Serine/arginine-rich Proteins Research Articles

Discovery and Expression Analysis of Alternative Splicing Events Conserved among Plant SR Proteins

The high frequency of alternative splicing among the serine/arginine-rich (SR) family of proteins in plants has been linked to important roles in gene regulation during development and in response to environmental stress. In this article, we have searched and manually annotated all the SR proteins in the genomes of maize and sorghum. The experimental validation of gene structure by reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed, with few exceptions, that SR genes produced multiple isoforms of transcripts by alternative splicing. Despite sharing high structural similarity and conserved positions of the introns, the profile of alternative splicing diverged significantly between maize and sorghum for the vast majority of SR genes. These include many transcript isoforms discovered by RT-PCR and not represented in extant expressed sequence tag (EST) collection. However, we report the occurrence of various maize and sorghum SR mRNA isoforms that display evolutionary conservation of splicing events with their homologous SR genes in Arabidopsis and moss. Our data also indicate an important role of both 5' and 3' untranslated regions in the regulation of SR gene expression. These observations have potentially important implications for the processes of evolution and adaptation of plants to land.

P.5.12 A mutation in TNPO3 causes LGMD1F and characteristic nuclear pathology

Limb-girdle muscular dystrophy 1F (LGMD1F) is an autosomal dominant muscular disease affecting a Spanish family. Using whole genome sequencing, we identified a single nucleotide deletion (c.2771del) in transportin-3 gene ( TNPO3 ) in a LGMD1F patient. The mutation disrupts the termination codon of TNPO3 and causes a reading frame shift. Transportin-3 is a nuclear protein, and mediates import of serine–arginine rich proteins into nucleus, which is important for mRNA splicing. This study aimed to investigate the significance of transportin-3 in the pathogenesis of LGMD1F. We performed dideoxy-sequencing of TNPO3 in 24 affected and 23 unaffected family members. Muscle specimens from 4 patients were analyzed by conventional stains and immunohistochemistry. Direct sequence of TNPO3 revealed that all patients carried a heterozygous mutation, and none of the unaffected subjects had the mutation. Hematoxylin-eosin (HE) stained muscle revealed nuclei (10.7 ± 3.0%; mean ± SD) with central pallor in all patients studied. Immunohistochemistry with anti-transportin-3 antibody showed colocalization with nuclei in control subjects. In patients, transportin-3 was also observed within nuclei, but was often unevenly distributed in periphery, a staining pattern similar to that seen by HE. Genetic and histological studies in a Spanish family strongly support the hypothesis that TNPO3 is the causative gene of LGMD1F. Pathological study also indicates that the subcellular distribution of transportin-3 is disrupted and affects the structure of nuclei.

Characterization of Nuclear Localization Signals (NLSs) and Function of NLSs and Phosphorylation of Serine Residues in Subcellular and Subnuclear Localization of Transformer-2β (Tra2β)

The serine/arginine-rich (SR) proteins are one type of major actors in regulation of pre-mRNA splicing. Their functions are closely related to the intracellular spatial organization. The RS domain and phosphorylation status of SR proteins are two critical factors in determining the subcellular distribution. Mammalian Transformer-2β (Tra2β) protein, a member of SR proteins, is known to play multiple important roles in development and diseases. In the present study, we characterized the subcellular and subnuclear localization of Tra2β protein and its related mechanisms. The results demonstrated that in the brain the nuclear and cytoplasmic localization of Tra2β were correlated with its phosphorylation status. Using deletional mutation analysis, we showed that the nuclear localization of Tra2β was determined by multiple nuclear localization signals (NLSs) in the RS domains. The point-mutation analysis disclosed that phosphorylation of serine residues in the NLSs inhibited the function of NLS in directing Tra2β to the nucleus. In addition, we identified at least two nuclear speckle localization signals within the RS1 domain, but not in the RS2 domain. The nuclear speckle localization signals determined the localization of RS1 domain-contained proteins to the nuclear speckle. The function of the signals did not depend on the presence of serine residues. The results provide new insight into the mechanisms by which the subcellular and subnuclear localization of Tra2β proteins are regulated.

