the sr protein family of splicing factors master regulators

Biochem. J. (2009) 417, 15–27 (Printed in Great Britain) doi:10.1042/BJ20081501 15

REVIEW ARTICLEThe SR protein family of splicing factors: master regulatorsof gene expressionJennifer C. LONG and Javier F. CACERES1

Medical Research Council Human Genetics Unit, Institute of Genetics and Molecular Medicine, Western General Hospital, Edinburgh EH4 2XU, U.K.

The SR protein family comprises a number of phylogeneticallyconserved and structurally related proteins with a characteristicdomain rich in arginine and serine residues, known as the RS do-main. They play significant roles in constitutive pre-mRNA spli-cing and are also important regulators of alternative splicing. Inaddition they participate in post-splicing activities, such as mRNAnuclearexport,nonsense-mediatedmRNAdecayandmRNAtrans-lation. These wide-ranging roles of SR proteins highlight theirimportance as pivotal regulators of mRNA metabolism, and if

these functions are disrupted, developmental defects or diseasemay result. Furthermore, animal models have shown a highlyspecific, non-redundant role for individual SR proteins in the regu-lation of developmental processes. Here, we will review thecurrent literature to demonstrate how SR proteins are emergingas one of the master regulators of gene expression.

Key words: alternative splicing, pre-mRNA splicing, RS domain,SR proteins, SR-related proteins, translation regulation.

INTRODUCTION

Pre-mRNA splicing was discovered in the late 1970s when itwas demonstrated that eukaryotic genes contained interveningsequences, or introns, that were not present in the maturemRNA [1,2]. Subsequent studies showed that introns are removedby a macromolecular complex, termed the spliceosome, whichconsists of five snRNPs [small nuclear RNPs (ribonucleoproteinparticles)], U1, U2, U4, U5 and U6, and a large number ofprotein components (reviewed in [3,4]). Spliceosomal assemblyis initiated by the recognition of the 5′ and 3′ ss (splice sites) bythe U1 snRNP and the heterodimeric U2AF (U2 snRNP auxiliaryfactor) respectively, forming the E complex. Recruitment of theU2 snRNP to the BP (branch-point), in an ATP-dependent manner,results in the formation of the A complex. Subsequent recruitmentof the U4/U6 · U5 tri-snRNP forms the B complex, which isfollowed by a series of structural rearrangements leading to theformation of the catalytically active spliceosomal C complex(reviewed in [5]). The spliceosome is a dynamic structure andmore than 300 proteins have been identified in active splicingcomplexes [6–8] (reviewed in [9]).

The aim of this article is to review the contribution of SR proteinfamily members to pre-mRNA splicing, as well as reviewingmore recent studies expanding their role in post-splicing activities.Finally, we will discuss how misregulation of SR protein functionscan lead to human disease.

THE SR PROTEIN FAMILY

The SR proteins were first discovered as splicing factors in theearly 1990s (reviewed in [10–12]). A protein domain rich inarginine and serine dipeptides, termed the RS domain, wasoriginally observed in three Drosophila splicing regulators, SWAP

(suppressor-of-white-apricot) [13], Tra (transformer) [14] andTra-2 (transformer-2) [15]. Subsequent identification of SF2/ASF(splicing factor 2/alternative splicing factor) [16,17] and SC35(spliceosomal component 35) [18] revealed that these proteinscontained an RS domain, which is also present in the U1 snRNP-associated protein, U1-70K [19,20].

SF2/ASF was the first SR protein to be identified as an activityrequired to complement an otherwise splicing-deficient HeLa(human cervical carcinoma cell) S100 extract [21] and was alsopurified from HEK (human embryonic kidney)-293 cells as afactor which could alter 5′ ss selection of an SV40 (simian virus40) early pre-mRNA [22]. The term ‘SR protein’ was coinedfollowing identification of additional RS domain-containing pro-teins that were recognized by a monoclonal antibody, mAb 104,which binds to active sites of RNA polymerase II transcription[23]. These novel proteins, which were active in splicing comple-mentation, included the SR proteins SRp20, SRp40, SRp55 andSRp75, named after their apparent molecular mass on an SDS/PAGE gel, and are conserved across vertebrates and invertebrates[24]. They have a modular structure containing one or two copiesof an RRM (RNA recognition motif) at the N-terminus thatprovides RNA-binding specificity and a C-terminal RS domainthat acts to promote protein–protein interactions that facilitaterecruitment of the spliceosome [25,26]. The RS domain canalso contact the pre-mRNA directly via the BP and the 5′ ss,suggesting an alternative way to promote spliceosome assembly[27,28] (reviewed in [29]). Furthermore, the RS domain acts asan NLS (nuclear localization signal), affecting the subcellularlocalization of SR proteins by mediating the interaction with theSR protein nuclear import receptor, transportin-SR [30–32].

The prototypical SR protein, SF2/ASF, functions in constitutivesplicing and also modulates alternative splicing [22,33]. Furtherstudies demonstrated that other SR protein family members

Abbreviations used: BP, branch-point; CLIP, cross-linking and immunoprecipitation; CTD, C-terminal domain; Dscam, Down’s syndrome cell adhesionmolecule; E1αPDH, E1α pyruvate dehydrogenase; eIF, eukaryotic initiation factor; ESE, exonic splicing enhancer; ESS, exonic splicing silencer; hnRNP,heterogeneous nuclear RNP; ISS, intronic splicing silencer; mTOR, mammalian target of rapamycin; NMD, non-sense-mediated decay; PESE, putativeexonic splicing enhancer; RESCUE, relative enhancer and silencer classification by unanimous enrichment; RNAi, RNA interference; RNAP II, RNApolymerase II; RNP, ribonucleoprotein particle; RRM, RNA recognition motif; S6K1, S6 kinase; SC35, spliceosomal component 35; SELEX, selectedevolution of ligands through exponential enrichment; SF2/ASF, splicing factor 2/alternative splicing factor; SMA, spinal muscular atrophy; SMN, survival ofmotor neuron; snRNP, small nuclear RNP; 3′/5′ ss, 3′/5′ splice site; Tra, transformer; U2AF, U2 snRNP auxiliary factor.

