Spliceosome Components Research Articles

Dynamics and functional roles of splicing factor autoregulation.

SUMMARYNon-core spliceosome components are essential, conserved regulators of alternative splicing. They provide concentration-dependent control of diverse pre-mRNAs. Many splicing factors direct unproductive splicing of their own pre-mRNAs through negative autoregulation. However, the impact of such feedback loops on splicing dynamics at the single-cell level remains unclear. Here, we developed a system to quantitatively analyze negative autoregulatory splicing dynamics by splicing factor SRSF1 in response to perturbations in single HEK293 cells. We show that negative autoregulatory splicing provides critical functions for gene regulation, establishing a ceiling of SRSF1 protein concentration, reducing cell-cell heterogeneity in SRSF1 levels, and buffering variation in transcription. Most important, it adapts SRSF1 splicing activity to variations in demand from other pre-mRNA substrates. A minimal mathematical model of autoregulatory splicing explains these experimentally observed features and provides values for effective biochemical parameters. These results reveal the unique functional roles that splicing negative autoregulation plays in homeostatically regulating transcriptional programs.

Border cell polarity and collective migration require the spliceosome component Cactin.

Border cells are an in vivo model for collective cell migration. Here, we identify the gene cactin as essential for border cell cluster organization, delamination, and migration. In Cactin-depleted cells, the apical proteins aPKC and Crumbs (Crb) become abnormally concentrated, and overall cluster polarity is lost. Apically tethering excess aPKC is sufficient to cause delamination defects, and relocalizing apical aPKC partially rescues delamination. Cactin is conserved from yeast to humans and has been implicated in diverse processes. In border cells, Cactin's evolutionarily conserved spliceosome function is required. Whole transcriptome analysis revealed alterations in isoform expression in Cactin-depleted cells. Mutations in two affected genes, Sec23 and Sec24CD, which traffic Crb to the apical cell surface, partially rescue border cell cluster organization and migration. Overexpression of Rab5 or Rab11, which promote Crb and aPKC recycling, similarly rescues. Thus, a general splicing factor is specifically required for coordination of cluster polarity and migration, and migrating border cells are particularly sensitive to splicing and cell polarity disruptions.

Ubiquitination and Ubiquitin-Like Modifications as Mediators of Alternative Pre-mRNA Splicing in Arabidopsis thaliana.

Alternative splicing (AS) is a common post-transcriptional regulatory process in eukaryotes. AS has an irreplaceable role during plant development and in response to environmental stress as it evokes differential expression of downstream genes or splicing factors (e.g., serine/arginine-rich proteins). Numerous studies have reported that loss of AS capacity leads to defects in plant growth and development, and induction of stress-sensitive phenotypes. A role for post-translational modification (PTM) of AS components has emerged in recent years. These modifications are capable of regulating the activity, stability, localization, interaction, and folding of spliceosomal proteins in human cells and yeast, indicating that PTMs represent another layer of AS regulation. In this review, we summarize the recent reports concerning ubiquitin and ubiquitin-like modification of spliceosome components and analyze the relationship between spliceosome and the ubiquitin/26S proteasome pathway in plants. Based on the totality of the evidence presented, we further speculate on the roles of protein ubiquitination mediated AS in plant development and environmental response.

Transcriptome analysis of clock disrupted cancer cells reveals differential alternative splicing of cancer hallmarks genes