Serine Arginine Splicing Factor 3 Is Involved in Enhanced Splicing of Glucose-6-phosphate Dehydrogenase RNA in Response to Nutrients and Hormones in Liver

Expression of G6PD is controlled by changes in the degree of splicing of the G6PD mRNA in response to nutrients in the diet. This regulation involves an exonic splicing enhancer (ESE) in exon 12 of the mRNA. Using the G6PD model, we demonstrate that nutrients and hormones control the activity of serine-arginine-rich (SR) proteins, a family of splicing co-activators, and thereby regulate the splicing of G6PD mRNA. In primary rat hepatocyte cultures, insulin increased the amount of phosphorylated SR proteins, and this effect was counteracted by arachidonic acid. The results of RNA affinity analysis with nuclear extracts from intact liver demonstrated that the SR splicing factor proteins SRSF3 and SRSF4 bound to the G6PD ESE. Consequently, siRNA-mediated depletion of SRSF3, but not SRSF4, in liver cells inhibited accumulation of both mRNA expressed from a minigene containing exon 12 and the endogenous G6PD mRNA. Consistent with the functional role of SRSF3 in regulating splicing, SRSF3 was observed to bind to the ESE in both intact cells and in animals using RNA immunoprecipitation analysis. Furthermore, refeeding significantly increased the binding of SRSF3 coincident with increased splicing and expression of G6PD. Together, these data establish that nutritional regulation of SRSF3 activity is involved in the differential splicing of the G6PD transcript in response to nutrients. Nutritional regulation of other SR proteins presents a regulatory mechanism that could cause widespread changes in mRNA splicing. Nutrients are therefore novel regulators of mRNA splicing.

Phosphoproteome dynamics reveal novel ERK1/2 MAP kinase substrates with broad spectrum of functions

The ERK1/2 MAP kinase pathway is an evolutionarily conserved signaling module that controls many fundamental physiological processes. Deregulated activity of ERK1/2 MAP kinases is associated with developmental syndromes and several human diseases. Despite the importance of this pathway, a comprehensive picture of the natural substrate repertoire and biochemical mechanisms regulated by ERK1/2 is still lacking. In this study, we used large-scale quantitative phosphoproteomics and bioinformatics analyses to identify novel candidate ERK1/2 substrates based on their phosphorylation signature and kinetic profiles in epithelial cells. We identified a total of 7936 phosphorylation sites within 1861 proteins, of which 155 classify as candidate ERK1/2 substrates, including 128 new targets. Candidate ERK1/2 substrates are involved in diverse cellular processes including transcriptional regulation, chromatin remodeling, RNA splicing, cytoskeleton dynamics, cellular junctions and cell signaling. Detailed characterization of one newly identified substrate, the transcriptional regulator JunB, revealed that ERK1/2 phosphorylate JunB on a serine adjacent to the DNA-binding domain, resulting in increased DNA-binding affinity and transcriptional activity. Our study expands the spectrum of cellular functions controlled by ERK1/2 kinases.

Structure of Phosphorylated SF1 Bound to U2AF65 in an Essential Splicing Factor Complex

The essential splicing factors U2AF⁶⁵ and SF1 cooperatively bind consensus sequences at the 3' end of introns. Phosphorylation of SF1 on a highly conserved "SPSP" motif enhances its interaction with U2AF⁶⁵ and the pre-mRNA. Here, we reveal that phosphorylation induces essential conformational changes in SF1 and in the SF1/U2AF⁶⁵/3' splice site complex. Crystal structures of the phosphorylated (P)SF1 domain bound to the C-terminal domain of U2AF⁶⁵ at 2.29 Å resolution and of the unphosphorylated SF1 domain at 2.48 Å resolution demonstrate that phosphorylation induces a disorder-to-order transition within a previously unknown SF1/U2AF⁶⁵ interface. We find by small-angle X-ray scattering that the local folding of the SPSP motif transduces into global conformational changes in the nearly full-length (P)SF1/U2AF⁶⁵/3' splice site assembly. We further determine that SPSP phosphorylation and the SF1/U2AF⁶⁵ interface are essential in vivo. These results offer a structural prototype for phosphorylation-dependent control of pre-mRNA splicing factors.

