Serine/arginine-rich Proteins Research Articles

Emerging Functions of Plant Serine/Arginine-Rich (SR) Proteins: Lessons from Animals

Serine/arginine-rich (SR) proteins are widely found in higher eukaryotes, including metazoans and plants. They form a major family of essential splicing factors that function in both constitutive splicing (CS) and alternative splicing (AS). Some SR proteins can also shuttle between the nucleus and cytoplasm and have roles beyond splicing. The activities of SR proteins are required for many living organisms to maintain normal growth and development. Although SR genes have been identified in numerous plants, they are less studied than those found in animals. The results of existing research suggest that plant SR proteins share many similar properties with their animal counterparts. However, plants generally have a higher number of SR proteins that display largely redundant functions under normal conditions but participate in specific stress responses. Moreover, many animal and plant SR genes are alternatively spliced and can be regulated by themselves or other SR proteins. These AS events, often coupled to nonsense-mediated decay (NMD), enable a mechanism for protein amount control under different conditions but sometimes also lead to translated protein isoforms with different functions. Here, we summarize the key findings of SR proteins in animals and plants and compare the essential characteristics of SR studies in these two research areas.

Downregulation of SRPK2 promotes cell cycle arrest though E2F1 in non-small cell lung cancer.

Serine-arginine protein kinase (SRPK) belongs to a class of cell cycle regulating kinases that can phosphorylate proteins containing serine/arginine-Rich (SR) regions. SR proteins are a family of RNAbinding phosphoproteins that control both constitutive and alternative pre-mRNA splicing events. However, little is known about their role in non-small cell lung cancer (NSCLC). In the present study, we found that serine-arginine protein kinase 2 (SRPK2) expression was upregulated in NSCLC tissues compared with adjacent normal tissues. Kaplan-Meier curve analyses showed that the overall survival time of NSCLC patients with high SRPK2 expression was shorter than those with low SRPK2 expression. Overexpression of SRPK2 promoted NSCLC cell proliferation and cell cycle arrest, while knockdown of SRPK2 inhibited proliferation and promoted cell cycle arrest in NSCLC cell lines. SRPK2 promoted the transcriptional regulation of E2F1 on downstream cell cycle related genes through phosphorylation of SC35. Xenograft model showed that SRPK2 promoted tumor growth in vivo. SRPK2 phosphorylated SC35 and phosphorylated SC35 activated E2F1 transcription of cyclin-related proteins, thereby promoting the cycle progression of NSCLC. Our findings demonstrated that SRPK2 may be a potential therapeutic target for NSCLC clinical therapy, which plays an important role in the progression of NSCLC.