Emerging evidence points towards a regulatory role of the circadian clock in alternative splicing (AS). Whether alterations in core-clock components may contribute to differential AS events is largely unknown. To address this, we carried out a computational analysis on recently generated time-series RNA-seq datasets from three core-clock knockout (KO) genes (ARNTL, NR1D1, PER2) and WT of a colorectal cancer (CRC) cell line, and time-series RNA-seq datasets for additional CRC and Hodgkin’s lymphoma (HL) cells, murine WT, Arntl KO, and Nr1d1/2 KO, and murine SCN WT tissue. The deletion of individual core-clock genes resulted in the loss of circadian expression in crucial spliceosome components such as SF3A1 (in ARNTLKO), SNW1 (in NR1D1KO), and HNRNPC (in PER2KO), which led to a differential pattern of KO-specific AS events. All HCT116KO cells showed a rhythmicity loss of a crucial spliceosome gene U2AF1, which was also not rhythmic in higher progression stage CRC and HL cancer cells. AS analysis revealed an increase in alternative first exon events specific to PER2 and NR1D1 KO in HCT116 cells, and a KO-specific change in expression and rhythmicity pattern of AS transcripts related to cancer hallmarks genes including FGFR2 in HCT116_ARNTLKO, CD44 in HCT116_NR1D1KO, and MET in HCT116_PER2KO. KO-specific changes in rhythmic properties of known spliced variants of these genes (e.g. FGFR2 IIIb/FGFR2 IIIc) correlated with epithelial-mesenchymal-transition signalling. Altogether, our bioinformatic analysis highlights a role for the circadian clock in the regulation of AS, and reveals a potential impact of clock disruption in aberrant splicing in cancer hallmark genes.

The Deubiquitinase USP39 Promotes Esophageal Squamous Cell Carcinoma Malignancy as a Splicing Factor.

Esophageal squamous cell carcinoma (ESCC) is an aggressive epithelial malignancy and the underlying molecular mechanisms remain elusive. Here, we identify that the ubiquitin-specific protease 39 (USP39) drives cell growth and chemoresistance by functional screening in ESCC, and that high expression of USP39 correlates with shorter overall survival and progression-free survival. Mechanistically, we provide evidence for the role of USP39 in alternative splicing regulation. USP39 interacts with several spliceosome components. Integrated analysis of RNA-seq and RIP-seq reveals that USP39 regulates the alternative splicing events. Taken together, our results indicate that USP39 functions as an oncogenic splicing factor and acts as a potential therapeutic target for ESCC.

Contribution of stress granules and spliceosomal components to the formation of chemoresistance in ovarian cancer cells

The emergence of chemoresistance in cancer cells is one of the main reasons for the high mortality from oncological diseases. We have shown that signaling molecules from dying cancer cells contribute to a more aggressive therapy‐resistant phenotype. However, the molecular mechanisms underlying this effect remain elusive.Here we aimed to find out which proteins secreted by dying tumor cells get into recipient cells and to determine the molecular cascades triggered in response to incubation with therapy‐induced secretomes. We modified the SILAC (Stable Isotope Labeling by Amino acids in Cell culture) technology. A heavy‐labeled primary culture of ovarian cancer cells was treated or untreated with cisplatin for 48 h. Then using ultrafiltration cartridges, a fraction of extracellular vesicles (EV) were isolated from culture media. The obtained EV were resuspended in a medium containing Medium‐labeled amino acids and added to the recipient chemonaive unlabeled tumor cells for 24 h. During incubation, recipient cells absorbed EV with Heavy‐labeled proteins from donor cells and synthesized new proteins with Medium label. Then, we performed proteomic analysis of recipient cells. In total, we identified 4224 proteins. Interestingly, dying tumor cells secrete and recipient cells absorb a large number of spliceosomal and ribosomal proteins along with translation initiation complex. These proteins are often presented in stress granules. In response to EV from dying tumor cells, recipient cells upregulated the translation of genes responsible for the regulation of the cell cycle and oxidative phosphorylation.To our knowledge, this is the first report about the secretion of stress granules and spliceosomal components into the extracellular space and their internalization in other cells. To confirm these data we performed transfection of donor cells with constructs encoding the fluorescent proteins TIA1 (protein of stress granules) and SRSF4 (protein of the spliceosome). We showed that both proteins are re‐located together from the nucleus into the cytoplasm under the cisplatin treatment. Then, we demostrated the absorption of fluorescent‐labeled proteins TIA1 and SRSF4 by recipient cells after incubation with therapy‐induced EV. Our findings demonstrate that in response to cisplatin treatment tumor cells secrete spliceosomal components into the extracellular space (presumably as a part of stress granules), then these components are absorbed by recipient chemonaive cells and trigger a restructuring of metabolism and cell cycle.

