remain associated with the spliced mRNA after intron infection [60]. In this case, splicing is repressed by the removal [66,67], and shuttle between the nucleus and the binding of the SR protein SF2/ASF to an intronic repressor cytoplasm [62], it seems highly likely that SR proteins also element located upstream of the 3’ splice site branchpoint play an important part in the export of spliced mRNAs. As sequence in the adenovirus pre-mRNA. When bound to the shown recently, 9G8 and SRp20 are involved in mediating repressor element, SF2/ASF prevents the recruitment of the efficient handover of mRNA to Tip-associated protein the snRNP U2 to the branchpoint sequence, thereby (TAP), which is an essential nuclear export factor [68]. in activat ing the 3’ splice site (Figure 5c). Other studies have provided further support for the idea that SR proteins SR protein involvement in translation bound to introns generally interfere with the productive SR proteins have been shown to influence translation assembly of spliceosomes [61]. These observations show either indirectly or directly. For example, the splicing that exonic splicing enhancers not only function in exon activity of SF2/ASF influences alternative splicing of the and splice-site recognition, but also act as barriers to pre-mRNA for the protein kinase MNK2, a kinase that prevent exon skipping. regulates translation initiation. High levels of SF2/ASF promote the production of an MNK2 mRNA isoform that Role of SR proteins in mRNA export enhances cap-dependent translation, whereas low levels Some SR proteins - SF2/ASF, SRp20, and 9G8 - shuttle achieve the opposite [69]. SF2/ASF is also involved in continuously between the nucleus and the cytoplasm [62]. regulating translation directly. It has been shown to The movement of these proteins requires the phosphory- associate with polyribosome fractions isolated from cyto- lation of specific residues in the RS domain and the RNA- plasmic extracts and to enhance the translation efficiency binding domain. These unique intracellular transport of an ESE-containing luciferase reporter [70], apparently properties suggest that a subset of SR proteins functions through mediating the recruitment of components of not only in pre-mRNA processing but also in mRNA export mTOR (mammalian target of rapamycin) signaling pathway [62]. In fact, the SR proteins 9G8 and SRp20 promote (Figure 6b). As a result of this recruitment, a competitive nuclear export of the intronless histone H2A mRNA in inhibitor of cap-dependent translation is released [71]. http://genomebiology.com/2009/10/10/242 Shepard and Hertel: Genome Biology 2009, 10:242 242.6 (a) (a) Nucleus 70K SR U1 Intronless mRNA Pre-mRNA protein snRNP U2AF U2AF65 5′ 3′ 5′ 3′ SR SR SR SR Py AG Exon ESE (b) SR SR SR SR U4/U6•U5 ALY/REF ALY/REF tri-snRNP TAP Export Export 100K machinery machinery 27K SR (b) 4EBP protein mTOR 70K SF2/ASF U1 4EBP m7G ESE AAAAAAAA snRNP U2AF U2 snRNP eIF4E eIF4E U2AF65 35 Exon 1 Py AG Exon 2 (c) U2AF Figure 6 U2AF65 35 SR protein functions other than splicing. (a) mRNA export. SR proteins associate site-specifically with intronless mRNAs, such as SR histone H2A mRNA [63], to promote their export (left-hand side). protein The export machinery is as yet unknown. For intron-containing pre- Py AG Exon mRNAs (right-hand side), SR protein association with the spliced mRNA has also been suggested to mediate nuclear export through interactions with the RNA export factor ALY/REF and Tip-containing protein (TAP). (b) Translation initiation. Interactions between Figure 5 mRNA-bound SF2/ASF and the protein kinase mTOR trigger phosphorylation of 4E-BP (eIF4E-binding protein). In its Splicing functions of SR proteins. (a) SR proteins (green) bound to phosphorylated form 4E-BP dissociates from the translation an exonic splicing enhancer (ESE) may function in constitutive initiation factor eIF4E, thereby releasing eIF4E and activating splicing by interacting with the splicing factors U2AF bound at the initiation of cap-dependent translation (green arrow) [71]. upstream 3’ splice site and U1 snRNP bound to the downstream 5’ splice site. Py represents the polypyrimidine tract, the binding site for U2AF. (b) Exon-independent functions of SR proteins. SR proteins may have two exon-independent functions. SR proteins facilitate splice-site pairing by simultaneously interacting with U1 occasional redundancies. Given the enormous functional snRNP and U2AF across the intron. SR proteins also assist in real estate this family of proteins covers, one is now recruiting the U4/U6•U5 