CTX-712, a Novel Clk Inhibitor Targeting Myeloid Neoplasms with SRSF2 Mutation

Splicing factors (SFs) are among the most frequent mutational targets in myeloid neoplasms, particularly in myelodysplastic syndromes (MDS) and a subset of acute myeloid leukemia (AML), designated as 'chromatin/spliceosome-mutated AML, where major SFs mutated include SF3B1, SRSF2, U2AF1, and ZRSR2. These SF mutations are largely mutually exclusive and except for ZRSR2 mutations, are invariably heterozygous, showing prominent hotspots, suggesting that mutations have neomorphic functions and might cause a synthetic lethality when they are homozygous or two SFs are mutated. Thus, SF functions might be a plausible target of therapy for MDS/AML. Of potential interest in this regard is serine/arginine-rich (SR) domains ubiquitously shared by many SFs, including U2AF1, SRSF2, and ZRSR2, which need to be phosphorylated for their nuclear translocation by evolutionally conserved kinases, known as CLK family of proteins. CLK family kinases regulate mRNA splicing by phosphorylating various SR proteins, and inhibition of CLK family kinases resulted in reduction of phosphorylation levels of SR proteins, induction of splicing alterations and protein depletion for multiple genes, including those involved in growth and survival pathways such as S6K, EGFR, EIF3D, and PARP. In addition, a recent report showed that CLK inhibition can induce skipped exons, cell death, and cell growth suppression, which are dependent of CLK2 expression levels. Thus, CLK family kinases are possible targets of inhibition by small molecules to induce synthetic lethality in SF-mutated MDS/AML and for this purpose, we have recently developed an orally available and highly potent CLK inhibitor, CTX-712 and evaluated its anti-leukemic activities both in vitro and in vivo. When tested in human myeloid cell lines (K562 and THP1), CTX-712, strongly inhibited phosphorylation of multiple SR proteins including SRSF3, SRSF4, SRSF5, and SRSF6 that bind to SRSF2. To further investigate the efficacy of CTX-712 in vivo, we established 5 AML-derived xenograft (PDX) models, which treated with varying doses of CTX-712. Among these 5 PDX models, SRSF2 mutation was found in only one case, which had a SRSF2 p.P95H, mutation, while others (a subcutaneous and 3 leukemia model) were negative for SRSF2 mutations. The SRSF2-mutated model showed a significant response to CTX-712 in a dose-dependent manner. Of note, 4 out of 5 mice treated using a high dose protocol (12.5 mg/kg) achieved complete remission (the tumor shrank completely to unmeasurable size). Two-week after treatment, tumor volumes (mm3) were 762 ± 147 (vehicle), 331 ± 64 (low dose of CTX-712: 6.25mg/kg, P=0.028), and 39 ± 39 (high dose, P=0.0014) (N=5 each, mean ± SEM). CTX-712 also significantly improved the survival of PDX #1. Median survivals (days after engraftment) were 34.5 (vehicle) vs. 93.5 (12.5mg/kg, P=0.015) (N=2 each). Interestingly, another leukemic model carrying KRAS, NF1, and TP53 but not SRSF2 mutations also showed a significant reduction of leukemic burden 2 weeks after CTX-712 treatment; leukemic burden after therapy, as measured by frequency of hCD45+cells in PB (%), were 82 ± 2.2 (vehicle), 17 ± 3.6 (low dose, P<0.0001), and 0.89 ± 0.43 (complete remission, high dose, P<0.0001), (N=4 each, mean ± SEM). In the third PDX model with mutations of FLT3, RAD21, RUNX1, and WT1, CTX-712 administration reduced subcutaneous AML tumors in a dose-dependent manner and achieved partial remission (high dose, P=0.0008) (N=6 each). CTX-712 also significantly improved the survival of the PDX #3 model (high dose, P=0.0069) (N=6 each). In PDX #4, leukemic model with mutations of ASXL1, BCOR, and TET2, high dose CTX-712 therapy strongly reduced the leukemic cell burden than vehicle control (P=0.0027), (N=4 each). CTX-712 also significantly improved the survival of this model (P=0.016) (N=5 each). The last AML PDX #5 model with U2AF1, BCOR, DNMT3A, IDH1, KDM6A, RUNX1, and TET2 mutations was refractory for CTX-712 therapy. Overall, 4 out of 5 PDX AML models showed anti-tumor effect of CTX-712. Complete disappearances of tumors were obtained in 2 cases, including an SRSF2-mutated model. These results provide mechanistic insights of CLK inhibition and a rationale for further investigation of the novel CLK inhibitor in MDS/AML. CTX-712 is currently in clinical phase 1 trials for relapsed and refractory malignancies. Disclosures Yoda: Chordia Therapeutics Inc.: Research Funding. Morishita:Chordia Therapeutics Inc.: Employment, Equity Ownership. Mizutani:Chordia Therapeutics Inc.: Employment, Equity Ownership. Satoh:Chordia Therapeutics Inc: Employment, Equity Ownership. Miyake:Chordia Therapeutics Inc.: Employment, Equity Ownership. Ogawa:Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Kan Research Laboratory, Inc.: Consultancy; RegCell Corporation: Equity Ownership; Asahi Genomics: Equity Ownership; Qiagen Corporation: Patents & Royalties.

Multiplex CRISPR Mutagenesis of the Serine/Arginine-Rich (SR) Gene Family in Rice.