Effects of spliceosomal mutations on brain patterning and morphogenesis

The major spliceosome consists of U1, U2, U5, and U4/U6 small nuclear ribonucleoprotein (snRNPs), and each snRNP has distinct and sequential roles during the mRNA splicing process. Mutations in the core components of the spliceosome are associated with brain and neurological defects. The goal of this study is to examine the role of three core spliceosomal components, SNRPB, SF3B4, and EFTUD2, in brain patterning and morphogenesis, and whether or not they are required in the neural tube or neural crest cells for brain development. Previously, we identified brain defects associated with the mutation of these spliceosomal components in the neural tube and neural crest cells. Structures derived from the midbrain and hindbrain region, such as the diencephalon, pons, and cerebellum, were found to be absent in E12.5 and E14.5 mutant embryos. In this study, we aim to use mutant mouse models to identify shared transcripts and pathways disrupted by mutations in these spliceosomal genes. Through RNA‐seq analysis, we identified a number of mis‐expressed RNA‐binding proteins that may contribute to the malformationis found in these mutants. We plan to examine level and expression of these RNA binding proteins in wild type and mutant embryos to evaluate how they contribute to the phenotypes found. Furthermore, to assess the potential clinical application of our findings, we will use human embryonic stem cells (hESC) to generate human neural crest cells with mutatioin in these splicing factors; shared transcripts and pathways identified in the mouse model will be compared to those found in induced human neural crest cells. Distinct spliceosomophathies are difficult to diagnose due to phenotypic overlaps. The significance of this work lies in its potential to identify a disrupted pathway shared by mutations in various splicing factors, a therapeutic alternative across multiple spliceosomopathies.

Multiomics analysis of the NAD+-PARP1 axis reveals a role for site-specific ADP-ribosylation in splicing in embryonic stem cells.

The differentiation of embryonic stem cells (ESCs) into a lineage-committed state is a dynamic process involving changes in cellular metabolism, epigenetic modifications, post-translational modifications, gene expression, and RNA processing. Here we integrated data from metabolomic, proteomic, and transcriptomic assays to characterize how alterations in NAD+ metabolism during the differentiation of mouse ESCs lead to alteration of the PARP1-mediated ADP-ribosylated (ADPRylated) proteome and mRNA isoform specialization. Our metabolomic analyses indicate that mESCs use distinct NAD+ biosynthetic pathways in different cell states: the de novo pathway in the pluripotent state, and the salvage and Preiss-Handler pathways as differentiation progresses. We observed a dramatic induction of PARP1 catalytic activity driven by enhanced nuclear NAD+ biosynthesis during the early stages of mESC differentiation (e.g., within 12 h of LIF removal). PARP1-modified proteins in mESCs are enriched for biological processes related to stem cell maintenance, transcriptional regulation, and RNA processing. The PARP1 substrates include core spliceosome components, such as U2AF35 and U2AF65, whose splicing functions are modulated by PARP1-mediated site-specific ADP-ribosylation. Finally, we observed that splicing is dysregulated genome-wide in Parp1 knockout mESCs. Together, these results demonstrate a role for the NAD+-PARP1 axis in the maintenance of mESC state, specifically in the splicing program during differentiation.

RNPS1 inhibits excessive tumor necrosis factor/tumor necrosis factor receptor signaling to support hematopoiesis in mice

Null mutations of spliceosome components or cofactors are homozygous lethal in eukaryotes, but viable hypomorphic mutations provide an opportunity to understand the physiological impact of individual splicing proteins. We describe a viable missense allele (F181I) of Rnps1 encoding an essential regulator of splicing and nonsense-mediated decay (NMD), identified in a mouse genetic screen for altered immune cell development. Homozygous mice displayed a stem cell–intrinsic defect in hematopoiesis of all lineages due to excessive apoptosis induced by tumor necrosis factor (TNF)–dependent death signaling. Numerous transcript splice variants containing retained introns and skipped exons were detected at elevated frequencies in Rnps1F181I/F181I splenic CD8+ T cells and hematopoietic stem cells (HSCs), but NMD appeared normal. Strikingly, Tnf knockout rescued all hematopoietic cells to normal or near-normal levels in Rnps1F181I/F181I mice and dramatically reduced intron retention in Rnps1F181I/F181I CD8+ T cells and HSCs. Thus, RNPS1 is necessary for accurate splicing, without which disinhibited TNF signaling triggers hematopoietic cell death.