Biological Analysis of SRSF2 Mutations in Leukemogenesis

Abstract 1282Emerging evidence is establishing a connection between MDS and spliceosome mutations. Spliceosome including SF3b1, U2AF1 and SRSF2 are frequently and exclusively mutated in myelodysplastic syndromes (MDS) and related myeloid neoplasms. Spliceosome mutations occur at varying frequencies in different disease subtypes. SF3B1 was shown to be highly associated with MDS characterized by increased ring sideroblasts and SRSF2 mutations are more prevalent in chronic myelomonocytic leukemia. In spite of the fact that the recent discovery constitutes a novel class of genomic lesions and defines an entirely new pathogenic pathway of leukaemogenesis, the pathogenesis of spliceosome mutation is not largely understood. To understanding the biological consequences of spliceosomal mutations, we previously reported mutant U2AF1 cause altered RNA splicing, and overexpressed mutant U2AF1 decrease in cell proliferarion. However, currently, no functional analysis of SRSF2 mutation has been published.SRSF2 belongs to the serine/arginine-rich (SR) protein family. SR proteins are a family of RNA binding proteins characterized by one or two RNA recognition motifs (RRMs) and a signature RS domain enriched with arginine and serine repeats (RS domain).Growing body of evidence suggests that SR protein may be directly involved in the process of carcinogenesis. Gene knockout experiment indicated SRSF2 is involved with specific pathways in regulating cell proliferation and genomic stability during mammalian organogenesis. In neck and head tumor, SRSF2 is frequently overexpressed. And upregulated SRSF2 increases missplicing and downregulates E-cadherin expression, which is an important tumor suppressor gene. Therefore SRSF2 potential function in tumorigenesis is suggested in epithelial cancers. SRSF2 mutations with MDS exclusively occur at P95 within an intervening sequence between RRM and RS domains, indicating a gain-of-function nature of these mutations.So, to clarify the biological role of SRSF2 mutations in leukemogenesis, we evaluated the oncogenic role of SRSF mutations by expressing a mutant SRSF2 allele in Jurkat cells. The cells transduced with a tumor-derived SRSF2 allele showed reduced cell proliferation and increased apoptosis compared to the mock and wild type SRSF2-transduced cells. Next we performed in vitro colony assay using a highly purified hematopoietic stem cell population (CD34-c-Kit+ScaI+ Lin-(CD34-KSL) cells) collected from C57BL/6 (B6)-Ly5.1 mouse that was retrovirally transduced with mock, mutant or wild-type SRSF2 construct. The mutant SRSF2-transduced cells showed reduced cell proliferation compared with mock- or wild-type SRSF2 transduced cells. Subsequently, we conducted bone marrow transplantaion assay. We collected CD34-KSL cells from B6-Ly5.1 mouse, and retrovirally transduce mock, mutant or wild-type SRSF2 construct, each harbouring the EGFP marker gene. And these cells were sorted by EGFP marker, and transplanted with competitor cells (B6-Ly5.1/5.2 F1 mice origin) into lethally irradiated B6-Ly5.2 mice. The wild-type SRSF2-transduced cells showed a lower reconstitution capacity than the mock-transduced cells. On the other hand, the recipients of the cells transduced with the mutant SRSF2 showed lower EGFP-positive cell chimaerism than those of the mock- or the wild-type SRSF2-transduced. Therefore, the mutant SRSF2 was indicated to have a negative effect on cellular proliferation capacity in vitro and in vivo, and a gain-of-function nature of these mutations is suggested. These results are similar to the effect of U2AF1 mutant, which we reported mutant U2AF1 transduced TF-1 and HeLa cells present with a decrease in cell proliferation and hematopoietic stem cells expressing mutant U2AF1 also displayed lower reconstitution capacity by competitive reconstitution assay in mice. So far, the mechanism responsible for the growth advantage of mutant cells in patient is unclear. We furthermore observe hematopoietic phenotype of the bone marrow transplanted model mouse. SRSF2 mutations can coexist with mutations in TET2, ASXL1 and RUNX1. Therefore we performed additionally bone marrow transplantation assay, utilizing hematopoietic cells derived from TET2 knockdown mice, as a model of multistep carcinogenesis. We will present the results of our biological assay on the SRSF2 mutations and discuss the pathogenesis of MDS. Disclosures:No relevant conflicts of interest to declare.