Plant growth responds to various environmental and developmental cues via signaling cascades that influence gene expression at the level of transcription and pre-mRNA splicing. Alternative splicing of pre-mRNA increases the coding potential of the genome from multiexon genes and regulates gene expression through multiple mechanisms. Serine/arginine-rich (SR) proteins, a conserved family of splicing factors, are the key players of alternative splicing and regulate pre-mRNA splicing under stress conditions. The rice (Oryza sativa) genome encodes 22 SR proteins categorized into six subfamilies. Three of the subfamilies are plant-specific with no mammalian orthologues, and the functions of these SR proteins are not well known. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a genome engineering tool that cleaves the target DNA at specific locations directed by a guide RNA (gRNA). Recent advances in CRISPR/Cas9-mediated plant genome engineering make it possible to generate single and multiple functional knockout mutants in diverse plant species. In this study, we targeted each rice SR locus and produced single knockouts. To overcome the functional redundancy within each subfamily of SR genes, we utilized a polycistronic tRNA-gRNA multiplex targeting system and targeted all loci of each subfamily. Sanger sequencing results indicated that most of the targeted loci had knockout mutations. This study provides useful resource materials for understanding the molecular role of SR proteins in plant development and biotic and abiotic stress responses.

Srrm234, but not canonical SR and hnRNP proteins, drive inclusion of Dscam exon 9 variable exons.

Alternative splicing of pre-mRNA is a major mechanism to diversify protein functionality in metazoans from a limited number of genes. The Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam) gene, which is important for neuronal wiring and phagocytosis of bacteria, can generate up to 38,016 isoforms by mutually exclusive alternative splicing in four clusters of variable exons. However, it is not understood how a specific exon is chosen from the many variables and how variable exons are prevented from being spliced together. A main role in the regulation of Dscam alternative splicing has been attributed to RNA binding proteins (RBPs), but how they impact on exon selection is not well understood. Serine-arginine rich (SR) proteins and hnRNP proteins are the two main types of RBPs with major roles in exon definition and splice site selection. Here, we analyzed the role of SR and hnRNP proteins in Dscam exon 9 alternative splicing in mutant Drosophila melanogaster embryos because of their essential function for development. Strikingly, loss or overexpression of canonical SR and hnRNP proteins even when multiple proteins are depleted together, does not affect Dscam alternative exon selection very dramatically. Conversely, noncanonical SR protein Serine–arginine repetitive matrix 2/3/4 (Srrm234) is a main determinant of exon inclusion in the Dscam exon 9 cluster. Since long-range base-pairings are absent in the exon 9 cluster, our data argue for a small complement of regulatory factors as main determinants of exon inclusion in the Dscam exon 9 cluster.

Genome-Wide Analysis of Serine/Arginine-Rich Protein Family in Wheat and Brachypodium distachyon.

By regulating the pre-mRNA splicing of other genes and themselves, plant serine/arginine-rich (SR) proteins play important roles in development and in response to abiotic stresses. Presently, the functions of most plant SR protein genes remain unclear. Wheat (Triticum aestivum) and Brachypodium distachyon are closely related species. In this study, 40 TaSR and 18 BdSR proteins were identified respectively, and they were classified into seven subfamilies: SR, RS, SCL, RSZ, RS2Z, SC35, and SR45. Similar to Arabidopsis and rice SR protein genes, most TaSR and BdSR protein genes are expressed extensively. Surprisingly, real-time polymerase chain reaction (RT-PCR) analyses showed that no alternative splicing event was found in TaSR protein genes, and only six BdSR protein genes are alternatively spliced genes. The detected alternatively spliced BdSR protein genes and transcripts are much fewer than in Arabidopsis, rice, maize, and sorghum. In the promoter regions, 92 development-related, stress-related, and hormone-related cis-elements were detected, indicating their functions in development and in response to environmental stresses. Meanwhile, 19 TaSR and 16 BdSR proteins were predicted to interact with other SR proteins or non-SR proteins, implying that they are involved in other functions in addition to modulating pre-mRNA splicing as essential components of the spliceosome. These results lay a foundation for further analyses of these genes.

