SRSF3: Newly discovered functions and roles in human health and diseases

The serine/arginine rich proteins (SR proteins) are members of a family of RNA binding proteins involved in regulating various features of RNA metabolism, including pre-mRNA constitutive and alternative splicing. In humans, a total of 12 SR splicing factors (SRSFs) namely SRSF1-SRSF12 have been reported. SRSF3, the smallest member of the SR family and the focus of this review, regulates critical steps in mRNA metabolism and has been shown to have mRNA-independent functions as well. Recent studies on SRSF3 have uncovered its role in a wide array of complex biological processes. We have also reviewed the involvement of SRSF3 in disease conditions like cancer, ageing, neurological and cardiac disorders. Finally, we have discussed in detail the autoregulation of SRSF3 and its implications in cancer and commented on the potential of SRSF3 as a therapeutic target, especially in the context of cancer.     

RNA-dependent dynamic histone acetylation regulates MCL1 alternative splicing

Histone deacetylases (HDACs) and lysine acetyltransferases (KATs) catalyze dynamic histone acetylation at regulatory and coding regions of transcribed genes. Highly phosphorylated HDAC2 is recruited within corepressor complexes to regulatory regions, while the nonphosphorylated form is associated with the gene body. In this study, we characterized the nonphosphorylated HDAC2 complexes recruited to the transcribed gene body and explored the function of HDAC-complex-mediated dynamic histone acetylation. HDAC1 and 2 were coimmunoprecipitated with several splicing factors, including serine/arginine-rich splicing factor 1 (SRSF1) which has roles in alternative splicing. The co-chromatin immunoprecipitation of HDAC1/2 and SRSF1 to the gene body was RNA-dependent. Inhibition of HDAC activity and knockdown of HDAC1, HDAC2 or SRSF1 showed that these proteins were involved in alternative splicing of MCL1. HDAC1/2 and KAT2B were associated with nascent pre-mRNA in general and with MCL1 pre-mRNA specifically. Inhibition of HDAC activity increased the occupancy of KAT2B and acetylation of H3 and H4 of the H3K4 methylated alternative MCL1 exon 2 nucleosome. Thus, nonphosphorylated HDAC1/2 is recruited to pre-mRNA by splicing factors to act at the RNA level with KAT2B and other KATs to catalyze dynamic histone acetylation of the MCL1 alternative exon and alter the splicing of MCL1 pre-mRNA.     

Phosphorylation regulates arginine-rich RNA-binding protein solubility and oligomerization

Posttranslational modifications (PTMs) such as phosphorylation of RNA-binding proteins (RBPs) regulate several critical steps in RNA metabolism, including spliceosome assembly, alternative splicing, and mRNA export. Notably, serine-/arginine- (SR)-rich RBPs are densely phosphorylated compared with the remainder of the proteome. Previously, we showed that dephosphorylation of the splicing factor SRSF2 regulated increased interactions with similar arginine-rich RBPs U1-70K and LUC7L3. However, the large-scale functional and structural impact of these modifications on RBPs remains unclear. In this work, we dephosphorylated nuclear extracts using phosphatase in vitro and analyzed equal amounts of detergent-soluble and -insoluble fractions by mass-spectrometry-based proteomics. Correlation network analysis resolved 27 distinct modules of differentially soluble nucleoplasm proteins. We found classes of arginine-rich RBPs that decrease in solubility following dephosphorylation and enrich the insoluble pelleted fraction, including the SR protein family and the SR-like LUC7L RBP family. Importantly, increased insolubility was not observed across broad classes of RBPs. We determined that phosphorylation regulated SRSF2 structure, as dephosphorylated SRSF2 formed high-molecular-weight oligomeric species in vitro. Reciprocally, phosphorylation of SRSF2 by serine/arginine protein kinase 2 (SRPK2) in vitro decreased high-molecular-weight SRSF2 species formation. Furthermore, upon pharmacological inhibition of SRPKs in mammalian cells, we observed SRSF2 cytoplasmic mislocalization and increased formation of cytoplasmic granules as well as cytoplasmic tubular structures that associated with microtubules by immunocytochemical staining. Collectively, these findings demonstrate that phosphorylation may be a critical modification that prevents arginine-rich RBP insolubility and oligomerization.     

