Splicing factor SRSF3 represses the translation of programmed cell death 4 mRNA by associating with the 5′-UTR region

Serine/arginine-rich splicing factor 3 (SRSF3), a member of the serine/arginine (SR)-rich family of proteins, regulates both alternative splicing of pre-mRNA and export of mature mRNA from the nucleus. Although its role in nuclear mRNA processing is well understood, the mechanism by which it alters the fate of cytoplasmic mRNA molecules remains elusive. Here, we provide evidence that SRSF3 not only regulates the alternative splicing pattern of programmed cell death 4 (PDCD4) mRNA, but also modulates its translational efficiency in the cytoplasm by lowering translation levels. We observed a marked increase in PDCD4 mRNA in translating polysome fractions upon silencing of SRSF3, and, conversely, ectopic overexpression of SRSF3 shifted PDCD4 mRNA into non-translating ribosomal fractions. In live cells, SRSF3 colocalized with PDCD4 mRNA in P-bodies (PBs), where translationally silenced mRNAs are deposited, and this localization was abrogated upon SRSF3 silencing. Furthermore, using two different reporter systems, we showed that SRSF3 interacts directly with PDCD4 mRNA and mediates translational repression by binding to the 5′-untranslated region (5′-UTR). In summary, our data suggest that the oncogenic potential of SRSF3 might be realized, in part, through the translational repression of PDCD4 mRNA.     

The MKK(3/6)-p38-signaling cascade alters the subcellular distribution of hnRNP A1 and modulates alternative splicing regulation

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

The distribution of phosphorylated SR proteins and alternative splicing are regulated by RANBP2

The mammalian cell nucleus is functionally compartmentalized into various substructures. Nuclear speckles, also known as interchromatin granule clusters, are enriched with SR splicing factors and are implicated in gene expression. Here we report that nuclear speckle formation is developmentally regulated; in certain cases phosphorylated SR proteins are absent from the nucleus and are instead localized at granular structures in the cytoplasm. To investigate how the nuclear architecture is formed, we performed a phenotypic screen of HeLa cells treated with a series of small interfering RNAs. Depletion of Ran-binding protein 2 induced cytoplasmic intermediates of nuclear speckles in G1 phase. Detailed analyses of these structures suggested that a late step in the sequential nuclear entry of mitotic interchromatin granule components was disrupted and that phosphorylated SR proteins were sequestered in an SR protein kinase-dependent manner. As a result, the cells had an imbalanced subcellular distribution of phosphorylated and hypophosphorylated SR proteins, which affected alternative splicing patterns. This study demonstrates that the speckled distribution of phosphorylated pre-mRNA processing factors is regulated by the nucleocytoplasmic transport system in mammalian cells and that it is important for alternative splicing.     

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.     

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.     

The centrosomal kinase NEK2 is a novel splicing factor kinase involved in cell survival

NEK2 is a serine/threonine kinase that promotes centrosome splitting and ensures correct chromosome segregation during the G2/M phase of the cell cycle, through phosphorylation of specific substrates. Aberrant expression and activity of NEK2 in cancer cells lead to dysregulation of the centrosome cycle and aneuploidy. Thus, a tight regulation of NEK2 function is needed during cell cycle progression. In this study, we found that NEK2 localizes in the nucleus of cancer cells derived from several tissues. In particular, NEK2 co-localizes in splicing speckles with SRSF1 and SRSF2. Moreover, NEK2 interacts with several splicing factors and phosphorylates some of them, including the oncogenic SRSF1 protein. Overexpression of NEK2 induces phosphorylation of endogenous SR proteins and affects the splicing activity of SRSF1 toward reporter minigenes and endogenous targets, independently of SRPK1. Conversely, knockdown of NEK2, like that of SRSF1, induces expression of pro-apoptotic variants from SRSF1-target genes and sensitizes cells to apoptosis. Our results identify NEK2 as a novel splicing factor kinase and suggest that part of its oncogenic activity may be ascribed to its ability to modulate alternative splicing, a key step in gene expression regulation that is frequently altered in cancer cells.     