The Arabidopsis splicing regulator SR45 confers salt tolerance in a splice isoform-dependent manner.

Functions of most splice isoforms that are generated by alternative splicing are unknown. We show that two splice variants that encode proteins differing in only eight amino acids have distinct functions in a stress response. Serine/arginine-rich (SR) and SR-like proteins, a conserved family of RNA binding proteins across eukaryotes, play important roles in pre-mRNA splicing and other post-transcriptional processes. Pre-mRNAs of SR and SR-like proteins undergo extensive alternative splicing in response to diverse stresses and produce multiple splice isoforms. However, the functions of most splice isoforms remain elusive. Alternative splicing of pre-mRNA of Arabidopsis SR45, which encodes an SR-like splicing regulator, generates two isoforms (long-SR45.1 and short-SR45.2). The proteins encoded by these two isoforms differ in eight amino acids. Here, we investigated the role of SR45 and its splice variants in salt stress tolerance. The loss of SR45 resulted in enhanced sensitivity to salt stress and changes in expression and splicing of genes involved in regulating salt stress response. Interestingly, only the long isoform (SR45.1) rescued the salt-sensitive phenotype as well as the altered gene expression and splicing patterns in the mutant. These results suggest that SR45 positively regulates salt tolerance. Furthermore, only the long isoform is required for SR45-mediated salt tolerance.

Differential Alternative Splicing Genes in Response to Boron Deficiency in Brassica napus.

Alternative splicing (AS) can increase transcriptome diversity, protein diversity and protein yield, and is an important mechanism to regulate plant responses to stress. Oilseed rape (Brassica napus L.), one of the main oil crops in China, shows higher sensitivity to boron (B) deficiency than other species. Here, we demonstrated AS changes that largely increased the diversity of the mRNA expressed in response to B deficiency in B. napus. Each gene had two or more transcripts on average. A total of 33.3% genes in both Qingyou10 (QY10, B-efficient cultivar) and Westar10 (W10, B-inefficient cultivar) showed AS in both B conditions. The types of AS events were mainly intron retention, 3′ alternative splice site, 5′ alternative splice site and exon skipping. The tolerance ability of QY10 was higher than that of W10, possibly because there were far more differential alternative splicing (DAS) genes identified in QY10 at low B conditions than in W10. The number of genes with both DAS and differentially expressed (DE) was far lower than that of the genes that were either with DAS or DE in QY10 and W10, suggesting that the DAS and DE genes were independent. Four Serine/Arginine-rich (SR) splicing factors, BnaC06g14780D, BnaA01g14750D, BnaA06g15930D and BnaC01g41640D, underwent differentially alternative splicing in both cultivars. There existed gene–gene interactions between BnaC06g14780D and the genes associated with the function of B in oilseed rape at low B supply. This suggests that oilseed rape could regulate the alterative pre-mRNA splicing of SR protein related genes to increase the plant tolerance to B deficiency.

Hepatitis B virus core protein phosphorylation: Identification of the SRPK1 target sites and impact of their occupancy on RNA binding and capsid structure.

Hepatitis B virus (HBV) replicates its 3 kb DNA genome through capsid-internal reverse transcription, initiated by assembly of 120 core protein (HBc) dimers around a complex of viral pregenomic (pg) RNA and polymerase. Following synthesis of relaxed circular (RC) DNA capsids can be enveloped and secreted as stable virions. Upon infection of a new cell, however, the capsid disintegrates to release the RC-DNA into the nucleus for conversion into covalently closed circular (ccc) DNA. HBc´s interactions with nucleic acids are mediated by an arginine-rich C terminal domain (CTD) with intrinsically strong non-specific RNA binding activity. Adaptation to the changing demands for nucleic acid binding during the viral life cycle is thought to involve dynamic phosphorylation / dephosphorylation events. However, neither the relevant enzymes nor their target sites in HBc are firmly established. Here we developed a bacterial coexpression system enabling access to definably phosphorylated HBc. Combining Phos-tag gel electrophoresis, mass spectrometry and mutagenesis we identified seven of the eight hydroxy amino acids in the CTD as target sites for serine-arginine rich protein kinase 1 (SRPK1); fewer sites were phosphorylated by PKA and PKC. Phosphorylation of all seven sites reduced nonspecific RNA encapsidation as drastically as deletion of the entire CTD and altered CTD surface accessibility, without major structure changes in the capsid shell. The bulk of capsids from human hepatoma cells was similarly highly, yet non-identically, phosphorylated as by SRPK1. While not proving SRPK1 as the infection-relevant HBc kinase the data suggest a mechanism whereby high-level HBc phosphorylation principally suppresses RNA binding whereas one or few strategic dephosphorylation events enable selective packaging of the pgRNA/polymerase complex. The tools developed in this study should greatly facilitate the further deciphering of the role of HBc phosphorylation in HBV infection and its evaluation as a potential new therapeutic target.