Identification of an intronic splicing regulatory element involved in auto‐regulation of alternative splicing of SCL33 pre‐mRNA

In Arabidopsis, pre-mRNAs of serine/arginine-rich (SR) proteins undergo extensive alternative splicing (AS). However, little is known about the cis-elements and trans-acting proteins involved in regulating AS. Using a splicing reporter (GFP-intron-GFP), consisting of the GFP coding sequence interrupted by an alternatively spliced intron of SCL33, we investigated whether cis-elements within this intron are sufficient for AS, and which SR proteins are necessary for regulated AS. Expression of the splicing reporter in protoplasts faithfully produced all splice variants from the intron, suggesting that cis-elements required for AS reside within the intron. To determine which SR proteins are responsible for AS, the splicing pattern of the GFP-intron-GFP reporter was investigated in protoplasts of three single and three double mutants of SR genes. These analyses revealed that SCL33 and a closely related paralog, SCL30a, are functionally redundant in generating specific splice variants from this intron. Furthermore, SCL33 protein bound to a conserved sequence in this intron, indicating auto-regulation of AS. Mutations in four GAAG repeats within the conserved region impaired generation of the same splice variants that are affected in the scl33 scl30a double mutant. In conclusion, we have identified the first intronic cis-element involved in AS of a plant SR gene, and elucidated a mechanism for auto-regulation of AS of this intron.

The Cellular EJC Interactome Reveals Higher-Order mRNP Structure and an EJC-SR Protein Nexus

In addition to sculpting eukaryotic transcripts by removing introns, pre-mRNA splicing greatly impacts protein composition of the emerging mRNP. The exon junction complex (EJC), deposited upstream of exon-exon junctions after splicing, is a major constituent of spliced mRNPs. Here, we report comprehensive analysis of the endogenous human EJC protein and RNA interactomes. We confirm that the major "canonical" EJC occupancy site in vivo lies 24 nucleotides upstream of exon junctions and that the majority of exon junctions carry an EJC. Unexpectedly, we find that endogenous EJCs multimerize with one another and with numerous SR proteins to form megadalton sized complexes in which SR proteins are super-stoichiometric to EJC core factors. This tight physical association may explain known functional parallels between EJCs and SR proteins. Further, their protection of long mRNA stretches from nuclease digestion suggests that endogenous EJCs and SR proteins cooperate to promote mRNA packaging and compaction.

Regulating the regulators: Serine/arginine‐rich proteins under scrutiny

Serine/arginine-rich (SR) proteins are among the most studied splicing regulators. They constitute a family of evolutionarily conserved proteins that, apart from their initially identified and deeply studied role in splicing regulation, have been implicated in genome stability, chromatin binding, transcription elongation, mRNA stability, mRNA export and mRNA translation. Remarkably, this list of SR protein activities seems far from complete, as unexpected functions keep being unraveled. An intriguing aspect that awaits further investigation is how the multiple tasks of SR proteins are concertedly regulated within mammalian cells. In this article, we first discuss recent findings regarding the regulation of SR protein expression, activity and accessibility. We dive into recent studies describing SR protein auto-regulatory feedback loops involving different molecular mechanisms such asunproductive splicing, microRNA-mediated regulation and translational repression. In addition, we take into account another step of regulation of SR proteins, presenting new findings about a variety of post-translational modifications by proteomics approaches and how some of these modifications can regulate SR protein sub-cellular localization or stability. Towards the end, we focus in two recently revealed functions of SR proteins beyond mRNA biogenesis and metabolism, the regulation of micro-RNA processing and the regulation of small ubiquitin-like modifier (SUMO) conjugation.

Human papillomavirus gene expression is controlled by host cell splicing factors

HPVs (human papillomaviruses) infect stratified epithelia and cause a variety of lesions ranging from benign warts to invasive tumours. The virus life cycle is tightly linked to differentiation of the keratinocyte it infects: papillomaviruses modulate host gene expression to ensure efficient virus replication. For example, the viral transcription factor E2 can directly up-regulate, in an epithelial differentiation-dependent manner, cellular SRSFs [SR (serine/arginine-rich) splicing factors] that control constitutive and alternative splicing. Changes in alternative splicing and the mechanisms controlling this for viral mRNAs have been the subject of intense exploration. However, to date experiments have only been carried out in model systems because the genetic systems suitable for studying alternative splicing of viral RNAs in the context of the virus life cycle are relatively recent and technically challenging. Now using these life cycle-supporting systems, our laboratory has identified SR proteins as important players in differentiation-dependent regulation of HPV gene expression. Better understanding of the role of cellular factors in regulating the virus life cycle is needed as it may help development of novel diagnostic approaches and antiviral therapies in the future.