A developmentally programmed splicing failure contributes to DNA damage response attenuation during mammalian zygotic genome activation.

Transition from maternal to embryonic transcriptional control is crucial for embryogenesis. However, alternative splicing regulation during this process remains understudied. Using transcriptomic data from human, mouse, and cow preimplantation development, we show that the stage of zygotic genome activation (ZGA) exhibits the highest levels of exon skipping diversity reported for any cell or tissue type. Much of this exon skipping is temporary, leads to disruptive noncanonical isoforms, and occurs in genes enriched for DNA damage response in the three species. Two core spliceosomal components, Snrpb and Snrpd2, regulate these patterns. These genes have low maternal expression at ZGA and increase sharply thereafter. Microinjection of Snrpb/d2 messenger RNA into mouse zygotes reduces the levels of exon skipping at ZGA and leads to increased p53-mediated DNA damage response. We propose that mammalian embryos undergo an evolutionarily conserved, developmentally programmed splicing failure at ZGA that contributes to the attenuation of cellular responses to DNA damage.

TR3 Enhances AR Variant Production and Transactivation, Promoting Androgen Independence of Prostate Cancer Cells.

Simple SummaryAdvanced prostate cancer development is associated with androgen-independent AR signaling. TR3 overexpression alters AR expression, splicing process, and transactivation towards increasing the androgen independence of AR signaling in prostate cancer cells. These results suggest that TR3 is a pivotal factor in the progression of prostate cancer to advanced form.The pro-oncogenic function of TR3, an orphan nuclear receptor, has been reported in prostate cancer. However, the roles of TR3 in androgen receptor (AR) expression and signaling in prostate cancer cells are poorly understood. Database analysis revealed that TR3 expression level is elevated in prostate tumors, and is positively, although weakly, correlated with that of AR. TR3 overexpression increased the production of AR splice variants in addition to general upregulation of AR expression. TR3 interacted with some spliceosomal complex components and AR precursor mRNA, altering the splice junction rates between exons. TR3 also enhanced androgen-independent AR function. Furthermore, TR3 overexpression increased cell proliferation and mobility of AR-positive prostate cancer cells and stimulated tumorigenesis of androgen-independent prostate cancer cells in mouse xenograft models. This is the first study to report that TR3 is a multifunctional regulator of AR signaling in prostate cancer cells. TR3 alters AR expression, splicing process, and activity in prostate cancer cells, increasing the androgen independence of AR signaling. Therefore, TR3 may play a crucial role in the progression of prostate cancer to an advanced castration-resistant form.

Mutations in FgPrp6 suppressive to the Fgprp4 mutant in Fusarium graminearum

The pre-mRNA processing factor Prp6 is an essential component of the U4/U6.U5 tri-small nuclear ribonucleoprotein (snRNP). In a previous study, mutations were identified in the PRP6 ortholog in four suppressors of Fgprp4 that was deleted of the only kinase FgPrp4 among the spliceosome components in the plant pathogenic fungus Fusarium graminearum. In this study, we identified additional suppressor mutations in FgPrp6 and determined the suppressive effects of selected mutations. In total, 12 mutations of FgPRP6 were identified in 20 suppressors of Fgprp4 by sequencing analysis. Whereas three mutation sites are in the linker region of FgPrp6, seven are in the first two HAT repeats. RNA-seq analysis showed that suppressor mutations on different sites caused different splicing efficiency recovery. The suppressive effects of E308K and R230H were verified. Similar to human and fission yeast, the FgPrp6 was phosphorylated by the FgPrp4 kinase. Interestingly, the conserved Prp4-phosphorylation sites T261, T219&T221, and predicted phosphorylation sites T199&T200 on FgPrp6 were dispensable for the function of FgPrp6 in hyphal growth and sexual reproduction but important in plant infection. They are required for the infectious growth of F. graminearum in wheat lemma. RNA-seq analysis of the wheat lemma infected with Fgprp6/FgPRP6Δ199–221-GFP or Fgprp6/FgPRP6Δ250–262-GFP showed that 28 and 35% introns had splicing defects, respectively, which may be responsible for their defects in plant infection.