SRSF6 is upregulated in asthmatic horses and involved in the MYH11 SMB expression.

Smooth muscle has a central role in bronchospasm‐induced airway obstruction in asthma. Alternative mRNA splicing of the smooth muscle myosin heavy chain (myh11) gene produces four different isoforms, one of which (SMB) is characterized by the inclusion of the exon5b, which doubles the smooth muscle cells contraction velocity. Deciphering the regulation of the expression levels of the SMB isoform would represent a major step for the understanding of the triggers and pathways leading to airway smooth muscle contraction in asthma. Our objective was therefore, to study the splicing regulation mechanisms of the exon5b in airway smooth muscle cells. Bioinformatics analysis was performed to identify the cis‐regulatory elements present in the exon5b using HSF finder 3 tool. The expression of the corresponding serine/arginine rich protein (SR) genes thus identified was evaluated by quantitative RT‐PCR (qPCR). SRSF1, SRSF6, and hnRNPA1 cis‐acting elements were identified by in silico analysis of the exon5b sequence as splicing regulator candidates. QPCR analyses showed that SRSF1 and SRSF6 are upregulated in ASM cells from asthmatic horses in exacerbation (n = 5) compared to controls (n = 5). The inhibition of the identified splicing factors by small interfering RNA allowed identifying the regulation of the SMB isoform by SRSF6. Our results implicate for the first time the upregulation of SRSF6 and SRSF1 in the asthmatic ASM cells and indicate that SRSF6 induces the exon5b inclusion. This study provides an important first step for the understanding of the triggers and pathways leading to ASM hypercontraction and identifies a possible new target for asthma.

Molecular interactions connecting the function of the serine-arginine–rich protein SRSF1 to protein phosphatase 1

Splicing generates many mRNA strands from a single precursor mRNA, expanding the proteome and enhancing intracellular diversity. Both initial assembly and activation of the spliceosome require an essential family of splicing factors called serine-arginine (SR) proteins. Protein phosphatase 1 (PP1) regulates the SR proteins by controlling phosphorylation of a C-terminal arginine-serine-rich (RS) domain. These modifications are vital for the subcellular localization and mRNA splicing function of the SR protein. Although PP1 has been shown to dephosphorylate the prototype SR protein splicing factor 1 (SRSF1), the molecular nature of this interaction is not understood. Here, using NMR spectroscopy, we identified two electrostatic residues in helix α2 and a hydrophobic residue in helix α1 in the RNA recognition motif 1 (RRM1) of SRSF1 that constitute a binding surface for PP1. Substitution of these residues dissociated SRSF1 from PP1 and enhanced phosphatase activity, reducing phosphorylation in the RS domain. These effects lead to shifts in alternative splicing patterns that parallel increases in SRSF1 diffusion from speckles to the nucleoplasm brought on by regiospecific decreases in RS domain phosphorylation. Overall, these findings establish a molecular and biological connection between PP1-targeted amino acids in an RRM with the phosphorylation state and mRNA-processing function of an SR protein.

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.

SRSF7 knockdown promotes apoptosis of colon and lung cancer cells.