P3‐352: Leucettines, a family of pharmacological inhibitors of DYRKs and CLKs derived from the marine sponge: Natural product Leucettamine B

We here report on the synthesis, optimization and biological characterization of leucettines, a family of kinase inhibitors derived from the marine sponge leucettamine B. Stepwise synthesis of analogues starting from the natural structure, guided by activity testing on 8 purified kinases, led to highly potent inhibitors of CLKs and DYRKs, two families of kinases involved in alternative premRNA splicing and Alzheimer’s disease / Down syndrome. Leucettine L41 was co-crystallized with DYRK1A, DYRK2, CLK3 and PIM1. It interacts with key residues located within the ATPbinding pocket of the kinase. Leucettine L41 inhibits the phosphorylation of serine/arginine-rich proteins (SRp), a family of proteins regulating pre-RNA splicing. Indeed leucettine L41 was demonstrated to modulate alternative pre-mRNA splicing in a cell based reporting system. The selectivity of Leucettine L41 was extensively studied (kinase activity and interaction assays, affinity chromatography). Leucettine L41 provides protection against glutamate -induced cell death in cultured mouse HT22 hippocampal cells. It also provides neuroprotection against APP-induced cell death in mouse brain slices. Finally it prevents in vivo cognitive impairments due to intracerebroventricular injection of amyloid-# 25-35. Leucettines should be further explored as pharmacological tools to study and modulate preRNA splicing. Leucettines should also be investigated as potential therapeutic drugs in Alzheimer’s disease and in diseases involving abnormal pre-mRNA splicing.

Alternative splicing in human tumour viruses: a therapeutic target?

Persistent infection with cancer risk-related viruses leads to molecular, cellular and immune response changes in host organisms that in some cases direct cellular transformation. Alternative splicing is a conserved cellular process that increases the coding complexity of genomes at the pre-mRNA processing stage. Human and other animal tumour viruses use alternative splicing as a process to maximize their transcriptomes and proteomes. Medical therapeutics to clear persistent viral infections are still limited. However, specific lessons learned in some viruses [e.g. HIV and HCV (hepatitis C virus)] suggest that drug-directed inhibition of alternative splicing could be useful for this purpose. The present review describes the basic mechanisms of constitutive and alternative splicing in a cellular context and known splicing patterns and the mechanisms by which these might be achieved for the major human infective tumour viruses. The roles of splicing-related proteins expressed by these viruses in cellular and viral gene regulation are explored. Moreover, we discuss some currently available drugs targeting SR (serine/arginine-rich) proteins that are the main regulators of constitutive and alternative splicing, and their potential use in treatment for so-called persistent viral infections.

Spliceosome Protein (SRp) Regulation of Glucocorticoid Receptor Isoforms and Glucocorticoid Response in Human Trabecular Meshwork Cells

Glaucoma is a leading cause of visual impairment and blindness, with elevated intraocular pressure (IOP) as a major causative risk factor. Glucocorticoid (GC) therapy causes morphologic and biochemical changes in the trabecular meshwork (TM), an ocular tissue involved in regulating IOP, which can lead to the development of glaucoma in susceptible individuals (steroid responders). Steroid responders comprise 40% of the general population and are at higher risk of developing glaucoma. In addition, a majority of glaucoma patients are steroid responders. Differential distribution of various isoforms of GC receptor (GR) may be responsible for this heterogeneity in the steroid response. The alternatively spliced GRβ isoform acts as dominant negative regulator of classical GRα transcriptional activity. mRNA splicing is mediated by spliceosomes, which include serine-arginine rich proteins (SRps). The purpose of this study was to determine whether specific SRps regulate levels of these isoforms and thereby GC response in TM cells. Quantitative RT-PCR, Western blot analysis, and immunocytochemistry were used to determine the differential expression of different SRps (SRp20, 30c, and 40) in human normal and glaucomatous TM cell strains. Bioinformatics was used to find putative binding sites for SRp20 and SRp40 on exon 9 of the GR gene. A peptide modulator of splicing (bombesin) and SRp expression vectors were used to modulate SRp levels and determine their effects on GRα/GRβ ratios as well as dexamethasone (DEX) responsiveness via GRE- luciferase reporter activity, fibronectin, and myocilin induction in TM cells. SRp20, SRp30c, and SRp40 regulate GR splicing and the GC response in TM cells. Modulation of SRp levels altered the GRβ/α ratio that correlated with DEX responsiveness. Bombesin decreased SRp20; increased SRp30c, SRp40 levels, and GRβ/α ratio, and suppressed DEX response in TM cells. Relative levels of SRp20, SRp30c, and SRp40 in TM cells control differential expression of the two alternatively spliced isoforms of the GR and thereby regulate GC responsiveness. Different levels and/or activities of these SRps may account for differential GC sensitivity among the normal and glaucoma populations.