Somatic Mutations in Core Spliceosome Components Promote Tumorigenesis and Generate an Exploitable Vulnerability in Human Cancer

Simple SummaryHigh throughput exome sequencing approaches have disclosed recurrent cancer-associated mutations in spliceosomal components, which drive aberrant pre-mRNA processing events and support the tumor phenotype. At the same time, mutations in spliceosome genes and aberrant splicing regulation establish a selective vulnerability of cancer cells to splicing-targeting approaches, which could be exploited therapeutically. It is conceivable that a better understanding of the mechanisms and roles of abnormal splicing in tumor metabolism will facilitate the development of a novel generation of tumor-targeting drugs. In this review, we describe recent advances in the elucidation of the biological impact and biochemical effects of somatic mutations in core spliceosome components on splicing choices and their associated targetable vulnerabilities.Alternative pre-mRNA processing enables the production of distinct mRNA and protein isoforms from a single gene, thus greatly expanding the coding potential of eukaryotic genomes and fine-tuning gene expression programs. Splicing is carried out by the spliceosome, a complex molecular machinery which assembles step-wise on mRNA precursors in the nucleus of eukaryotic cells. In the last decade, exome sequencing technologies have allowed the identification of point mutations in genes encoding splicing factors as a recurrent hallmark of human cancers, with higher incidence in hematological malignancies. These mutations lead to production of splicing factors that reduce the fidelity of the splicing process and yield splicing variants that are often advantageous for cancer cells. However, at the same time, these mutations increase the sensitivity of transformed cells to splicing inhibitors, thus offering a therapeutic opportunity for novel targeted strategies. Herein, we review the recent literature documenting cancer-associated mutations in components of the early spliceosome complex and discuss novel therapeutic strategies based on small-molecule spliceosome inhibitors that exhibit strong anti-tumor effects, particularly against cancer cells harboring mutations in spliceosomal components.

Human Antigen R (HuR) Facilitates miR-19 Synthesis and Affects Cellular Kinetics in Papillary Thyroid Cancer.

Pre-mRNA splicing is an essential step in eukaryotic gene expression regulation. Genes are composed of exons that remain in the mature mRNAs and intervening sequences named introns. Splicing is the removal of introns and ligation of exons in a mature transcript. Splice site or spliceosome component mutations can lead to different diseases, including neurodegenerative diseases and several cancer types. HuR is an RNA-binding protein that preferentially binds to U- and AU-rich elements, usually found at the 3' UTRs of some mRNAs. We previously observed HuR specifically associated with spliceosomes assembled on introns containing miR-18a and miR-19a. miR-18a and miR-19a are components of the intronic miR-17-92 cluster, along with other five miRNAs. This cluster has been reported to regulate proliferation, migration, and angiogenesis in cells. In this context, we reasoned HuR could be controlling the splicing and processing of these miRNAs, leading to altered cellular phenotypes. We induced HuR overexpression in BCPAP and HEK-293T and analyzed the expression of miRNAs using qPCR, as well as the phenotypic effects in those cells. Cell counting to analyze cell growth was performed after trypan blue staining. Migration and invasion assays were performed using transwell filters and cells were counted after staining with crystal violet. We knocked down HuR using a specific siRNA and analyzed expression of miRNAs by qPCR, as well as cellular kinetics. Our results revealed HuR is associated with miR-19a in BCPAP and HEK-293T cells. Conversely, silencing HuR led to reduced miR-17-5p and miR-19a in BCPAP cells. Our data support that HuR stimulates the expression of miR-19, which is further processed and capable of finding its target sequence in a reporter plasmid. Cells overexpressing HuR showed increased cellular proliferation, migration, and invasion rates. Notably, under the presence of antimiR-19a, BCPAP-HuR cells showed reduced cell growth. Taken together, these results indicate the molecular alterations observed are associated with upregulation of miR-19a, leading to cellular processes involved in cancer development. Our findings propose a connection between HuR, miRNA biogenesis and cellular modifications. HuR stimulates miR-19a and miR-19b expression, which leads to up-regulation of cell proliferation, migration and invasion, promoting cancer development.