1 To whom correspondence should be addressed (email [email protected]).

c© The Authors Journal compilation c© 2009 Biochemical Society

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Table 1 ‘Classical’ SR proteins

Protein name Gene name Key domains Splicing role UniProt

SF2/ASF SFRS1 RRM × 2, RS Constitutive and alternative splicing activator Q07955SC35 SFRS2 RRM, RS Constitutive and alternative splicing activator Q01130SRp20 SFRS3 RRM, RS Constitutive and alternative splicing activator P84103SRp75 SFRS4 RRM × 2, RS Constitutive and alternative splicing activator Q08170SRp40 SFRS5 RRM × 2, RS Constitutive and alternative splicing activator Q13243SRp55 SFRS6 RRM × 2, RS Constitutive and alternative splicing activator Q132479G8 SFRS7 RRM, RS, CCHC-type zinc finger Constitutive and alternative splicing activator Q16629

Table 2 Additional SR proteins


p54 SFRS11 RRM, RS Alternative splicing repressor Q05519SRp30c SFRS9 RRM × 2, RS Constitutive and alternative splicing regulator Q13242SRp38, TASR FUSIP1 RRM, RS General splicing repressor O75494hTra2α TRA2A RRM, RS × 2 Splicing activator Q13595hTra2β SFRS10 RRM, RS × 2 Splicing activator P62995RNPS1 RNPS1 RRM, RS Constitutive and alternative splicing regulator Q15287SRrp35 SRRP35 RRM, RS Negative regulator of alternative splicing Q8WXF0SRrp86, SRrp508 SFRS12 RRM, RS Positive and negative regulator of alternative splicing Q8WXA9U2AF35 U2AF1 RRM, RS, C3H1-type zinc finger × 2 Constitutive splicing factor Q01081U2AF65 U2AF2 RRM × 3, RS Constitutive splicing factor P26368U1-70K SNRP70 RRM, RS Constitutive splicing factor P08621XE7 SFRS17A RRM, RS Alternative splicing regulator Q02040SRp46 SFRS2B RRM, RS Constitutive and alternative splicing regulator Q9BRL6

Table 3 RNA-binding SR-related factors


Urp ZRSR2 RRM, RS Splicing factor Q15696HCC1/CAPER RBM39 RRM, RS Alternative splicing regulator Q14498hSWAP SFRS16 RS Alternative splicing regulator Q8N2M8Pinin PNN RS Alternative splicing regulator Q9H307SRrp129 SFRS2IP RS Splicing factor Q99590U4/U6 · U5 tri-snRNP-associated 27 kDa protein RY-1 RS Unknown Q8WVK2LUC7B1 LUC7L RS, C2H2-type zinc finger Unknown Q9NQ29Acinus ACIN1 RRM, RS, SAP Unknown Q9UKV3SR-A1 SFRS19/ SCAF1 RS Unknown Q9H7N4ZNF265 ZRANB2 RS, RANBP2-type zinc finger × 2 Alternative splicing regulator O95218SRm160 SRRM1 RS, PWI Constitutive and alternative splicing co-activator Q8IYB3SRm300 SRRM2 RS Constitutive and alternative splicing co-activator Q9UQ35RBM5 RBM5 RRM × 2, RS, RANBP2- and C2H2-type zinc fingers Unknown P52756U2-associated protein SR140 SR140 RRM, RS Unknown O15042RBM23 RBM23 RRM × 2, RS Unknown Q86U06SFRS15 SFRS15 RRM, RS Unknown O95104

could also affect alternative splicing in vitro [34,35]. Thus, thecriteria used to define ‘classical’ SR protein family membersare (i) structural similarity, (ii) dual function in constitutive andalternative splicing, (iii) the presence of a phosphoepitope recog-nised by mAb104; and (iv) their purification using magnesiumchloride (Table 1).

A genome-wide survey in metazoans identified a large numberof RS domain-containing proteins with a role not only in splicingbut also in other cellular processes such as chromatin remodelling,transcription and cell cycle progression [36]. These relatedproteins contain an RS domain but may lack a defined RRM,however a subset can bind RNA through other domains such asthe PWI motif found in the splicing activator SRm160 [37,38](Tables 2–4). These factors are collectively known as SR-likeor SR-related proteins and include both subunits of the U2AF

heterodimer, U1-70K and the splicing coactivators SRm 160/300,among others [39]. It was recently proposed that SR proteinsshould be redefined based on their common structural featuresand their role in pre-mRNA splicing [40]. Based on this, a ‘bonafide’ SR protein has to contain at least one RRM and an RSdomain (irrespective of their positions within the protein) and tofunction in constitutive or alternative splicing, as assayed by eithercomplementation of splicing-deficient S100 HeLa cytoplasmicextracts or in an alternative splicing assay respectively. The humanhomologues of the Drosophila splicing regulators Tra2α [41]and Tra2β [42] contain an RRM flanked by two RS domains,which is not the domain structure found in classical SR proteins(Figure 1). Since both proteins function as sequence-specificsplicing activators [43], they could be classified as SR proteins(Table 2). Other proteins may contain an RS domain but also have


Functional roles of SR proteins in RNA processing 17

Table 4 Other RS-domain containing proteins


SRrp53 RSRC1 RS, coiled-coil domain Unknown Q96IZ7hPRP5 DDX46 RS, DEAH box Spliceosomal rearrangement Q7L014hPRP16 DHX38 RS, DEAH box Splicing factor Q92620Prp22/HRH1 DHX8 RS, DEAH box Spliceosomal rearrangement Q14562U5-100k/hPRP28 DDX23 RS, DEAD box Spliceosomal rearrangement Q9BUQ8ClkSty-1 CLK1 RS, kinase domain SR protein kinase P49759ClkSty-2 CLK2 RS, kinase domain SR protein kinase P49760ClkSty-3 CLK3 RS, kinase domain SR protein kinase P49761Prp4k PRPF4B RS, kinase domain SR protein kinase Q13523CrkRS CRKRS RS, kinase domain SR protein kinase Q9NYV4CDC2L5 CDC2L5 RS, kinase domain Alternative splicing regulator Q14004Cyclin-L1 CCNL1 RS, cyclin-like domain × 2 Alternative splicing regulator Q9UK58Cyclin-L2 CCNL2 RS, cyclin-like domain × 2 Alternative splicing regulator Q96S94SR-cyp PPIG RS, PPIase cyclophilin-type domain Regulates localisation of SR proteins Q13427CIR CIR RS Alternative splicing regulator Q86X95SRrp130 SFRS18 RS × 2 Unknown Q8TF01