Dynamic Distribution and Interaction of the Arabidopsis SRSF1 Subfamily Splicing Factors

Ser/Arg-rich (SR) proteins are essential nucleus-localized splicing factors. Our prior studies showed that Arabidopsis (Arabidopsis thaliana) RSZ22, a homolog of the human SRSF7 SR factor, exits the nucleus through two pathways, either dependent or independent on the XPO1 receptor. Here, we examined the expression profiles and shuttling dynamics of the Arabidopsis SRSF1 subfamily (SR30, SR34, SR34a, and SR34b) under control of their endogenous promoter in Arabidopsis and in transient expression assay. Due to its rapid nucleocytoplasmic shuttling and high expression level in transient assay, we analyzed the multiple determinants that regulate the localization and shuttling dynamics of SR34. By site-directed mutagenesis of SR34 RNA-binding sequences and Arg/Ser-rich (RS) domain, we further show that functional RRM1 or RRM2 are dispensable for the exclusive protein nuclear localization and speckle-like distribution. However, mutations of both RRMs induced aggregation of the protein whereas mutation in the RS domain decreased the stability of the protein and suppressed its nuclear accumulation. Furthermore, the RNA-binding motif mutants are defective for their export through the XPO1 (CRM1/Exportin-1) receptor pathway, but retain nucleocytoplasmic mobility. We performed a yeast two hybrid screen with SR34 as bait and discovered SR45 as a new interactor. SR45 is an unusual SR splicing factor bearing two RS domains. These interactions were confirmed in planta by FLIM-FRET and BiFC and the roles of SR34 domains in protein-protein interactions were further studied. Altogether, our report extends our understanding of shuttling dynamics of Arabidopsis SR splicing factors.Ser/Arg-rich (SR) protein is the collective name given to a family of highly conserved splicing factors in Eukaryotes that regulate constitutive and alternative precursor mRNA splicing. SR proteins contain at least one RNA recognition motif (RRM) and an Arg/Ser-rich (RS) C-terminal domain (Manley and Krainer, 2010; Califice et al., 2012). The RRM appears to determine RNA-binding specificity, while the RS domain is involved in protein-protein and protein-RNA interactions (Shen et al., 2004). In human, twelve SR proteins have been described based on a set of formal criteria (Manley and Krainer, 2010). SR proteins have a modular organization: some SR proteins contain two RRMs while others contain a Zn-knuckle, which contributes to RNA binding. The activity of SR proteins is regulated by posttranslational modifications, such as Ser phosphorylation/dephosphorylation and Arg methylation. At steady-state, SR proteins accumulate in subnuclear speckles, which correspond to storage, assembly, and/or modification compartments for splicing factors. Several human SR proteins shuttle between the nucleus and the cytoplasm, and this dynamic shuttling is linked to their postsplicing activities in mRNA export, stability, and translation (Long and Caceres, 2009). The multiple roles and mechanisms of action of mammalian SR proteins have been extensively studied (for review, see Long and Caceres, 2009; Zhong et al., 2009; Kornblihtt et al., 2013; Änkö, 2014).The number of genes encoding SR proteins is higher in plants compared with metazoan. Plant genomes contain SR proteins homologous to the animal prototypes SRSF1/SRSF2/SRSF7, as well as plant-specific ones (Barta et al., 2010; Califice et al., 2012). Arabidopsis SR splicing factors localize into nuclear irregular dynamic domains similar to speckles, with no, only partial or complete colocalization (Tillemans et al., 2005; Lorković et al., 2008; Reddy et al., 2012). The functions of plant SR factors in postsplicing events remain unknown, though a nucleocytoplasmic shuttling activity has been described for RSZ22, a prototypic member of the SRSF7 subgroup (1 RRM, 1 Zn-knuckle) of Arabidopsis (Arabidopsis thaliana) SR protein family (Tillemans et al., 2006; Rausin et al., 2010).The nucleocytoplasmic transport of RNA and