Cancer-Associated SF3B1 Mutations Confer a BRCA-Like Cellular Phenotype and Synthetic Lethality to PARP Inhibitors.

The cancer-associated SF3B1K700E mutation induces DNA damage via generation of genotoxic R-loops and stalled replication forks, defective homologous recombination, and increased replication fork degradation, which can be targeted with PARP inhibitors.

A novel role for nucleolin in splice site selection

ABSTRACT Latent 5ʹ splice sites, not normally used, are highly abundant in human introns, but are activated under stress and in cancer, generating thousands of nonsense mRNAs. A previously proposed mechanism to suppress latent splicing was shown to be independent of NMD, with a pivotal role for initiator-tRNA independent of protein translation. To further elucidate this mechanism, we searched for nuclear proteins directly bound to initiator-tRNA. Starting with UV-crosslinking, we identified nucleolin (NCL) interacting directly and specifically with initiator-tRNA in the nucleus, but not in the cytoplasm. Next, we show the association of initiator-tRNA and NCL with pre-mRNA. We further show that recovery of suppression of latent splicing by initiator-tRNA complementation is NCL dependent. Finally, upon nucleolin knockdown we show activation of latent splicing in hundreds of coding transcripts having important cellular functions. We thus propose nucleolin, a component of the endogenous spliceosome, through its direct binding to initiator-tRNA and its effect on latent splicing, as the first protein of a nuclear quality control mechanism regulating splice site selection to protect cells from latent splicing that can generate defective mRNAs.

Elevated SNRPA1, as a Promising Predictor Reflecting Severe Clinical Outcome via Effecting Tumor Immunity for ccRCC, Is Related to Cell Invasion, Metastasis, and Sunitinib Sensitivity.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal carcinoma and is associated with poor prognosis and notorious for its immune dysfunction characteristic. SNRPA1 is a spliceosome component responsible for processing pre-mRNA into mRNA, while the biological effect of SNRPA1 in ccRCC remains elusive. The aim of this study was to decipher the effect of SNRPA1 on clinical effect and tumor immunity for ccRCC patients. Multi-databases were collected to evaluate the different expression, prognostic value, DNA methylation, tumor immune microenvironment, and drug sensitivity of SNRPA1 on ccRCC. IHC was utilized to validate the expression and prognostic value of SNRPA1 in ccRCC patients from the SMMU cohort. The knockout expression of SNRPA by sgRNA plasmid inhibited the cell proliferation, migration, and metastasis ability and significantly increased the sensitivity of sunitinib treatment. In addition, we explored the role of SNRPA1 in pan-cancer level. The results indicated that SNRPA1 was differentially expressed in most cancer types. SNRPA1 may significantly influence the prognosis of multiple cancer types, especially in ccRCC patients. Notably, SNRPA1 was significantly correlated with immune cell infiltration and immune checkpoint inhibitory genes. In addition, the aggressive and immune inhibitory effects shown in SNRPA1 overexpression and the effect of SNRPA1 on ccRCC cell line invasion, metastasis, and drug sensitivity in vitro were observed. Moreover, SNRPA1 was related to Myc, MTORC, G2M, E2F, and DNA repair pathways in various cancer types. In all, SNRPA1 may prove to be a new biomarker for prognostic prediction, effect tumor immunity, and drug susceptibility in ccRCC.

The spliceosome component Usp39 controls B cell development by regulating immunoglobulin gene rearrangement.