Figure 1 Schematic diagram of SR and SR-related proteins

The domain structures are depicted. DEAH Box, motif characteristic of RNA helicases; RS: arginine/serine-rich domain; PWI: an alternative RNA binding motif; Zn, zinc finger motif. With the exceptionof SRm160 and hPRP5, all proteins are drawn to scale.

other domains required for their enzymatic activities, as is thecase for the RNA helicases HRH1 and hPRP16, that contain aDEAH box domain [44,45] (Table 4).

SR PROTEINS AND TRANSCRIPTION

SR proteins are concentrated in nuclear speckles and are recruitedfrom these sites to nascent sites of RNAP II (RNA polymerase II)transcription [46]. It is well documented that RNA splicing occursco-transcriptionally [47,48]. Interactions between SR-relatedproteins and the CTD (C-terminal domain) of RNAP II havebeen reported [49], and members of the SR protein family wereidentified among the hundreds of proteins present in the RNAP II

complex [50]. It was recently reported that SC35 promotes RNAPII elongation in a subset of genes, confirming the existenceof coupling between transcription and splicing, and perhapssurprisingly, showing that this coupling can be bidirectional [51](reviewed in [52]). In this study [51] it was demonstrated thatSC35 interacts not only with the CTD but also with CDK9(cyclin-dependent kinase 9), which is the kinase component of thetranscriptional elongation factor P-TEFb (positive transcriptionelongation factor b), resulting in phosphorylation of Ser2 in theCTD and leading to transcriptional elongation. This activity ofSR proteins in transcriptional elongation may be functionallyrelated to their reported effect in the maintenance of genomestability. It has been shown that depletion of SF2/ASF, SC35and the SR-related protein RNSP1 results in the formation of



R-loops (RNA:DNA hybrid structures) leading to a hypermutationphenotype [53–55].

The co-transcriptional nature of pre-mRNA splicing underliesa role for the transcriptional machinery in alternative splicingregulation [56]. A kinetic coupling model proposed that changesin the rate of transcriptional elongation affect the timing in whichsplice sites are presented to the splicing machinery, leading todifferential splice site selection [57]. Furthermore, differentialrecruitment of splicing factors to the CTD of RNAP II may alsoinfluence this process [58] (reviewed in [59]).

ROLES OF CLASSICAL SR PROTEINS IN CONSTITUTIVE ANDALTERNATIVE SPLICING

Splice site consensus sequences are generally not sufficient todirect assembly of a functional spliceosome, and auxiliary ele-ments known as ESEs and ISEs (exonic and intronic splicingenhancers respectively) and ESSs and ISSs (exonic and intronicsilencers respectively) are involved in both constitutive and, toperhaps a greater extent, alternative splicing. Binding of SRproteins to ESEs acts as a barrier that prevents exon skipping,thus ensuring the correct 5′ to 3′ linear order of exons in splicedmRNA [60]. Two main models have been proposed to explainthe mechanism by which SR proteins regulate exon inclusion. The‘recruitment model’ focuses on the ability of ESE-bound SRproteins to recruit and stabilize interactions between the U1snRNP at the 5′ ss and U2AF65 at the 3′ ss [61], in a process knownas exon definition [62] (Figure 2A). The hnRNP (heterogeneousnuclear RNP) family comprises a structurally diverse group ofRNA-binding proteins with roles in many aspects of RNA bio-genesis, including pre-mRNA splicing (reviewed in [63]). Inthe ‘inhibitor model’, ESE-bound SR proteins may act byantagonizing the negative activity of hnRNP proteins recognizingESSs [64] (Figure 2B). The SR proteins may also form a networkof protein–protein interactions across introns to juxtapose the 5′

and 3′ ss early in spliceosomal assembly, as shown by the reportedinteractions of SF2/ASF and SC35 with U1-70K at the 5′ ss andwith U2AF35 at the 3′ ss in an RS domain-dependent manner [25](Figure 2C). Additionally, the enhancer-bound RS domain of theSR protein SF2/ASF has been shown to interact directly withRNA at the BP to promote pre-spliceosomal assembly [27,28].SR proteins may also facilitate the recruitment of the U4/U6 · U5tri-snRNP to the pre-spliceosome [65] via RS domain-mediatedinteractions with the SR-related proteins SRrp65 and SRrp110[66]. The function of SF2/ASF in pre-mRNA splicing dependson the context of the pre-mRNA sequence to which it binds,as shown by the fact that SF2/ASF inhibits adenovirus IIIa pre-mRNA splicing when bound to an intronic repressor element [67].The second RRM of SF2/ASF, and in particular a phylogeneticallyconserved heptapeptide, SWQDLKD, which is located in the firstα-helix of this domain [68], is essential for the splice site selectionactivity of SF2/ASF [69,70]. The structure of this domain revealedan atypical RRM fold that binds to RNA in a novel manner [71].

Use of FRAP (fluorescence recovery after photobleaching)approaches revealed a high mobility for SF2/ASF within thenucleus, with kinetics compatible with a diffusion mechanism[72,73]. Advances in imaging have allowed analysis of splicingfactors both in speckles and at other sites in the nucleoplasm byFRET (fluorescence resonance energy transfer) [74,75]. A recentstudy provided a map of SR protein splicing complexes in thenucleus, and showed that they act in exon and intron definitionin vivo [76].