Serine/arginine-rich (SR) proteins are a family of important splicing factors, which are involved in multiple aspects of RNA processing, including splicing, mRNA nuclear export, mRNA stability and translation. Previous studies have identified a number of SR proteins that exhibit abnormal expression in various tumor types. In the present study, the expression and function of serine/arginine-rich splicing factor 7 (SRSF7) were investigated in colon and lung cancer. Using tissue immunohistochemistry, it was observed that SRSF7 was overexpressed in colon and lung cancer tissues. As the role of SRSF7 in cancer remains to be fully elucidated, the expression of SRSF7 was knocked down in the present study by transfecting SRSF7-specific small interfering RNAs (siRNAs) into the HCT116 colon cancer cell line and A549 lung cancer cell line, which exhibited elevated expression of SRSF7. MTS assays, western blot analysis, flow cytometry and spectrofluorometer analyses were performed to assess the effects of SRSF7 knockdown on the proliferation and apoptosis of cells. The results demonstrated that the expression of SRSF7 was efficiently knocked down by SRSF7 siRNA, and that SRSF7 knockdown inhibited proliferation and enhanced apoptosis of HCT116 and A549 cells. Further experiments involving BEAS-2B cells stably overexpressing SRSF7, and A549 cells with stable knockdown of SRSF7 revealed that SRSF7 regulated the splicing of the apoptosis regulator Fas. Collectively, these data indicated that SRSF7 is critical for the survival of colon and lung cancer cells, and may be a potential therapeutic target for the treatment of colon and lung cancer.

The mechanisms of a mammalian splicing enhancer.

Exonic splicing enhancer (ESE) sequences are bound by serine & arginine-rich (SR) proteins, which in turn enhance the recruitment of splicing factors. It was inferred from measurements of splicing around twenty years ago that Drosophila doublesex ESEs are bound stably by SR proteins, and that the bound proteins interact directly but with low probability with their targets. However, it has not been possible with conventional methods to demonstrate whether mammalian ESEs behave likewise. Using single molecule multi-colour colocalization methods to study SRSF1-dependent ESEs, we have found that that the proportion of RNA molecules bound by SRSF1 increases with the number of ESE repeats, but only a single molecule of SRSF1 is bound. We conclude that initial interactions between SRSF1 and an ESE are weak and transient, and that these limit the activity of a mammalian ESE. We tested whether the activation step involves the propagation of proteins along the RNA or direct interactions with 3′ splice site components by inserting hexaethylene glycol or abasic RNA between the ESE and the target 3′ splice site. These insertions did not block activation, and we conclude that the activation step involves direct interactions. These results support a model in which regulatory proteins bind transiently and in dynamic competition, with the result that each ESE in an exon contributes independently to the probability that an activator protein is bound and in close proximity to a splice site.

The SR protein B52/SRp55 regulates splicing of the period thermosensitive intron and mid-day siesta in Drosophila

Similar to many diurnal animals, Drosophila melanogaster exhibits a mid-day siesta that is more robust as temperature increases, an adaptive response that aims to minimize the deleterious effects from exposure to heat. This temperature-dependent plasticity in mid-day sleep levels is partly based on the thermal sensitive splicing of an intron in the 3′ untranslated region (UTR) of the circadian clock gene termed period (per). In this study, we evaluated a possible role for the serine/arginine-rich (SR) splicing factors in the regulation of dmpi8 splicing efficiency and mid-day siesta. Using a Drosophila cell culture assay we show that B52/SRp55 increases dmpi8 splicing efficiency, whereas other SR proteins have little to no effect. The magnitude of the stimulatory effect of B52 on dmpi8 splicing efficiency is modulated by natural variation in single nucleotide polymorphisms (SNPs) in the per 3′ UTR that correlate with B52 binding levels. Down-regulating B52 expression in clock neurons increases mid-day siesta and reduces dmpi8 splicing efficiency. Our results establish a novel role for SR proteins in sleep and suggest that polymorphisms in the per 3′ UTR contribute to natural variation in sleep behavior by modulating the binding efficiencies of SR proteins.