Shuttling SR proteins: more than splicing factors

Serine-arginine (SR) proteins commonly designate a family of eukaryotic RNA binding proteins containing a protein domain composed of several repeats of the arginine-serine dipeptide, termed the arginine-serine (RS) domain. This protein family is involved in essential nuclear processes such as constitutive and alternative splicing of mRNA precursors. Besides participating in crucial activities in the nuclear compartment, several SR proteins are able to shuttle between the nucleus and the cytoplasm and to exert regulatory functions in the latter compartment. This review aims at discussing the properties of shuttling SR proteins with particular emphasis on their nucleo-cytoplasmic traffic and their cytoplasmic functions. Indeed, recent findings have unravelled the complex regulation of SR protein nucleo-cytoplasmic distribution and the diversity of cytoplasmic mechanisms in which these proteins are involved.     

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.     

A conserved Drosophila transportin-serine/arginine-rich (SR) protein permits nuclear import of Drosophila SR protein splicing factors and their antagonist repressor splicing factor 1

Members of the highly conserved serine/arginine-rich (SR) protein family are nuclear factors involved in splicing of metazoan mRNA precursors. In mammals, two nuclear import receptors, transportin (TRN)-SR1 and TRN-SR2, are responsible for targeting SR proteins to the nucleus. Distinctive features in the nuclear localization signal between Drosophila and mammalian SR proteins prompted us to examine the mechanism by which Drosophila SR proteins and their antagonist repressor splicing factor 1 (RSF1) are imported into nucleus. Herein, we report the identification and characterization of a Drosophila importin beta-family protein (dTRN-SR), homologous to TRN-SR2, that specifically interacts with both SR proteins and RSF1. dTRN-SR has a broad localization in the cytoplasm and the nucleus, whereas an N-terminal deletion mutant colocalizes with SR proteins in nuclear speckles. Far Western experiments established that the RS domain of SR proteins and the GRS domain of RSF1 are required for the direct interaction with dTRN-SR, an interaction that can be modulated by phosphorylation. Using the yeast model system in which nuclear import of Drosophila SR proteins and RSF1 is impaired, we demonstrate that complementation with dTRN-SR is sufficient to target these proteins to the nucleus. Together, the results imply that the mechanism by which SR proteins are imported to the nucleus is conserved between Drosophila and humans.     

Acetylation and phosphorylation of SRSF2 control cell fate decision in response to cisplatin

SRSF2 is a serine/arginine-rich protein belonging to the family of SR proteins that are crucial regulators of constitutive and alternative pre-mRNA splicing. Although it is well known that phosphorylation inside RS domain controls activity of SR proteins, other post-translational modifications regulating SRSF2 functions have not been described to date. In this study, we provide the first evidence that the acetyltransferase Tip60 acetylates SRSF2 on its lysine 52 residue inside the RNA recognition motif, and promotes its proteasomal degradation. We also demonstrate that the deacetylase HDAC6 counters this acetylation and acts as a positive regulator of SRSF2 protein level. In addition, we show that Tip60 downregulates SRSF2 phosphorylation by inhibiting the nuclear translocation of both SRPK1 and SRPK2 kinases. Finally, we demonstrate that this acetylation/phosphorylation signalling network controls SRSF2 accumulation as well as caspase-8 pre-mRNA splicing in response to cisplatin and determines whether cells undergo apoptosis or G(2)/M cell cycle arrest. Taken together, these results unravel lysine acetylation as a crucial post-translational modification regulating SRSF2 protein level and activity in response to genotoxic stress.     

The Host Proteins Transportin SR2/TNPO3 and Cyclophilin A Exert Opposing Effects on HIV-1 Uncoating