The U12-type class of pre-mRNA introns, also known as AT-ACintrons, are spliced by the less abundant U12-dependent (minor)spliceosome. The 5′ ss and BP sequences are highly conserved in

Figure 2 Roles of SR proteins in splice site selection

(A) SR proteins bound to ESE elements recruit U2AF35 to an upstream 3′ ss and U1-70K tothe downstream 5′ ss. (B) ESSs recruit hnRNP proteins which block 3′ ss selection by U2AF.SR proteins bound to ESEs can antagonize the action of these splicing repressors, therebypromoting splice site selection. (C) SR proteins can facilitate intron bridging interactions bybinding, via the RS domain, to U1-70K and U2AF 35, thereby juxtaposing the 5′ and 3′ ss.

AT-AC introns, unlike the degenerate sequences found in GT-AGintrons (reviewed in [77]). The SR proteins have been shown toparticipate in AT-AC intron splicing where they promote bindingof the U11 and U12 snRNPs to the 5′ ss and BP respectively [78].There is also evidence that SR proteins contact the pre-mRNAof U12-type introns directly via their RS domain, again in ananalogous fashion to that seen in conventional splicing [79].

A delicate interplay of cis-acting sequences and trans-actingfactors modulate the splicing of regulated exons in a combinatorialfashion [80]. SR family proteins antagonize the activity of hnRNPA/B proteins in splice site selection, with an excess of hnRNPA1 favouring distal 5′ ss, whereas SF2/ASF promotes the use ofproximal 5′ ss [81–85]. Thus, the ratio of hnRNP A1 to SR proteinsin the nucleus is of great importance in alternative splicing regul-ation and may have a crucial role in the tissue-specific and devel-opmental control of regulated splicing. Accordingly, the proteinlevels of SF2/ASF and hnRNP A1 have been found to varynaturally over a very wide range in rat tissues and also in immortaland transformed cell lines [35,86]. SF2/ASF and hnRNP A1 havealso been found to have an antagonistic role in the regulation ofthe neuronal-specific N1 exon of the c-src gene [87]. Antagonismbetween hnRNP proteins and SR proteins has also been shownto regulate a highly complex pattern of mutually exclusive exonsin the Dscam (Down’s syndrome cell adhesion molecule) gene inDrosophila [88]. A subset of SR proteins has been shown toactivate alternative splicing of the cTNT (cardiac troponin T)exon 5 by directly interacting with a purine-rich ESE. Thusregulation of the levels of individual SR proteins may contributeto the developmental regulation of alternative splicing in cTNT[89]. Interestingly, individual SR proteins can sometimes haveantagonistic effects on splice site selection, as is the case withSRp20 and SF2/ASF in the regulation of SRp20 pre-mRNAalternative splicing [90] and of SF2/ASF and SC35 in the



regulation of β-tropomyosin [91] and human growth hormonepre-mRNA alternative splicing [92]. Other, non-classical, SRproteins, including p54, SRp38 and SRp86, function solely asnegative regulators of alternative splicing, antagonizing classicalSR proteins and promoting exon skipping [93–95].

SR PROTEIN TARGETS

Several approaches have been taken to identify physiologicalRNA targets of SR proteins. One such approach, termed SELEX(selected evolution of ligands through exponential enrichment)involves the selection of high-affinity binding sites from ran-domized pools of RNA sequences [96]. This has resulted in theidentification of high-affinity binding sites for SF2/ASF and SC35[97], SRp40 [98], 9G8 and SRp20 [99] (Table 5). These bindingsites consist of purine-rich sequences that resemble 5′ ss or exonicsequences, known to function as splicing enhancers. SELEX canbe used in conjunction with large-scale bioinformatic screensto identify further potential binding sites. An alternative to theSELEX approach was the development of a functional SELEXstrategy, which involves selection for a sequence that will promotesplicing, rather than binding alone [100]. This can be modifiedfurther by performing the splicing reactions in a S100 extractwith the addition of a single SR protein [101,102]. The motifsidentified for the SR proteins SF2/ASF, SC35, SRp40 and SRp55using functional SELEX were more redundant than those foundby conventional SELEX, suggesting that the specificity of bindingin vivo depends on factors other than just sequence recognition(Table 5). These motifs have been integrated into a web-basedprogram, known as ESE finder, where input sequences can bescanned for potential ESEs responsive to the above proteins [103].

A number of computational approaches have also been usedto define splicing sequence motifs that regulate exon inclusion.RESCUE (relative enhancer and silencer classification byunanimous enrichment)-ESE predicts sequences which couldfunction as ESEs by statistical analysis of exon–intron and splicesite composition [104]. This is based on the observation thatESEs function in a highly position-dependent fashion and arepresent in constitutively spliced exons and absent in introns.This approach identified 238 candidate ESEs that occurred morefrequently in exons with weak splice sites than in exons withstrong splice sites. By sequence similarity, these were condensedto ten RESCUE-ESE motif clusters and were shown to haveenhancer activity in vivo. Another computational study found2000 RNA octamers, identified as PESEs (putative ESEs), thatwere found more frequently in exons than in pseudo-exons orintronless genes [105]. Validation of a subset of these PESEsresulted in 82% exhibiting decreased splicing efficiency whenthe PESE was mutated [106].

ChIP (chromatin immunoprecipitation) can be used to studynascent RNA–protein interactions, but a variation of thistechnique named RIP (RNP immuno-precipitation) provides moreinformation on protein–RNA interactions in vivo [107]. RIP in-volves cross-linking the protein–RNA interactions using formal-dehyde, followed by immuno-precipitation of the protein–RNAcomplexes. After reversing the cross-links, the RNA can be amp-lified by RT–PCR (reverse transcription–PCR). This techniquerelies on random hexamer primers to identify unknown RNAs orcan be used in conjunction with microarray technology. Reasso-ciation of RNA-binding proteins after cell lysis can complicate theanalysis of these results, since observed protein–RNA interactionsmay not necessarily reflect true in vivo interactions [108]. Anadaptation of the SELEX method described above, known asgenomic SELEX, uses real genomic sequences rather than random

Table 5 RNA sequences identified as SR protein binding sites

N: any nucleotide; R: purine; Y: pyrimidine; D: A, G or U; K: U or G; M: A or C; S: G or C; W: Aor U.

