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The SR Protein Family Peter J Shepard and Klemens J Hertel

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The SR Protein Family Peter J Shepard and Klemens J Hertel Protein family review The SR protein family 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 family of serine/arginine (SR)-rich proteins are critical compo- splicing or RNA metabolism [10]. Because of differences in nents of the machineries carrying out these essential processing domain structure, lack of mAb104 recognition, or lack of a 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; thought to contact the RNA [17]. In the cases where it has RRMH, RRM homology; RS, arginine/serine-rich domain; Zn, Zinc been determined, SR proteins have specific, yet degenerate knuckle. 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 SRp30c Hs SC35 Dm SC35 Ce RSP4 0.05 0.17 Hs SRP75 Sp SRP Hs SRp20 0.04 Dm SFRS3 0.16 Hs SRP55 Hs 9G8 0.08 Dm xl6 0.04 Dm RBP 1 0.2 Hs SRP40 Ce RSP6 Hs SF2-ASF Hs SRp30c 0.2 Hs SRp20 Dm SF2 0.07 Ce RSP3 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.
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