proteins occurs through nuclear pore complexes (NPCs), which require importin and exportin receptors (karyopherins or Kap) for trafficking of molecules larger than 40–90 kD. Kap often binds to cargo molecules that carry either nuclear localization signals (NLS) for nuclear import or nuclear export signals (NES) for nuclear export (Boruc et al., 2012). The best-known import pathway is mediated by the importin-α/β Kap that binds to NLS. Kap-β2 (or Transportin-SR, TRN-SR) was shown to function as the nuclear import receptor for human SRSF1 and SRSF2, and several Arabidopsis SR proteins (Yun et al., 2003; Xu et al., 2011). The human TRN-SR has recently been shown to embrace both the RRM and RS domains of SRSF1 for nuclear import (Maertens et al., 2014).XPO1 (Exportin-1, also named CRM1 in yeast [Saccharomyces cerevisiae]) is a well-characterized mammalian nuclear export receptor which recognizes Leu-rich NES (φ-X_2-3-φ-X_2-3-φ-X-φ, where φ is L, V, I, F, or M and X is any amino acid) on proteins implicated in snRNA and rRNA export (Natalizio and Wente, 2013). XPO1/CRM1 was also shown to mediate the export of unspliced (or partially spliced) viral mRNAs and of a small subset of mRNAs. XPO1 recruitment to mRNA is mediated by single adaptor proteins including Leu-rich pentatricopeptide repeat proteins (LRPPRC) and HuR (Natalizio and Wente, 2013). Apart from this, the bulk of mRNA is exported by the nonkaryopherin heterodimer Nxf1-Nxt1 (TAP-p15) in metazoans (Mex67-Mtr2 in yeast). The shuttling SR proteins are known to promote messenger ribonucleoprotein (mRNP) export through NPCs when dephosphorylated by interacting with export factor Nxf1 (Huang et al., 2003). Several human SR proteins are also part of the exon junction complex (EJC) deposited upstream of exon-exon junctions after splicing, consistent with a role of SR proteins in mRNP export and nonsense mediated RNA decay (Singh et al., 2012). The RS domain is necessary but not sufficient for the cytoplasmic export of shuttling SR proteins (Cáceres et al., 1997).We previously identified RSZ22 as a shuttling splicing factor whose nuclear export is at least partly controlled by the XPO1-dependent export pathway (Tillemans et al., 2006; Rausin et al., 2010). Mutating conserved residues within the RNA-binding motifs of this specific SR protein highlighted the in vivo dependence of RNA binding for proper subcellular dynamics (Rausin et al., 2010). However, the role of the different protein domains in directing the cellular dynamics may vary among SR proteins, and the role of the RS domain of RSZ22 had not been investigated. It is also unknown whether XPO1-dependent nuclear export also includes other Arabidopsis SR proteins. A more global understanding of the molecular mechanisms underlying the nucleocytoplasmic transport of plant SR factors therefore required further investigation.Here, we functionally characterized the four Arabidopsis SR proteins of the SRSF1 subfamily (orthologs of mammalian SRSF1) that contain two conserved RRM domains (Califice et al., 2012). We studied the expression profiles of SR30, SR34, SR34a, and SR34b, and attempted to investigate their shuttling activity. Among these SR proteins, SR30 showed a less active nuclear export rate, and SR34b protein was not detectable in any expression assay. Because of its stability and rapid shuttling, we further focused on the SR34 protein by generating a series of mutant versions of the RRMs and RS domains. We established the overall requirement of these protein domains to retain nucleocytoplasmic shuttling activity. Yeast two-hybrid (Y2H) assays also revealed strong interactions between SRSF1 subfamily members (SR30, SR34, and SR34a) and SR45, an atypical SR protein (two RS domains). We also investigated the importance of SR34 domains in protein-protein interactions. Collectively, our findings provide a more detailed mechanistic understanding of the role of the structural determinants regulating SR proteins dynamics, and insights into protein domain function in in vivo interactions.     