SR protein Binding site Method Reference

SF2/ASF RGAAGAAC SELEX [97]AGGACRRAGC SELEX [97]SRSASGA Functional SELEX [102]UGRWG CLIP [114]

SC35 AGSAGAGUA SELEX [97]GUUCGAGUA SELEX [97]GRYYCSYR Functional SELEX [101]UGUUCSAGWU SELEX [99]GWUWCCUGCUA SELEX [99]GGGUAUGCUG SELEX [99]GAGCAGUAGKS SELEX [99]AGGAGAU SELEX [99]UGCNGYY Functional SELEX [211]

SRp20 GGUCCUCUUC Gel shift [214]WCWWC Splicing assay [99]CUCKUCY RNA affinity [211]

SRp75 GAAGGA UV cross-linking [187]

SRp40 GAGCAGUCGGCUC SELEX [98]ACDGS Functional SELEX [102]

SRp55/B52 USCGKM Functional SELEX [102]UCAACCAGGCGAC SELEX [213]

9G8 UCAACA UV cross-linking [215]ACGAGAGAY SELEX [99]GGACGACGAG Functional SELEX [211]

p54 C Rich UV cross-linking [218]

SRp30c GACGAC Functional SELEX [212]AAAGAGCUCGG Functional SELEX [212]CUGGAUU Gel shift [217]

hTra2β (GAA)n SELEX [43]

SRm160 Purine rich (GAA)n Splicing assay [216]

pools, which allows for identification of authentic protein-bindingRNA sequences [109,110].

Previously, a novel technique named CLIP (cross-linking andimmunoprecipitation) was developed in order to identify in vivoRNA targets [111]. CLIP involves an in vivo photo cross-linkingstep to capture the protein–RNA interactions, followed by partialRNase digestion to generate RNA tags of approximately 60nucleotides followed by specific immunoprecipitation of theprotein of interest. An advantage of this method is that by usingan in vivo photo cross-linking step, which induces a covalentprotein–nucleic acid bond, these interactions are preserved inan intact cell. CLIP was used to characterize the in vivo RNAbinding targets of the neuronal-specific splicing factor Nova andhas allowed the generation of an RNA map to predict splicingregulation dependent on this protein [112,113]. It has also beenused for other RNA binding proteins, including SF2/ASF [114](Table 5). The identification of SR protein targets and the studyof how tissue-specific patterns of splicing change, depending onthe complement of SR proteins present, can also be analysed byalternative splicing microarrays [115–117].

The additional importance of structural elements in splice siteselection should also be taken into account. For example, RNAstructure elements associated with alternative splice-site selectionhave been recently identified in the human genome [118]. Inaddition, RNA folding has been shown to affect the recruitment of



SR proteins to mouse and human ESE elements in the fibronectinEDA exon [119,120]. Finally, competing intronic RNA secondarystructures help to define a complex pattern of mutually exclusiveexons in the Dscam gene [121].

SR PROTEINS HAVE FUNCTIONAL SPECIFICITY

Initially, the ability of different individual SR proteins tocomplement splicing-deficient extracts suggested that SR proteinsmay have redundant functions. However, the sequence-specificRNA binding ability of individual SR proteins and differences intheir ability to regulate alternative splicing suggested otherwise[35,122,123].

A growing body of evidence showed that individual SR proteinswere not functionally equivalent in Drosophila, Caenorhabditiselegans and mouse models. For instance, SF2/ASF was shown tobe an essential factor for cell viability in a chicken cell line and itsdepletion could not be rescued by expression of SC35 or SRp40,indicating a non-redundant function of SF2/ASF [124]. Otherstudies have shown that the SR protein B52/SRp55 is essential forDrosophila development [125,126]. B52/SRp55 was shown notto be essential for the splicing of a number of substrates [127],but specific substrates that were mis-spliced in B52-deficient flieswere identified [128,129]. Furthermore, B52/SRp55 regulates theinclusion of alternative exon 2 in eyeless, a master regulatorof eye development in Drosophila, resulting in the production ofa protein isoform that gives rise to a small-eye phenotype. Conver-sely, the canonical eyeless isoform induces eye overgrowth [130].

Use of RNAi (RNA interference) to inhibit SR protein functionduring C. elegans development revealed that depletion of theorthologue of the mammalian SF2/ASF (CeSF2/ASF) resulted inembryonic lethality, which indicates an essential, non-redundant,role for this gene during nematode development. By contrast,RNAi-mediated depletion of other SR genes resulted in noobvious phenotype, which is indicative of functional redundancy[131–133]. The function of SR proteins has also been studied inmouse model systems (recently reviewed in [134]). All SR-nullmice for SRp20 [135], SC35 [136,137] and SF2/ASF [138] showan early embryonic phenotype indicating that SR proteins arenot redundant. However, these essential functions appear to betissue- or developmental stage-specific, as cultured cells fromthe knockout mice are viable. The generation of conditionalknockouts has allowed further characterization of SR proteinfunction in different tissue types or at various developmental timepoints. Deletion of SC35 in mice results in decreased thymus sizeand a major defect in T-cell maturation [136], whereas tissue-specific ablation of SC35 in the heart has been shown to causedilated cardiomyopathy [137]. SF2/ASF has also been shown tohave a role in cardiac function; however its main function is in thedevelopmental process of postnatal heart remodelling [138]. Miceknockouts of other splicing factors, including Nova, U2AF26,hnRNP U and hnRNP C, also result in embryonic lethalityor developmental defects, which highlights the importance ofsplicing for the correct regulation of biological processes such asembryogenesis and tissue maintenance [134].