Nuclear hyaluronidase 2 drives alternative splicing of CD44 pre-mRNA to determine profibrotic or antifibrotic cell phenotype.

The cell surface protein CD44 is involved in diverse physiological processes, and its aberrant function is linked to various pathologies such as cancer, immune dysregulation, and fibrosis. The diversity of CD44 biological activity is partly conferred by the generation of distinct CD44 isoforms through alternative splicing. We identified an unexpected function for the ubiquitous hyaluronan-degrading enzyme, hyaluronidase 2 (HYAL2), as a regulator of CD44 splicing. Standard CD44 is associated with fibrotic disease, and its production is promoted through serine-arginine-rich (SR) protein-mediated exon exclusion. HYAL2 nuclear translocation was stimulated by bone morphogenetic protein 7, which inhibits the myofibroblast phenotype. Nuclear HYAL2 displaced SR proteins from the spliceosome, thus enabling HYAL2, spliceosome components (U1 and U2 small nuclear ribonucleoproteins), and CD44 pre-mRNA to form a complex. This prevented double-exon splicing and facilitated the inclusion of CD44 exons 11 and 12, which promoted the accumulation of the antifibrotic CD44 isoform CD44v7/8 at the cell surface. These data demonstrate previously undescribed mechanisms regulating CD44 alternative splicing events that are relevant to the regulation of cellular phenotypes in progressive fibrosis.

Alignment of Mitotic Chromosomes in Human Cells Involves SR-Like Splicing Factors Btf and TRAP150.

Serine-arginine-rich (SR) or SR-like splicing factors interact with exon junction complex proteins during pre-mRNA processing to promote mRNA packaging into mature messenger ribonucleoproteins (mRNPs) and to dictate mRNA stability, nuclear export, and translation. The SR protein family is complex, and while many classical SR proteins have well-defined mRNA processing functions, those of other SR-like proteins is unclear. Here, we show that depletion of the homologous non-classical serine-arginine-rich (SR) splicing factors Bcl2-associated transcription factor (Btf or BCLAF) and thyroid hormone receptor-associated protein of 150 kDa (TRAP150) causes mitotic defects. We hypothesized that the depletion of these SR-like factors affects mitosis indirectly through an altered expression of mitotic checkpoint regulator transcripts. We observed an altered abundance of transcripts that encode mitotic regulators and mitotic chromosome misalignment defects following Btf and/or TRAP150 depletion. We propose that, in addition to their previously reported roles in maintaining mRNA distribution, Btf and TRAP150 control the abundance of transcripts encoding mitotic regulators, thereby affecting mitotic progression in human cells.

The FgSRP1 SR-protein gene is important for plant infection and pre-mRNA processing in Fusarium graminearum.

The versatile functions of SR (serine/arginine-rich) proteins in pre-mRNA splicing and processing are modulated by reversible phosphorylation. Previous studies showed that FgPrp4, the only protein kinase among spliceosome components, is important for intron splicing and the FgSrp1 SR protein is phosphorylated at five conserved sites in Fusarium graminearum. In this study, we showed that the Fgsrp1 deletion mutant rarely produced conidia and caused only limited symptoms on wheat heads and corn silks. Deletion of FgSRP1 also reduced ascospore ejection and deoxynivalenol (DON) production. Interestingly, FgSRP1 had two transcript isoforms due to alternative splicing and both of them were required for its normal functions in growth and DON biosynthesis. FgSrp1 localized to the nucleus and interacted with FgPrp4 in vivo. Deletion of all four conserved phosphorylation sites but not individual ones affected the FgSRP1 function, suggesting their overlapping functions. RNA-seq analysis showed that the expression of over thousands of genes and splicing efficiency in over 140 introns were affected. Taken together, FgSRP1 is important for conidiation, and pathogenesis and alternative splicing is important for its normal functions. The FgSrp1 SR protein is likely important for pre-mRNA processing or splicing of various genes in different developmental and infection processes.