tri-snRNP. (c) Splicing repression is pressed to find out how it is possible to transition these mediated when SR proteins associate with intronic sequences close to the splice sites. Recruitment of spliceosomal components is proteins between their involvements in the various steps of inhibited through steric hindrance or nonproductive spliceosomal mRNA processing. Clearly, reversible modification, such as assembly. Adapted with permission from [79]. serine phosphorylation within the RS domain, is likely to be the ticket for SR protein functional flexibility [51]. The challenge will be to determine the extent and dynamics of Importantly, other SR proteins have also been reported to such modifications within SR proteins specifically involved function in translation. SRp20 promotes translation of a in one of these activities and whether changes in viral RNA initiated at an internal ribosome entry site [72], modification lend support to the existence of an SR and 9G8 increases translation efficiency of unspliced protein-modification code, perhaps similar in principle to mRNA containing a constitutive transport element [73]. the now well-described histone-modification code [74]. Frontiers An old foe makes up another challenge: SR protein The functional characterization of SR proteins has revealed structure. For more than 15 years attempts have been made a wealth of information, placing SR proteins in the context to obtain high-resolution structures of SR proteins. So far, of regulating constitutive and alternative pre-mRNA these attempts have failed because of problems of low splicing, mediating efficient transport of mRNAs, and solubility and the likely heterogeneity of RS-domain modu lat ing mRNA translation. As such, SR proteins could modifications. As a first step towards gaining ground in easily be mistaken for ‘Jacks of all trades, masters of none’ this endeavor, clever modification approaches have been in mRNA metabolism. However, many studies have used to obtain a high-resolution structure of the SR protein demon strated their essential presence in the cell, even with RRM domain. This is a significant first step. However, the http://genomebiology.com/2009/10/10/242 Shepard and Hertel: Genome Biology 2009, 10:242 242.7 much more elusive RS domain is still the big prize, 4. 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Chasin LA: Searching for splicing motifs. Adv Exp Med Biol 2007, 623:85-106. © 2009 BioMed Central Ltd http://genomebiology.com/2009/10/10/242 Shepard and Hertel: Genome Biology 2009, 10:242 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Genome Biology Springer Journals

The SR protein family

Shepard, Peter; Hertel, Klemens
Genome Biology , Volume 10 (10) – Oct 27, 2009
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Life Sciences; Animal Genetics and Genomics; Human Genetics; Plant Genetics & Genomics; Microbial Genetics and Genomics; Bioinformatics; Evolutionary Biology
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19857271
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Abstract

Protein family review Peter J Shepard and Klemens J Hertel Address: Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697-4025, USA. Correspondence: Klemens J Hertel. Email: [email protected] More recent genome-wide studies have identified several Summary other RS-domain-containing proteins, most of which are The processing of pre-mRNAs is a fundamental step required conserved in higher eukaryotes and function in pre-mRNA for the expression of most metazoan genes. Members of the splicing or RNA metabolism [10]. Because of differences in family of serine/arginine (SR)-rich proteins are critical compo- domain structure, lack of mAb104 recognition, or lack of a nents of the machineries carrying out these essential processing events, highlighting their importance in maintaining efficient prototypical RRM, these proteins are referred to as SR-like gene expression. SR proteins are characterized by their ability or SR-related proteins. An extensive list of SR-related to interact simultaneously with RNA and other protein compo- proteins and their functional roles in RNA metabolism was nents via an RNA recognition motif (RRM) and through a recently discussed [11]. domain rich in arginine and serine residues, the RS domain. Their functional roles in gene expression are surprisingly diverse, ranging from their classical involvement in constitutive and While introns are common to all eukaryotes, the complex- alternative pre-mRNA splicing to various post-splicing activities, ity of