POST-SPLICING ACTIVITIES OF SR PROTEINS

SR proteins also function in mRNA processing reactions thatoccur after splicing, including mRNA nuclear export, NMD (non-sense-mediated decay) and translation (reviewed in [139]). SRproteins display a nuclear localization pattern and are foundto accumulate in splicing speckles [140]. However, a subset ofSR proteins, which includes SF2/ASF, SRp20 and 9G8, shuttle

continuously between the nucleus and the cytoplasm [141],reminiscent of what was found for a subset of hnRNP proteins[142]. This suggested that the shuttling SR proteins may functionin cytoplasmic processes, or be involved in the transport ofspliced mRNA. Indeed, SRp20, 9G8 and SF2/ASF function inthe nucleocytoplasmic export of mRNA by interacting with themRNA nuclear export receptor TAP/NFX1 [143,144], exhibitinga higher affinity when hypophosphorylated [145].

SR proteins have also been implicated in regulating the NMDpathway, whereby mRNAs containing premature terminationcodons are targeted for degradation. Increased expression of asubset of SR proteins, including SF2/ASF, SC35, SRp40 andSRp55, strongly enhanced NMD [146]. Interestingly, this effectdoes not appear to be dependent on their nucleocytoplasmicshuttling, suggesting a role for SR proteins in enhancing nuclearsteps of NMD. A recent study showed that SF2/ASF has the poten-tial to affect the cellular site of NMD, shifting this process to thenuclear compartment before mRNA release from nuclei [147].

SF2/ASF controls alternative splicing of pre-mRNAs encodingthe kinases MNK2 [MAPK (mitogen-activated protein kinase)-interacting kinase 2] and S6K1 (S6 kinase 1) that are involved intranslational regulation. Increased expression of SF2/ASF resultsin the production of an isoform of MNK2, which promotesMAPK-independent eIF4E (eukaryotic initiation factor 4E) phos-phorylation, and an unusual oncogenic isoform of S6K1, therebyenhancing cap-dependent translation [148]. SR proteins havealso been shown to directly affect translational regulation. SF2/ASF associates with polyribosomes in cytoplasmic extracts andenhances the translation of an ESE-containing luciferase reporterboth in vivo and in vitro [149]. This direct effect of SF2/ASFin the regulation of the translation of SF2/ASF-bound mRNAtargets is mediated by the recruitment of components of themTOR (mammalian target of rapamycin) signalling pathway,resulting in phosphorylation and release of 4E-BP, a competitiveinhibitor of cap-dependent translation [150]. The role of mTORin the activation of S6K1, which phosphorylates eIF4B and S6,promoting translation initiation, may also be enhanced by SF2/ASF [151] (Figure 3). Other SR proteins have also been reportedto function in translation. SRp20 has been shown to function inIRES (internal ribosome entry site)-mediated translation of a viralRNA [152], whereas 9G8 plays a role in translation of unsplicedmRNA containing a CTE (constitutive transport element) [153].

The results described in this section demonstrate that SRprotein function is not restricted to nuclear mRNA splicing, and itseems sensible that proteins already bound to spliced mRNA mayfunction in subsequent processing events as they are already inplace to facilitate future interactions. However, it also highlightsthe requirement for exquisite regulation of SR proteins in order tomaintain their role in cytoplasmic processing of mRNAs withoutdisrupting nuclear processes, which are highly sensitive to therelative concentration of splicing factors.

SR PROTEIN REGULATION

A dynamic cycle of phosphorylation and dephosphorylation isrequired for pre-mRNA splicing [154], this being related, at leastin part, to the phosphorylation status of SR proteins. The RSdomain of SR proteins is extensively phosphorylated on serineresidues and this plays an important role in regulating the sub-cellular localization and activity of SR proteins (reviewed in [40]).For instance, phosphorylation of the RS domain in SF2/ASF actsto enhance protein–protein interactions with other RS domain-containing splicing factors, such as U1-70K [155], whereasdephosphorylation of SR and SR-related proteins is required forsplicing catalysis to proceed [156,157].



Figure 3 Role of SF2/ASF in translation

SF2/ASF-bound mRNAs recruit the mTOR kinase resulting in the phosphorylation and release of 4E-BP, leading to in enhanced translation initiation. The mTOR kinase phosphorylates S6K1, whichpromotes translation initiation, and this may also be enhanced by SF2/ASF. The Mnk2b isoform induced by SF2/ASF-dependent alternative splicing also leads to translation activation.

Several protein kinase families have been shown to phos-phorylate the RS domain of SR proteins, including the SRPK(SR protein kinase) family [158,159], the Clk/Sty family of dual-specificity kinases [160] and topoisomerase I [161]. SRPK1 is aserine-specific kinase that binds to a ‘docking motif’ in SF2/ASFthat restricts phosphorylation to the N-terminus of the RS domain[162]. In contrast, Clk/Sty can phosphorylate the whole of the RSdomain, resulting in a hyperphosphorylated state [163]. The phos-phorylation status of the RS domain of SR proteins is alsoimportant in the post-splicing activities of SR proteins. A hypo-phosphorylated RS domain is required for the interaction of nuc-leocytoplasmic shuttling SR proteins with the TAP/NFX1 nuclearexport receptor [145]. SR protein kinases present in the cytoplasmare required to re-phosphorylate the RS domain before the SRprotein can return to the nucleus [164]. RS domain dephosphoryl-ation also plays an important role in sorting SR proteins in thenucleus, where shuttling SR proteins and non-shuttling SR pro-teins are recycled via different pathways [165]. In the cytoplasm,dephosphorylation of the RS domain enhances mRNA binding ofSF2/ASF and contributes to its role in translation [166]. SR proteinphosphorylation is also important in developmental regulation, asdemonstrated in the nematode Ascaris lumbricoides [167].