SIRT1 ACTS ON SPLICING FACTOR SC35 TO REGULATE THE ALTERNATIVE SPLICING OF TAU EXON 10

Approximately equal amount of 3R-tau and 4R-tau on account of normal alternative splicing of tau exon 10 is extremely necessary for maintaining the normal brain function. the abnormal alternative splicing of tau exon 10 causes the imbalance of 3R-tau to 4R-tau, resulting in the hyperphosphorylation and aggregation of tau and consequently the neurofibrillary degeneration. ASF (also name as SRSF1) and SC35 (also name as SRSF2), members of the superfamily of the serine/arginine-rich (SR) proteins, play an important role in promoting the inclusion of tau exon 10. Plenty of researches indicate that phosphorylation highly regulates the functions of SR proteins. High resolution mass spectrum reveals that there are a great many of acetylation sites on the splicing proteins including SR proteins and that the major acetylation region locates in the RRM domain. But up to now, little is known about the location and function of acetylation modification of SR proteins. Sirt1, one of the member in mammalian Sirtuin family removing acetyls from lysine residues of histones or nonhistones by hydrolysis, is extensively studied and well known to associate closely with age-related diseases such as AD. By co-immunoprecipitation, GST pull down and confocal, we investigate the interaction between Sirt1 and SC35. By mini-tau gene, pCI/SI9-SI10 which consists of exons 9, 10, 11, a part of intron 9 and intron 10 and RT-PCR, we elucidated whether and how Sirt1 regulates SC35-promoted tau exon 10 inclusion in cultured cells. We observed that Sirt1 interacts with and deacetylates SC35. By comparative analysis, we found that Sirt1 regulates the SC35-promoted alternative splicing of tau exon 10 but not ASF. Overexpression or knocking down of Sirt1 affected its role in suppression SC35-promoted tau exon 10 inclusion. Dysregulation of Sirt1 in Alzheimer's disease brain may cause aberrant splicing of tau exon 10 through SC35, leading to the imbalance in 3R-tau and 4R-tau expression, which may initiate or accelerate tau pathology and cause neurofibrillary degeneration in AD brain.

Abstract 2361: Pim kinase inhibition alters mRNA splicing in AML cell lines

Abstract Oncogenic kinase activity is a common feature of nearly all cancers and kinases are major targets for therapeutic intervention. Pim kinases are deregulated in hematopoietic cancers including Acute Myeloid Leukemia (AML), as well as prostate cancer. While cancer cells can become dependent on Pim activity to sustain proliferation, in normal adult tissues, Pim kinase activity appears to be dispensable. These features make Pim kinases an attractive target for cancer therapy, however, their physiological roles have not been fully characterized. Using the reverse in-gel kinase assay (RIKA), we identified a battery of novel Pim substrates that are involved in mRNA splicing regulation. We hypothesized that Pim family kinases regulate mRNA splicing through phosphorylation of splicing factors. Microarray analysis revealed more than 10,000 splicing changes in AML cells treated with the highly selective small molecule Pim kinase inhibitor AZD1208. Using RT-PCR analysis, multiple AZD1208-induced splicing changes were validated. To discern the mechanisms whereby Pim kinases regulate splicing, we determined whether Pim inhibition alters phosphorylation of serine/arginine-rich (SR) proteins. Inhibition of serine arginine protein kinase (SRPK) activity using a small molecular inhibitor (SRPIN340) reduced SR protein phosphorylation, whereas Pim kinase inhibition did not, suggesting that Pim kinases regulate splicing through an SRPK independent pathway. Comparison of splicing changes in AZD1208 and SRPIN340 treated AML cells demonstrated distinct patterns, providing further evidence of SRPK-independent splicing regulation by Pim kinases. Biomarkers of kinase inhibitor efficacy are critical components of clinical trials. These data suggest that changes in mRNA splicing may serve as a biomarker for assessing patient responsiveness to Pim inhibition therapy. Citation Format: Tejashree A. Joglekar, Xiang Li, Charles J. Bieberich. Pim kinase inhibition alters mRNA splicing in AML cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2361. doi:10.1158/1538-7445.AM2017-2361