Exon 9 skipping of apoptotic caspase-2 pre-mRNA is promoted by SRSF3 through interaction with exon 8

Alternative splicing plays an important role in gene expression by producing different proteins from a gene. Caspase-2 pre-mRNA produces anti-apoptotic Casp-2S and pro-apoptotic Casp-2L proteins through exon 9 inclusion or skipping. However, the molecular mechanisms of exon 9 splicing are not well understood. Here we show that knockdown of SRSF3 (also known as SRp20) with siRNA induced significant increase of endogenous exon 9 inclusion. In addition, overexpression of SRSF3 promoted exon 9 skipping. Thus we conclude that SRSF3 promotes exon 9 skipping. In order to understand the functional target of SRSF3 on caspase-2 pre-mRNA, we performed substitution and deletion mutagenesis on the potential SRSF3 binding sites that were predicted from previous reports. We demonstrate that substitution mutagenesis of the potential SRSF3 binding site on exon 8 severely disrupted the effects of SRSF3 on exon 9 skipping. Furthermore, with the approach of RNA pulldown and immunoblotting analysis we show that SRSF3 interacts with the potential SRSF3 binding RNA sequence on exon 8 but not with the mutant RNA sequence. In addition, we show that a deletion of 26 nt RNA from 5′ end of exon 8, a 33 nt RNA from 3′ end of exon 10 and a 2225 nt RNA from intron 9 did not compromise the function of SRSF3 on exon 9 splicing. Therefore we conclude that SRSF3 promotes exon 9 skipping of caspase-2 pre-mRNA by interacting with exon 8. Our results reveal a novel mechanism of caspase-2 pre-mRNA splicing.     

Differential connectivity of splicing activators and repressors to the human spliceosome

BackgroundDuring spliceosome assembly, protein-protein interactions (PPI) are sequentially formed and disrupted to accommodate the spatial requirements of pre-mRNA substrate recognition and catalysis. Splicing activators and repressors, such as SR proteins and hnRNPs, modulate spliceosome assembly and regulate alternative splicing. However, it remains unclear how they differentially interact with the core spliceosome to perform their functions.ResultsHere, we investigate the protein connectivity of SR and hnRNP proteins to the core spliceosome using probabilistic network reconstruction based on the integration of interactome and gene expression data. We validate our model by immunoprecipitation and mass spectrometry of the prototypical splicing factors SRSF1 and hnRNPA1. Network analysis reveals that a factor’s properties as an activator or repressor can be predicted from its overall connectivity to the rest of the spliceosome. In addition, we discover and experimentally validate PPIs between the oncoprotein SRSF1 and members of the anti-tumor drug target SF3 complex. Our findings suggest that activators promote the formation of PPIs between spliceosomal sub-complexes, whereas repressors mostly operate through protein-RNA interactions.ConclusionsThis study demonstrates that combining in-silico modeling with biochemistry can significantly advance the understanding of structure and function relationships in the human spliceosome.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0682-5) contains supplementary material, which is available to authorized users.     

The structure and selectivity of the SR protein SRSF2 RRM domain with RNA

SRSF2 is a prototypical SR protein which plays important roles in the alternative splicing of pre-mRNA. It has been shown to be involved in regulatory pathways for maintaining genomic stability and play important roles in regulating key receptors in the heart. We report here the solution structure of the RNA recognition motifs (RRM) domain of free human SRSF2 (residues 9-101). Compared with other members of the SR protein family, SRSF2 structure has a longer L3 loop region. The conserved aromatic residue in the RNP2 motif is absent in SRSF2. Calorimetric titration shows that the RNA sequence 5'AGCAGAGUA3' binds SRSF2 with a K(d) of 61 ± 1 nM and a 1:1 stoichiometry. NMR and mutagenesis experiments reveal that for SFSF2, the canonical β1 and β3 interactions are themselves not sufficient for effective RNA binding; the additional loop L3 is crucial for RNA complex formation. A comparison is made between the structures of SRSF2-RNA complex with other known RNA complexes of SR proteins. We conclude that interactions involving the L3 loop, N- and C-termini of the RRM domain are collectively important for determining selectivity between the protein and RNA.     