Importantly, alternative splicing is extensively regulated bysignal transduction pathways, whereby signalling cascades canlink the splicing machinery to the exterior environment [168].For instance, the SR protein SRp38 is dephosphorylated uponheat shock by the phosphatase PP1 and becomes a potent splicingrepressor [169,170]. Two other well described examples are theinsulin-induced promotion of protein kinase C beta II alternativesplicing as a result of SRp40 phosphorylation by Akt [171], andthe growth factor induced alternative splicing of the fibronectinEDA exon, via phosphorylation of SF2/ASF and 9G8 by Akt[172]. Interestingly, growth factors not only modify the alternativesplicing pattern of the fibronectin gene but also affect its trans-lation in an SR protein-dependent fashion, providing an examplewhere modification of SR protein activity in response to extra-cellular stimulation leads to a concerted regulation of splicing andtranslation [173]. Caffeine regulates the alternative splicing of a

subset of cancer-associated genes, including the tumor suppressorKLF6. This response is mediated by the SR protein, SC35, whichis in turn induced by caffeine, and its overexpression is sufficientto recapitulate this regulated event [174]. Another example of thetight regulation of the SR protein family members is exemplifiedby the common existence of unproductive splicing of SR genes.This is associated with ultraconserved elements that overlap alter-natively spliced exons and target the resulting mRNAs fordegradation by NMD [175,176].

SR PROTEINS AND HUMAN DISEASE

Disruption of the many roles of SR family proteins can lead tohuman disease. Approximately 15% of mutations that cause ge-netic disease affect pre-mRNA splicing [177], targeting conservedsplicing signals including the 5′ ss, 3′ ss and BP, as well asenhancer and silencer sequences. Indeed, analysis of a databaseof 50 single-base substitutions associated with exon-skipping inhuman genes revealed that more than 50% of these mutationsdisrupted at least one of the target motifs for the SR proteinsSF2/ASF, SRp40, SRp55 and SC35 [178,179] (reviewed in [180]).

Cancer

There is emerging evidence that establishes a connection betweenthe mis-expression of SR proteins and the development of can-cerous tissues, mainly as a result of change in the alternativesplicing patterns of key transcripts. Increased expression of SRproteins usually correlates with cancer progression, as shown byelevated expression of SF2/ASF, SC35 and SRp20 in malignantovarian tissue [181] and of several classical SR proteins in breastcancer [182]. However, the mRNA levels of SF2/ASF, SRp40,SRp55 and SRp75 are lower in non-familial colon adenocarcino-mas than adjacent non-pathological tissue, suggesting the levelsof SR proteins in cancerous tissues may be tissue-specific [183].

SF2/ASF was found to be upregulated in several human tu-mours, including lung, colon, kidney, liver, pancreas and breasttumours [148]. Accordingly, gene amplification of SFRS1, which



codes for SF2/ASF, is commonly found in breast cancers [184].Furthermore, increased expression of SF2/ASF transformsimmortal rodent fibroblasts and leads to the formation of sarcomasin nude mice, whereas downregulation of SF2/ASF reverses thesephenotypes. Other SR proteins, such as SC35 and SRp55, didnot have transforming activity, indicating a highly specific role ofSF2/ASF in cancer development. Altogether, these results supportthe notion that SFRS1 is a proto-oncogene [148]. Another RNAtarget for SF2/ASF that can explain its transforming activity isthe proto-oncogene Ron (macrophage-stimulating 1 receptor).SF2/ASF regulates the alternative splicing of Ron pre-mRNAby binding to an ESE in exon 12 and promoting skipping of exon11 [185]. This results in production of �Ron, a constitutivelyactive isoform which confers increased motility on expressingcells, a characteristic required for tumour metastasis. Importantly,abnormal accumulation of �Ron occurs in breast and colontumours and the levels of SF2/ASF mirror those of �Ron [185].

HIV

HIV-1 uses a combination of several alternative 5′ and 3′ ssto generate more than 40 different mRNAs from its full-lengthgenomic pre-mRNA [186]. Several SR proteins have been shownto regulate different splicing events affecting the viral transcripts.For instance, SRp75 binds a viral ESE [187], whereas SF2/ASFand SRp40 bind a guanosine-adenosine-rich ESE identified inexon 5 of HIV-1 leading to its inclusion [188]. Furthermore,HIV infection induces changes in the levels of splicing factors,including SR proteins, that regulate viral alternative splicing andtherefore virus replication [189]. Current drugs used to treat HIV-infected patients involve the use of combinations of retroviralsthat specifically target viral proteins such as reverse transcrip-tase, protease and gp120 (reviewed by [190]). HIV viral produc-tion is tightly linked with alternative splicing of the viral HIV-1pre-mRNA. Therefore, an alternative and novel approach to cir-cumvent the problem of resistance of HIV-1 to current inhibitors isto target the role of SR proteins in HIV pre-mRNA splicing [191].A screen for chemical inhibitors of pre-mRNA splicing identifiedindole derivatives that specifically inhibit ESE-dependent splicingby interacting directly and selectively with individual SR proteins[192]. One such small chemical compound was shown to preventthe production of key viral HIV-1 regulatory proteins whosesplicing depends on weak 3′ ss [193].

SMA (spinal muscular atrophy)

SMA is a severe hereditary neurodegenerative disorder that resultsfrom the lack of a functional SMN1 (survival of motor neuron 1)gene product, which is a key component of the snRNP biogenesispathway. An SMN1 paralogue, the centromeric SMN2 gene,differs by a single nucleotide change, a C > T transition in exon7, that causes substantial skipping of this exon and results in theproduction of a non-functional protein. This exon-skipping eventhas been attributed either to the loss of an SF2/ASF-dependentexonic splicing enhancer [194] or to the creation of an hnRNPA/B-dependent exonic splicing silencer [195].

Several therapeutic approaches, which focus on altering thesplicing of SMN2 to induce exon 7 inclusion and would resultin functional SMN protein in affected patients, have made useof antisense technology [196]. The first uses bifunctional ASOs(antisense oligonucleotides) which are comprised of oligonuc-leotides complementary to exon 7, with a non-complementarytail containing exonic-splicing enhancer motifs recognized by SRproteins. This approach has been shown to mediate the bindingof SF2/ASF to SMN2 exon 7 and promote exon inclusion [197].