Loss of Pnn expression results in mouse early embryonic lethality and cellular apoptosis through SRSF1-mediated alternative expression of Bcl-xS and ICA**

Pinin (Pnn), a serine/arginine-rich (SR)-related protein, has been shown to play multiple roles within eukaryotic cells including cell-cell adhesion, cell migration, regulation of gene transcription, mRNA export and alternative splicing. In this study, an attempt to generate mice homozygously deficient in Pnn failed because of early embryonic lethality. To evaluate the effects of loss of Pnn expression on cell survival, RNA interference experiments were performed in MCF-7 cells. Depletion of Pnn resulted in cellular apoptosis and nuclear condensation. In addition, nuclear speckles were disrupted, and expression levels of SR proteins were diminished. RT-PCR analysis showed that alternative splicing patterns of SRSF1 as well as of apoptosis-related genes Bcl-x and ICAD were altered, and expression levels of Bim isoforms were modulated in Pnn-depleted cells. Cellular apoptosis induced by Pnn depletion was rescued by overexpression of SRSF1, which also restored generation of Bcl-xL and functionless ICAD. Pnn expression is, therefore, essential for survival of mouse embryos and the breast carcinoma cell line MCF-7. Moreover, Pnn depletion, modulated by SRSF1, determines cellular apoptosis through activation of the expression of pro-apoptotic Bcl-xS transcripts.

Son maintains accurate splicing for a subset of human pre-mRNAs

Serine-arginine-rich (SR) proteins play a key role in alternative pre-mRNA splicing in eukaryotes. We recently showed that a large SR protein called Son has unique repeat motifs that are essential for maintaining the subnuclear organization of pre-mRNA processing factors in nuclear speckles. Motif analysis of Son highlights putative RNA interaction domains that suggest a direct role for Son in pre-mRNA splicing. Here, we used in situ approaches to show that Son localizes to a reporter minigene transcription site, and that RNAi-mediated Son depletion causes exon skipping on reporter transcripts at this transcription site. A genome-wide exon microarray analysis was performed to identify human transcription and splicing targets of Son. Our data show that Son-regulated splicing encompasses all known types of alternative splicing, the most common being alternative splicing of cassette exons. We confirmed that knockdown of Son leads to exon skipping in pre-mRNAs for chromatin-modifying enzymes, including ADA, HDAC6 and SetD8. This study reports a comprehensive view of human transcription and splicing targets for Son in fundamental cellular pathways such as integrin-mediated cell adhesion, cell cycle regulation, cholesterol biosynthesis, apoptosis and epigenetic regulation of gene expression.

A single ancient origin for prototypical serine/arginine-rich splicing factors.

Eukaryotic precursor mRNA splicing is a process involving a very complex RNA-protein edifice. Serine/arginine-rich (SR) proteins play essential roles in precursor mRNA constitutive and alternative splicing and have been suggested to be crucial in plant-specific forms of developmental regulation and environmental adaptation. Despite their functional importance, little is known about their origin and evolutionary history. SR splicing factors have a modular organization featuring at least one RNA recognition motif (RRM) domain and a carboxyl-terminal region enriched in serine/arginine dipeptides. To investigate the evolution of SR proteins, we infer phylogenies for more than 12,000 RRM domains representing more than 200 broadly sampled organisms. Our analyses reveal that the RRM domain is not restricted to eukaryotes and that all prototypical SR proteins share a single ancient origin, including the plant-specific SR45 protein. Based on these findings, we propose a scenario for their diversification into four natural families, each corresponding to a main SR architecture, and a dozen subfamilies, of which we profile both sequence conservation and composition. Finally, using operational criteria for computational discovery and classification, we catalog SR proteins in 20 model organisms, with a focus on green algae and land plants. Altogether, our study confirms the homogeneity and antiquity of SR splicing factors while establishing robust phylogenetic relationships between animal and plant proteins, which should enable functional analyses of lesser characterized SR family members, especially in green plants.