alternative splicing varies among species. SR proteins including mRNA nuclear export, nonsense-mediated decay, and exist in all metazoan species [8] as well as in some lower mRNA translation. These activities point up the importance of SR eukaryotes, such as the fission yeast Schizosaccharomyces proteins during the regulation of mRNA metabolism. pombe [12,13]. However, classical SR proteins are not present in all eukaryotes and are apparently missing from Gene organization and evolutionary history the budding yeast Saccharomyces cerevisiae, which lacks The discovery of SR proteins goes back to studies in alternative splicing. Instead, three SR-like proteins have Drosophila where genetic screens identified SWAP been identified in S. cerevisiae, one of which has been (suppressor-of-white-apricot) [1], Tra (transformer) [2] shown to modulate the efficiency of pre-mRNA splicing and Tra-2 (transformer-2) [3] as splicing factors. Their [14]. In general, the species-specific presence of SR sequence characterization led to the identification of a proteins correlates with the presence of RS domains within protein domain rich in arginine and serine dipeptides, other components of the general splicing machinery. The termed the arginine/serine (RS) domain. Subsequent observation that the density of RS repeats correlates with identification of the splicing factors SF2/ASF and SC35 the conservation of the branch-point signal, a critical from human cell lines also revealed the presence of sequence element of the 3’ splice site, argues for an ancestral extended RS domains in addition to at least one RNA- origin of SR proteins [15]. As such, SR proteins appear to be binding domain of the RNA recognition motif (RRM)-type ancestral to eukaryotes and were subsequently lost [4-6]. The family of SR proteins was classified following independently in some lineages (Figure 2). Phylo genetic tree the identification of additional RS-domain-containing analyses further suggest that successive gene duplications proteins on the basis of the presence of a phosphoepitope played an important role in SR protein evolution [16]. These recognized by the monoclonal antibody mAb104 [7], their duplication events are coupled with high rates of conservation across vertebrates and invertebrates, and nonsynonymous substitutions that promoted positive their activity in splicing complementation assays [8]. In selection favoring the gain of new functions, supporting the humans, the SR protein family is encoded by nine genes, hypothesis that the expansion of RS repeats during designated splicing factor, arginine/serine-rich (SFRS) 1-7, evolution had a fundamental role in the relaxation of the 9, and 11 (Table 1). All nine members of the human SR splicing signals and in the evolution of regulated splicing. protein family - SF2/ASF, SC35, SRp20, SRp40, SRp55, SRp75, SRp30c, 9G8, and SRp54 - have a common Characteristic structural features structural organization (Figure 1), containing either one or All SR proteins share two main structural features: the RS two amino-terminal RNA-binding domains that provide domain and at least one RRM (Figure 1). For the majority RNA-binding specificity, and a variable-length RS domain of SR proteins with two RNA-binding domains, the second at their carboxyl terminus that functions as a protein is a poor match to the RRM consensus and is referred to as interaction domain [9]. an RRM homolog (RRMH). The only exception is 9G8, http://genomebiology.com/2009/10/10/242 Shepard and Hertel: Genome Biology 2009, 10:242 242.2 Table 1 SRp20 RRM RS Human genes encoding SR proteins SC35 RRM RS Gene name SR protein Chromosomal location UniProt SRp54 RRM RS SFRS1 SF2/ASF/SRp30a 17q21.3-q22 Q07955 SF2/ASF RRM RRMH RS SFRS2 SC35/SRp30b 17q25.1 Q01130 SRp30c RRM RRMH RS SFRS3 SRp20 6p21.31 P84103 SFRS4 SRp75 1p35.3 Q08170 SRp40 RRM RRMH RS SFRS5 SRp40 14q24.2 Q13243 SRp55 RRM RRMH RS SFRS6 SRp55 20q13.11 Q13247 SRp75 RRM RRMH RS SFRS7 9G8 2p22.1 Q16629 9G8 RRM Zn RS SFRS9 SRp30c 12q24.23 Q13242 SFRS11 SRp54 1p31.1 Q05519 Figure 1 The human SR protein family. The structural organization of the which contains an RRM and a zinc-knuckle domain that is nine human SR proteins is shown. RRM, RNA recognition motif; RRMH, RRM homology; RS, arginine/serine-rich domain; Zn, Zinc thought to contact the RNA [17]. In the cases where it has knuckle. been determined, SR proteins have specific, yet degenerate RNA-binding specificities [18,19]. The RS domains of SR proteins participate in protein interactions with a number of other RS-domain-containing splicing factors [20,21]. structural insights provided an explanation for the These include other SR proteins, SR-related proteins [22], seemingly low specificity of RNA binding exhibited by and components of the general splicing machinery [20,21, SRp20 [31,32]. 