Following entry of the HIV-1 core into target cells, productive infection depends on the proper disassembly of the viral capsid (uncoating). Although much is known regarding HIV-1 entry, the actions of host cell proteins that HIV-1 utilizes during early postentry steps are poorly understood. One such factor, transportin SR2 (TRN-SR2)/transportin 3 (TNPO3), promotes infection by HIV-1 and some other lentiviruses, and recent studies have genetically linked TNPO3 dependence of infection to the viral capsid protein (CA). Here we report that purified recombinant TNPO3 stimulates the uncoating of HIV-1 cores in vitro. The stimulatory effect was reduced by RanGTP, a known ligand for transportin family members. Depletion of TNPO3 in target cells rendered HIV-1 less susceptible to inhibition by PF74, a small-molecule HIV-1 inhibitor that induces premature uncoating. In contrast to the case for TNPO3, addition of the CA-binding host protein cyclophilin A (CypA) inhibited HIV-1 uncoating and reduced the stimulatory effect of TNPO3 on uncoating in vitro. In cells in which TNPO3 was depleted, HIV-1 infection was enhanced 4-fold by addition of cyclosporine, indicating that the requirement for TNPO3 in HIV-1 infection is modulated by CypA-CA interactions. Although TNPO3 was localized primarily to the cytoplasm, depletion of TNPO3 from target cells inhibited HIV-1 infection without reducing the accumulation of nuclear proviral DNA, suggesting that TNPO3 facilitates a stage of the virus life cycle subsequent to nuclear entry. Our results suggest that TNPO3 and cyclophilin A facilitate HIV-1 infection by coordinating proper uncoating of the core in target cells.     

Phosphorylation mechanism and structure of serine-arginine protein kinases

The splicing of mRNA requires a group of essential factors known as SR proteins, which participate in the maturation of the spliceosome. These proteins contain one or two RNA recognition motifs and a C-terminal domain rich in Arg-Ser repeats (RS domain). SR proteins are phosphorylated at numerous serines in the RS domain by the SR-specific protein kinase (SRPK) family of protein kinases. RS domain phosphorylation is necessary for entry of SR proteins into the nucleus, and may also play important roles in alternative splicing, mRNA export, and other processing events. Although SR proteins are polyphosphorylated in vivo, the mechanism underlying this complex reaction has only been recently elucidated. Human alternative splicing factor [serine/arginine-rich splicing factor 1 (SRSF1)], a prototype for the SR protein family, is regiospecifically phosphorylated by SRPK1, a post-translational modification that controls cytoplasmic-nuclear localization. SRPK1 binds SRSF1 with unusually high affinity, and rapidly modifies about 10-12 serines in the N-terminal region of the RS domain (RS1), using a mechanism that incorporates sequential, C-terminal to N-terminal phosphorylation and several processive steps. SRPK1 employs a highly dynamic feeding mechanism for RS domain phosphorylation in which the N-terminal portion of RS1 is initially bound to a docking groove in the large lobe of the kinase domain. Upon subsequent rounds of phosphorylation, this N-terminal segment translocates into the active site, and a β-strand in RNA recognition motif 2 unfolds and occupies the docking groove. These studies indicate that efficient regiospecific phosphorylation of SRSF1 is the result of a contoured binding cavity in SRPK1, a lengthy Arg-Ser repetitive segment in the RS domain, and a highly directional processing mechanism.     

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.     

The cargo-binding domain of transportin 3 is required for lentivirus nuclear import

Lentiviruses, unlike the gammaretroviruses, are able to infect nondividing cells by transiting through nuclear pores to access the host genomic DNA. Several nuclear import and nuclear pore components have been implicated as playing a role in nuclear import, including transportin 3 (TNPO3), a member of the importin-β family of nuclear import proteins. We demonstrated that TNPO3 was required by several lentiviruses, with simian immunodeficiency virus mac239 (SIVmac239) and equine infectious anemia virus (EIAV) the most dependent and human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) the least. Analysis of HIV-1/SIVmac239 chimeric viruses showed that dependence on TNPO3 mapped to the SIVmac239 capsid. Mutation of a single amino acid, A76V in the SIVmac239 capsid, rendered the virus TNPO3 independent and resistant to mCPSF6-358, a truncated splicing factor that prevents HIV-1 nuclear import. Using a complementation assay based on 293T cells that express a TNPO3-targeted short hairpin RNA (shRNA), we showed that the Drosophila TNPO3 homologue can substitute for its human counterpart and that it mapped a key functional domain of TNPO3 to the carboxy-terminal cargo-binding domain. Within the cargo-binding domain, two hydrophobic motifs were required for TNPO3-dependent infection. The mutated TNPO3 proteins maintained their ability to localize to the nucleus, suggesting that their inability to restore lentivirus infection resulted from an inability to bind to a host or viral cargo protein.