An alternative strategy has recently been developed based onbifunctional U7 snRNAs that contain both an antisense sequencetargeting exon 7 and a splicing enhancer sequence to improverecognition of the exon. These RNAs are stably introduced intocells and the U7 snRNAs become incorporated into snRNPs,inducing a prolonged restoration of SMN protein in SMA fibro-blasts [198]. Another approach uses ESSENCE (exon-specificsplicing enhancement by small chimaeric effectors) molecules,which also contain an antisense moiety complementary to thetarget exon and a minimal RS domain peptide designed to mimicthe effect of SR proteins. The ESSENCE molecules have alsobeen shown to restore SMN2 levels to that of wild-type SMN1levels by exon inclusion [199]. Interestingly, the antisense moietyalone stimulated exon 7 inclusion, and functional full-lengthSMN protein was produced in primary fibroblasts from a type ISMA patient [200,201]. An in vivo delivery system has beendeveloped for bifunctional RNAs using a viral vector [202].

Other human diseases

It has been demonstrated that SR proteins are autoantigens inpatients with systemic lupus erythematosus [203]. SR familyproteins have also been shown to have regulatory roles in thesplicing of several pre-mRNAs associated with human disease.For example, SF2/ASF and SRp40 bind to an ISS and promoteexclusion of exon 9 of CFTR (cystic fibrosis transmembraneconductance regulator) [204]. Lack of exon 9 correlates with theoccurrence of monosymptomatic and full forms of cystic fibrosisdisease [205]. SC35 has a role in the aberrant splicing of theE1αPDH (E1α pyruvate dehydrogenase) mRNA, resulting in adefect of mitochondrial energy metabolism. An intronic mutationof the E1αPDH gene that activates a cryptic 5′ ss leads to mis-spliced mRNA and defective protein [206]. SC35 has been shownto significantly activate splicing at this cryptic site. Accordingly,RNAi-mediated depletion of SC35 in primary fibroblasts fromthe affected patient could restore the normal E1αPDH splicingpattern [207]. It has also been recently reported that the expressionof SRp20 is elevated in bipolar patients, which may explain theaberrant splicing of glucocorticoid receptor α in these patients[208].

CONCLUSIONS AND PERSPECTIVES

This article has reviewed the many roles of SR proteins in geneexpression. An obvious question is, why are SR proteins involvedin so many cellular functions? These are very abundant nuclearproteins and a subset of them shuttle to the cytoplasm where theyare involved in NMD and translation regulation. Their functionin different biochemical activities may underlie the extensivenetwork of coupling amongst gene expression machines [209]. Itshould be noted that SR proteins are not the only proteins couplingnuclear and cytoplamic RNA processing events, since the EJC(exon junction complex), a multiprotein complex deposited asa consequence of pre-mRNA splicing, links pre-mRNA splicingwith mRNA export, NMD and translation (reviewed in [210]).Individual SR proteins regulate subsets of pre-mRNAs via spli-cing in the nucleus and post-splicing processes in the cytoplasm.The next few years will see considerable efforts to identify physio-logical RNA targets of SR proteins and gain a better understandingof the many cellular functions of these master regulators of RNAprocessing.

ACKNOWLEDGEMENTS

We are grateful to Sonia Guil (Barcelona) for critical reading of this manuscript.



FUNDING

This work was supported by the Medical Research Council and by the European AlternativeSplicing Network of Excellence, EURASNET [grant number S18238].

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207 Gabut, M., Mine, M., Marsac, C., Brivet, M., Tazi, J. and Soret, J. (2005) The SRprotein SC35 is responsible for aberrant splicing of the E1α pyruvate dehydrogenasemRNA in a case of mental retardation with lactic acidosis. Mol. Cell Biol. 25, 3286–3294

208 Watanuki, T., Funato, H., Uchida, S., Matsubara, T., Kobayashi, A., Wakabayashi, Y.,Otsuki, K., Nishida, A. and Watanabe, Y. (2008) Increased expression of splicing factorSRp20 mRNA in bipolar disorder patients. J. Affect. Disord. 110, 62–69

209 Maniatis, T. and Reed, R. (2002) An extensive network of coupling among geneexpression machines. Nature 416, 499–506

210 Tange, T. O., Nott, A. and Moore, M. J. (2004) The ever-increasing complexities of theexon junction complex. Curr. Opin. Cell Biol. 16, 279–284



211 Schaal, T. D. and Maniatis, T. (1999) Selection and characterization of pre-mRNAsplicing enhancers: identification of novel SR protein-specific enhancer sequences.Mol. Cell Biol. 19, 1705–1719

212 Tian, H. and Kole, R. (2001) Strong RNA splicing enhancers identified by a modifiedmethod of cycled selection interact with SR protein. J. Biol. Chem. 276,33833–33839

213 Shi, H., Hoffman, B. E. and Lis, J. T. (1997) A specific RNA hairpin loop structure bindsthe RNA recognition motifs of the Drosophila SR protein B52. Mol. Cell Biol. 17,2649–2657

214 Lou, H., Neugebauer, K. M., Gagel, R. F. and Berget, S. M. (1998) Regulation ofalternative polyadenylation by U1 snRNPs and SRp20. Mol. Cell Biol. 18, 4977–4985

215 Lynch, K. W. and Maniatis, T. (1996) Assembly of specific SR protein complexes ondistinct regulatory elements of the Drosophila doublesex splicing enhancer. Genes Dev.10, 2089–2101

216 Eldridge, A. G., Li, Y., Sharp, P. A. and Blencowe, B. J. (1999) The SRm160/300 splicingcoactivator is required for exon-enhancer function. Proc. Natl. Acad. Sci. U.S.A. 96,6125–6130

217 Simard, M. J. and Chabot, B. (2002) SRp30c is a repressor of 3′ splice site utilization.Mol. Cell Biol. 22, 4001–4010

218 Kennedy, C. F., Kramer, A. and Berget, S. M. (1998) A role for SRp54 during intronbridging of small introns with pyrimidine tracts upstream of the branch point.Mol. Cell Biol. 18, 5425–5434

Received 24 July 2008/4 September 2008; accepted 4 September 2008Published on the Internet 12 December 2008, doi:10.1042/BJ20081501


the sr protein family of splicing factors master regulators

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