23-25]. Furthermore, the RS domain can function as a nuclear localization signal by mediating the interaction Localization and function with the SR protein nuclear import receptor, trans- Many proteins involved in pre-mRNA splicing, including portin-SR [26-28]. the SR proteins, are enriched in nuclear compartments termed speckles, which occur throughout the nucleus. Structural characterization of a complete SR protein has Speckles are of two distinct structural types [33]: inter- not yet been achieved. Consequently, only isolated RRMs chromatin granule clusters (IGCs) about 20-25 nm in of SR proteins have been analyzed structurally by nuclear diameter, which are storage/reassembly sites for pre- magnetic resonance spectroscopy. Unfortunately, no mRNA splicing factors; and perichromatin fibrils approxi- structural information detailing the RS domain is available mately 5 nm in diameter, which are sites of actively to date. This may be explained by the poor solubility of transcribing genes and co-transcriptional splicing [34]. these proteins in their free state and the unknown The SR proteins are a prominent component of nuclear phosphorylation state of the serines within the RS domain. speckles (Figure 4) [35,36], and biochemical analyses have In addition, the degenerate RNA-binding sequences indicated that RS domains are responsible for targeting the recognized by SR proteins may have prevented their study SR proteins to these structures [26,37]. The intranuclear in the bound form. To tackle the solubility issues, the organization of SR proteins is dynamic, and they are RRMs of SRp20 and 9G8 were fused to the immuno- recruited from the IGC storage clusters to the sites of globulin G-binding domain 1 of Streptococcal protein G co-transcriptional splicing, the perichromatin fibrils (GB1) solubility tag [29] or overexpressed RRMs were [38,39]. Interestingly, both the RNA-binding domains and suspended in a solution containing charged amino acids RS domains are required for recruitment of SR proteins [30]. Using these manipulations it was possible to obtain from the IGCs to the perichromatin fibrils, as is phos- solution structures of the free 9G8 and SRp20 RRMs and phory lation of the RS domain [40]. of the SRp20 RRM in complex with the RNA sequence 5’-CAUC-3’ (Figure 3). When examining the unbound Splicing activation RRMs of SRp20 and 9G8, one is struck by an unusually In classic cases of alternative splicing, it has been shown large exposed hydrophobic surface, which could explain that cis-acting RNA sequence elements, known as splicing why the solubility of SR proteins is so low. The SRp20 enhancers, increase exon inclusion by serving as sites for RRM complex with RNA shows that although all four recruitment of the splicing machinery - the spliceosome - nucleotides present are contacted by the RRM, only the which is a complex of ribonucleoprotein splicing factors, 5’ cytosine is recognized in a specific manner. These such as U1 and U2 small nuclear ribonucleoproteins http://genomebiology.com/2009/10/10/242 Shepard and Hertel: Genome Biology 2009, 10:242 242.3 (a) Human SR proteins (b) Main species tree 0.083 0.1335 0.13 Hs p54 Hs SF2-ASF Dm SRP54 0.165 Ce RSP7 0.19 Hs SC35 Hs SRp30c Dm SC35 Ce RSP4 0.05 0.17 Sp SRP Hs SRP75 Hs SRp20 0.04 Dm SFRS3 0.16 Hs 9G8 Hs SRP55 0.08 Dm xl6 0.04 Dm RBP 1 0.2 Ce RSP6 Hs SRP40 Hs SF2-ASF Hs SRp30c 0.2 Hs SRp20 Dm SF2 Ce RSP3 0.07 Sp SRP2 0.22 Hs 9G8 Hs SRP75 Hs SRP55 Hs SRP40 0.27 Hs SC35 Dm B52 Ce RSP1 Ce RSP2 0.42 Hs_p54 Ce RSP5 Dm CG4266 Sp RNPS1 At SC35 At SR33 At SCL28 At RSZ33 At RSZp22 At SR1 At SRP34a At SRp30 At RSP31 At RSP 40 At RSP40 Figure 2 Evolutionary relationship between members of the SR family. The phylogeny was inferred using the neighbor-joining method. ClustalW was used to align sequences and perform phylogenetic analysis. Trees were drawn by CTree. The horizontal lines in each panel indicate the similarity between SR proteins. (a) Phylogenetic tree based on the alignment of the human (Hs) SR protein family. The numbers above each bar indicate the degree of similarity. (b) Phylogenetic tree based on the alignment of Homo sapiens (Hs), Drosophila melanogaster (Dm), Caenorhabditis elegans (Ce), Arabidopsis thaliana (At), and Schizosaccharomyces pombe (Sp) SR protein sequences. Green and blue