Daily temperature cycles promote alternative splicing of RNAs encoding SR45a,a splicing regulator in maize

Elevated temperatures enhance alternative RNA splicing in maize (Zea mays) with the potential to expand the repertoire of plant responses to heat stress. Alternative RNA splicing generates multiple RNA isoforms for many maize genes, and here we observed changes in the pattern of RNA isoforms with temperature changes. Increases in maximum daily temperature elevated the frequency of the major modes of alternative splices (AS), in particular retained introns and skipped exons. The genes most frequently targeted by increased AS with temperature encode factors involved in RNA processing and plant development. Genes encoding regulators of alternative RNA splicing were themselves among the principal AS targets in maize. Under controlled environmental conditions, daily changes in temperature comparable to field conditions altered the abundance of different RNA isoforms, including the RNAs encoding the splicing regulator SR45a, a member of the SR45 gene family. We established an “in protoplast” RNA splicing assay to show that during the afternoon on simulated hot summer days, SR45a RNA isoforms were produced with the potential to encode proteins efficient in splicing model substrates. With the RNA splicing assay, we also defined the exonic splicing enhancers that the splicing-efficient SR45a forms utilize to aid in the splicing of model substrates. Hence, with rising temperatures on hot summer days, SR45a RNA isoforms in maize are produced with the capability to encode proteins with greater RNA splicing potential.

RNA splicing patterns for SR45a, a major RNA splicing regulator in maize, change in response to maximum daily temperature and vary during the day in response to daily temperature cycles.     

SRSF5 functions as a novel oncogenic splicing factor and is upregulated by oncogene SRSF3 in oral squamous cell carcinoma

Alternative splicing of precursor messenger RNA has been increasingly associated with tumorigenesis. The serine/arginine-rich protein (SR) family plays key roles in the regulation of pre-mRNA alternative splicing. Increasing evidence has demonstrated that the SR protein family is involved in tumorigenesis. However, the functions and mechanisms of SR proteins in tumourigenesis remain largely unknown. In the present study, we discovered that serine/arginine-rich splicing factor 5 (SRSF5) is a novel oncogenic splicing factor that is overexpressed in oral squamous cell carcinoma (OSCC) tissues and cells, being crucial for OSCC cell proliferation and tumor formation. Overexpression of SRSF5 transformed immortal rodent fibroblasts to form tumors in nude mice, while downregulation of SRSF5 in oral squamous cell lines retarded cell growth, cell cycle progression, and tumor growth. The expression of SRSF5 is controlled by an autoregulation mechanism. Serine/arginine-rich splicing factor 3 (SRSF3) has been identified as an oncogene. We found that SRSF5 is a novel target of SRSF3. SRSF3 impairs the autoregulation of SRSF5 and promotes SRSF5 overexpression in cancer cells. Altogether, the present study demonstrated that SRSF5 is a novel oncogene that is upregulated by SRSF3 in OSCC cells.     

Proteasome-Mediated Proteolysis of SRSF5 Splicing Factor Intriguingly Co-occurs with SRSF5 mRNA Upregulation during Late Erythroid Differentiation

SR proteins exhibit diverse functions ranging from their role in constitutive and alternative splicing, to virtually all aspects of mRNA metabolism. These findings have attracted growing interest in deciphering the regulatory mechanisms that control the tissue-specific expression of these SR proteins. In this study, we show that SRSF5 protein decreases drastically during erythroid cell differentiation, contrasting with a concomitant upregulation of SRSF5 mRNA level. Proteasome chemical inhibition provided strong evidence that endogenous SRSF5 protein, as well as protein deriving from stably transfected SRSF5 cDNA, are both targeted to proteolysis as the cells undergo terminal differentiation. Consistently, functional experiments show that overexpression of SRSF5 enhances a specific endogenous pre-mRNA splicing event in proliferating cells, but not in differentiating cells, due to proteasome-mediated targeting of both endogenous and transfection-derived SRSF5. Further investigation of the relationship between SRSF5 structure and its post-translation regulation and function, suggested that the RNA recognition motifs of SRSF5 are sufficient to activate pre-mRNA splicing, whereas proteasome-mediated proteolysis of SRSF5 requires the presence of the C-terminal RS domain of the protein. Phosphorylation of SR proteins is a key post-translation regulation that promotes their activity and subcellular availability. We here show that inhibition of the CDC2-like kinase (CLK) family and mutation of the AKT phosphorylation site Ser86 on SRSF5, have no effect on SRSF5 stability. We reasoned that at least AKT and CLK signaling pathways are not involved in proteasome-induced turnover of SRSF5 during late erythroid development.