Tumor suppressive microRNA-1 mediated novel apoptosis pathways through direct inhibition of splicing factor serine/arginine-rich 9 (SRSF9/SRp30c) in bladder cancer

We have previously found that restoration of tumor suppressive microRNA-1 (miR-1), induced cell apoptosis in bladder cancer (BC) cell lines. However, the apoptosis mechanism induced by miR-1 was not fully elucidated. Alternative splicing of mRNA precursors provides cancer cells with opportunities to translate many oncogenic protein variants, which promote cell proliferation and survival under unpreferable condition for cancer development. Serine/arginine-rich (SR) protein family, which involved in alternative pre-mRNA splicing, plays a critical role for regulating apoptosis by splicing apoptosis-related genes. However, transcriptional regulation of SR proteins, themselves, has not been elucidated. In this study, we focused on splicing factor serine/arginine-rich 9 (SRSF9/SRp30c) on the basis of our previous genome-wide gene expression analysis using miR-1-transfected BC cell lines because putative target sites of miR-1 are existed in 3′-untranslated region (UTR) of SRSF9 mRNA. The expression levels of mRNA of SRSF9 were extremely reduced in the miR-1 transfectants. A luciferase activity significantly decreased in the transfectants suggesting that actual binding occurred between miR-1 and 3′UTR of SRSF9 mRNA. Loss-of-function assays demonstrated that significant inhibitions of cell proliferation, migration, and invasion were observed in the si-SRSF9 transfectants. Apoptosis assays demonstrated that cell apoptosis fraction increased and that caspase-3/7 was activated in the si-SRSF9 transfectants. Our data indicated that tumor suppressive miR-1 induces apoptosis through direct inhibition of SRSF9 in BC. The identification of molecular mechanisms between miRNAs and SR proteins could provide novel apoptosis pathways and their epigenetic regulations and offer new strategies for BC treatment.

VEGF Spliced Variants: Possible Role of Anti-Angiogenesis Therapy

Angiogenesis has been targeted in retinopathies, psoriasis, and a variety of cancers (colon, breast, lung, and kidney). Among these tumour types, clear cell renal cell carcinomas (RCCs) are the most vascularized tumours due to mutations of the von Hippel Lindau gene resulting in HIF-1 alpha stabilisation and overexpression of Vascular Endothelial Growth Factor (VEGF). Surgical nephrectomy remains the most efficient curative treatment for patients with noninvasive disease, while VEGF targeting has resulted in varying degrees of success for treating metastatic disease. VEGF pre-mRNA undergoes alternative splicing generating pro-angiogenic isoforms. However, the recent identification of novel splice variants of VEGF with anti-angiogenic properties has provided some insight for the lack of current treatment efficacy. Here we discuss an explanation for the relapse to anti-angiogenesis treatment as being due to either an initial or acquired resistance to the therapy. We also discuss targeting angiogenesis via SR (serine/arginine-rich) proteins implicated in VEGF splicing.

Plant serine/arginine‐rich proteins: roles in precursor messenger RNA splicing, plant development, and stress responses

Global analyses of splicing of precursor messenger RNAs (pre-mRNAs) have revealed that alternative splicing (AS) is highly pervasive in plants. Despite the widespread occurrence of AS in plants, the mechanisms that control splicing and the roles of splice variants generated from a gene are poorly understood. Studies on plant serine/arginine-rich (SR) proteins, a family of highly conserved proteins, suggest their role in both constitutive splicing and AS of pre-mRNAs. SR proteins have a characteristic domain structure consisting of one or two RNA recognition motifs at the N-terminus and a C-terminal RS domain rich in arginine/serine dipeptides. Plants have many more SR proteins compared to animals including several plant-specific subfamilies. Pre-mRNAs of plant SR proteins are extensively alternatively spliced to increase the transcript complexity by about six-fold. Some of this AS is controlled in a tissue- and development-specific manner. Furthermore, AS of SR pre-mRNAs is altered by various stresses, raising the possibility of rapid reprogramming of the whole transcriptome by external signals through regulation of the splicing of these master regulators of splicing. Most SR splice variants contain a premature termination codon and are degraded by up-frameshift 3 (UPF3)-mediated nonsense-mediated decay (NMD), suggesting a link between NMD and regulation of expression of the functional transcripts of SR proteins. Limited functional studies with plant SRs suggest key roles in growth and development and plant responses to the environment. Here, we discuss the current status of research on plant SRs and some promising approaches to address many unanswered questions about plant SRs.