lines indicate different clusters. Cluster set selection is based on minimizing the subtype diversity ratio, a measure that groups related subclasses. http://genomebiology.com/2009/10/10/242 Shepard and Hertel: Genome Biology 2009, 10:242 242.4 [9,41,42]. SR proteins act at several steps during the splicing reaction [4,5,8,43-45] and require phosphorylation for efficient splice-site recognition and dephosphorylation for splicing catalysis [46,47]. A number of SR protein kinases have been identified that specifically phosphorylate serine residues within the RS domain of SR proteins. These include SR protein kinase 1 (SRPK1) [48], Clk/Sty kinase [49], cdc2p34 [50], and topoisomerase [51]. Surprisingly, binding sites for SR proteins are not only limited to alternatively spliced exons, but have also been verified for exons of constitutively spliced pre-mRNAs [52,53]. It is therefore likely that SR proteins bind to sequences found in most, if not all, exons. One model for the mechanism of splicing activation proposes that the RS domain of an enhancer-bound SR protein interacts directly with other splicing factors contain ing an RS domain, thus facilitating the recruitment of spliceosomal components such as the snRNP U1 to the 5’ splice site or U2AF65 (the large subunit of the splicing factor U2AF) to the 3’ splice site [9]. An alternative mode of spliceosomal recruitment was suggested by experiments showing that RS domains of SR proteins contact the pre- mRNA within the functional spliceosome [54,55]. Irres pec- tive of the RS domain activation mode, SR proteins facili- tate the recruitment of spliceosomal components to the regulated splice site [42,56] (Figure 5a). Thus, SR proteins bound to ESEs function as general activators of exon definition [57]. Kinetic analyses have shown that the relative activity of ESE-bound SR proteins determines the magnitude of splicing promotion. This activity depended on the number of SR proteins assembled on an ESE and the distance between the ESE and the intron. It was also shown that activation of splicing was proportional to the number of serine-arginine repeats within the RS domain of the bound SR protein. Thus, the quantity of serine-arginine repeats appears to dictate the activation potential of SR proteins [58]. In addition to their exon-dependent functions, SR proteins Figure 3 have activities that do not require interaction with exon sequences [59]. The role of the exon-independent function Solution structure of an SR protein RRM from human SRp20 (blue) in may be to promote the pairing of 5’ and 3’ splice sites complex with the RNA sequence 5’-CAUC-3’ (red). All four nucleotides present are contacted by the RRM, but only the 5’ cytosine is across the intron or to facilitate the incorporation of the recognized specifically. The structure was generated using the Visual tri-snRNP U4/U6•U5 into the spliceosome [44] (Figure 5b). Molecular Dynamics program [78] from coordinates deposited in the U4/U6•U5 is a complex of snRNPs that contains the Brookhaven National Laboratory Protein Data Bank [30]. splicing activity. Although the RRM of the SR protein is essential for its exon-independent activity [59], it is likely that SR proteins interact with the partially assembled (snRNPs), and their associated proteins, such as U2 spliceosome or the tri-snRNP through RS domain contacts. auxiliary factor (U2AF), that splices exons together and releases the intron RNA. Splicing enhancers are usually Splicing repression located within the regulated exon, and are thus known as One striking feature of SR proteins is their prevalent exonic splicing enhancers (ESEs) [41,42]. ESEs are usually location within the pre-mRNA. In nearly all cases SR recognized by at least one member of the SR protein family proteins have been found to interact with exonic sequences and recruit the splicing machinery to the adjacent intron of the pre-mRNA. This is a surprising finding considering http://genomebiology.com/2009/10/10/242 Shepard and Hertel: Genome Biology 2009, 10:242 242.5 10 µm 10 µm 10 µm GFP-SRp20 SC35 Merge Figure 4 Localization of SR proteins within the nucleus. Left panel: HeLa cells transfected with GFP-SRp20. The GFP fluorescence is visualized directly. Middle panel: cells are also stained with anti-SC35 hybridoma supernatant to highlight clusters of SR proteins in the nucleus (red), which are referred to as nuclear speckles. Speckles are believed to be storage compartments for SR proteins and other splicing factors. Right panel: merge of GFP-SRp20 and SC35 images. The bar in each panel indicates the scale. Images courtesy of Lin Li and Rozanne Sandri-Goldin. the fact that their relatively promiscuous binding mammalian cells and Xenopus oocytes [63] by binding to a specificity predicts that introns are littered with potential 22-nucleotide sequence within the H2A mRNA (Figure SR-protein-binding sites. The fact that SR proteins are 6a). In addition, the S. cerevisiae protein Npl3p, which is mainly observed to bind within exonic sequences suggests closely related to the SR proteins, assists in mRNA export that additional requirements need to be met for functional in yeast [64]. Once again, phosphorylation of specific SR protein binding to the pre-mRNA. There are, however, serine residues within the RS domain seems to control the some instances of SR proteins binding within the intron, efficiency of the mRNA-export function of Npl3p [65]. where they function as negative regulators of splicing. The Given the fact that SR proteins are essential for splicing best-characterized example occurs during adenovirus [9], remain associated with the spliced mRNA after intron infection [60]. In this case, splicing is repressed by the removal [66,67], and shuttle between the nucleus and the binding of the SR protein SF2/ASF to an intronic repressor cytoplasm [62], it seems highly likely that SR proteins also element located upstream of the 3’ splice site branchpoint play an important part in the export of spliced mRNAs. As sequence in the adenovirus pre-mRNA. When bound to the shown recently, 9G8 and SRp20 are involved in mediating repressor element, SF2/ASF prevents the recruitment of the efficient handover of mRNA to Tip-associated protein the snRNP U2 to the branchpoint sequence, thereby (TAP), which is an essential nuclear export factor [68]. in activat ing the 3’ splice site (Figure 5c). Other studies have provided further support for the idea that SR proteins SR protein involvement in translation bound to introns generally interfere with the productive SR proteins have been shown to influence translation assembly of spliceosomes [61]. These observations show either indirectly or directly. For example, the splicing that exonic splicing enhancers not only function in exon activity of SF2/ASF influences alternative splicing of the and splice-site recognition, but also act as barriers to pre-mRNA for the protein kinase MNK2, a kinase that prevent exon skipping. regulates translation initiation. High levels of SF2/ASF promote the production of an MNK2 mRNA isoform that Role of SR proteins in mRNA export enhances cap-dependent translation, whereas low levels Some SR proteins - SF2/ASF, SRp20, and 9G8 - shuttle achieve the opposite [69]. SF2/ASF is also involved in continuously between the nucleus and the cytoplasm [62]. regulating translation directly. It has been shown to The movement of these proteins requires the phosphory- associate with polyribosome fractions isolated from cyto- lation of specific residues in the RS domain and the RNA- plasmic extracts and to enhance the translation efficiency binding domain. These unique intracellular transport of an ESE-containing luciferase reporter [70], apparently properties suggest that a subset of SR proteins functions through mediating the recruitment of components of not only in pre-mRNA processing but also in mRNA export mTOR (mammalian target of rapamycin) signaling pathway [62]. In fact, the SR proteins 9G8 and SRp20 promote (Figure 6b). As a result of this recruitment, a competitive nuclear export of the intronless histone H2A mRNA in inhibitor of cap-dependent translation is released [71]. http://genomebiology.com/2009/10/10/242 Shepard and Hertel: Genome Biology 2009, 10:242 242.6 (a) (a) Nucleus 70K SR U1 Intronless mRNA Pre-mRNA protein snRNP U2AF U2AF65 5′ 3′ 5′ 3′ SR SR SR SR Py AG Exon ESE (b) SR SR SR SR U4/U6•U5 ALY/REF ALY/REF tri-snRNP TAP Export Export 100K machinery machinery 27K SR (b) 4EBP protein mTOR 70K SF2/ASF U1 4EBP m7G ESE AAAAAAAA snRNP U2AF U2 snRNP eIF4E eIF4E U2AF65 35 Exon 1 Py AG Exon 2 (c) U2AF Figure 6 U2AF65 35 SR protein functions other than splicing. (a) mRNA export. SR proteins associate site-specifically with intronless mRNAs, such as SR histone H2A mRNA [63], to promote their export (left-hand side). protein The export machinery is as yet unknown. For intron-containing pre- Py AG Exon mRNAs (right-hand side), SR protein association with the spliced mRNA has also been suggested to mediate nuclear export through interactions with the RNA export factor ALY/REF and Tip-containing protein (TAP). (b) Translation initiation. Interactions between Figure 5 mRNA-bound SF2/ASF and the protein kinase mTOR trigger phosphorylation of 4E-BP (eIF4E-binding protein). In its Splicing functions of SR proteins. (a) SR proteins (green) bound to phosphorylated form 4E-BP dissociates from the translation an exonic splicing enhancer (ESE) may function in constitutive initiation factor eIF4E, thereby releasing eIF4E and activating splicing by interacting with the splicing factors U2AF bound at the initiation of cap-dependent translation (green arrow) [71]. upstream 3’ splice site and U1 snRNP bound to the downstream 5’ splice site. Py represents the polypyrimidine tract, the binding site for U2AF. (b) Exon-independent functions of SR proteins. SR proteins may have two exon-independent functions. SR proteins facilitate splice-site pairing by simultaneously interacting with U1 occasional redundancies. Given the enormous functional snRNP and U2AF across the intron. SR proteins also assist in real estate this family of proteins covers, one is now recruiting the U4/U6•U5 tri-snRNP. (c) Splicing repression is pressed to find out how it is possible to transition these mediated when SR proteins associate with intronic sequences close to the splice sites. Recruitment of spliceosomal components is proteins between their involvements in the various steps of inhibited through steric hindrance or nonproductive spliceosomal mRNA processing. Clearly, reversible modification, such as assembly. Adapted with permission from [79]. serine phosphorylation within the RS domain, is likely to be the ticket for SR protein functional flexibility [51]. The challenge will be to determine the extent and dynamics of Importantly, other SR proteins have also been reported to such modifications within SR proteins specifically involved function in translation. SRp20 promotes translation of a in one of these activities and whether changes in viral RNA initiated at an internal ribosome entry site [72], modification lend support to the existence of an SR and 9G8 increases translation efficiency of unspliced protein-modification code, perhaps similar in principle to mRNA containing a constitutive transport element [73]. the now well-described histone-modification code [74]. Frontiers An old foe makes up another challenge: SR protein The functional characterization of SR proteins has revealed structure. For more than 15 years attempts have been made a wealth of information, placing SR proteins in the context to obtain high-resolution structures of SR proteins. So far, of regulating constitutive and alternative pre-mRNA these attempts have failed because of problems of low splicing, mediating efficient transport of mRNAs, and solubility and the likely heterogeneity of RS-domain modu lat ing mRNA translation. As such, SR proteins could modifications. As a first step towards gaining ground in easily be mistaken for ‘Jacks of all trades, masters of none’ this endeavor, clever modification approaches have been in mRNA metabolism. However, many studies have used to obtain a high-resolution structure of the SR protein demon strated their essential presence in the cell, even with RRM domain. This is a significant first step. However, the http://genomebiology.com/2009/10/10/242 Shepard and Hertel: Genome Biology 2009, 10:242 242.7 much more elusive RS domain is still the big prize, 4. Ge H, Manley JL: A protein factor, ASF, controls cell-spe- cific alternative splicing of SV40 early pre-mRNA in vitro. requiring further creative approaches and manipulations Cell 1990, 62:25-34. to freeze this seemingly unstructured domain in a 5. Krainer AR, Conway GC, Kozak D: Purification and charac- conformation that permits its structural elucidation. terization of pre-mRNA splicing factor SF2 from HeLa cells. Genes Dev 1990, 4:1158-1171. 6. Fu XD, Maniatis T: The 35-kDa mammalian splicing factor A different and experimentally challenging puzzle to SC35 mediates specific interactions between U1 and U2 address is the balance between the relatively low RNA- small nuclear ribonucleoprotein particles at the 3’ splice site. Proc Natl Acad Sci USA 1992, 89:1725-1729. binding specificity exhibited by SR proteins and their 7. Roth MB, Zahler AM, Stolk JA: A conserved family of nuclear usually specific functional impact. 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Chasin LA: Searching for splicing motifs. Adv Exp Med Biol 2007, 623:85-106. © 2009 BioMed Central Ltd http://genomebiology.com/2009/10/10/242 Shepard and Hertel: Genome Biology 2009, 10:242

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