The spliceosome is a large ribonucleoprotein complex responsible for pre-mRNA splicing and genome stability maintenance. Disruption of the spliceosome activity may lead to developmental disorders and tumorigenesis. However, the physiological role that the spliceosome plays in B cell development and function is still poorly defined. Here, we demonstrate that ubiquitin-specific peptidase 39 (Usp39), a spliceosome component of the U4/U6.U5 tri-snRNP complex, is essential for B cell development. Ablation of Usp39 in B cell lineage blocks pre-pro-B to pro-B cell transition in the bone marrow, leading to a profound reduction of mature B cells in the periphery. We show that Usp39 specifically regulates immunoglobulin gene rearrangement in a spliceosome-dependent manner, which involves modulating chromatin interactions at the Igh locus. Moreover, our results indicate that Usp39 deletion reduces the pre-malignant B cells in Eμ-Myc transgenic mice and significantly improves their survival.

SF3B1 inhibition disrupts malignancy and prolongs survival in glioblastoma patients through BCL2L1\xa0splicing and mTOR/\xdf-catenin pathways imbalances

BackgroundGlioblastoma is one of the most devastating cancer worldwide based on its locally aggressive behavior and because it cannot be cured by current therapies. Defects in alternative splicing process are frequent in cancer. Recently, we demonstrated that dysregulation of the spliceosome is directly associated with glioma development, progression, and aggressiveness.MethodsDifferent human cohorts and a dataset from different glioma mouse models were analyzed to determine the mutation frequency as well as the gene and protein expression levels between tumor and control samples of the splicing-factor-3B-subunit-1 (SF3B1), an essential and druggable spliceosome component. SF3B1 expression was also explored at the single-cell level across all cell subpopulations and transcriptomic programs. The association of SF3B1 expression with relevant clinical data (e.g., overall survival) in different human cohorts was also analyzed. Different functional (proliferation/migration/tumorspheres and colonies formation/VEGF secretion/apoptosis) and mechanistic (gene expression/signaling pathways) assays were performed in three different glioblastomas cell models (human primary cultures and cell lines) in response to SF3B1 blockade (using pladienolide B treatment). Moreover, tumor progression and formation were monitored in response to SF3B1 blockade in two preclinical xenograft glioblastoma mouse models.ResultsOur data provide novel evidence demonstrating that the splicing-factor-3B-subunit-1 (SF3B1, an essential and druggable spliceosome component) is low-frequency mutated in human gliomas (~ 1 %) but widely overexpressed in glioblastoma compared with control samples from the different human cohorts and mouse models included in the present study, wherein SF3B1 levels are associated with key molecular and clinical features (e.g., overall survival, poor prognosis and/or drug resistance). Remarkably, in vitro and in vivo blockade of SF3B1 activity with pladienolide B drastically altered multiple glioblastoma pathophysiological processes (i.e., reduction in proliferation, migration, tumorspheres formation, VEGF secretion, tumor initiation and increased apoptosis) likely by suppressing AKT/mTOR/ß-catenin pathways, and an imbalance of BCL2L1 splicing.ConclusionsTogether, we highlight SF3B1 as a potential diagnostic and prognostic biomarker and an efficient pharmacological target in glioblastoma, offering a clinically relevant opportunity worth to be explored in humans.

XAP5 CIRCADIAN TIMEKEEPER specifically modulates 3’ splice site recognition and is important for circadian clock regulation partly by alternative splicing of LHY and TIC

Pre-mRNA splicing is an essential step during gene expression, which takes place in the spliceosome, a large dynamic ribonucleoprotein complex assembled in a stepwise manner. During the last decade, several spliceosomal mutants were functionally identified to cause a lengthened circadian period by introducing intron retention defects into circadian clock genes in Arabidopsis. However, the spliceosomal components that play opposite roles in the circadian period via alternative 3′ splice site (Alt 3'ss) are largely unknown. Here, we demonstrated that XCT (XAP5 CIRCADIAN TIMEKEEPER) is a key spliceosomal component associated with multiple splicing factors. Moreover, genome-wide analysis revealed that inactivation of XCT particularly results in defects in Alt 3'ss recognition by RNA sequencing. Further analysis indicated that a strong alteration in the 3′ splice sites of LHY and TIC partly accounts for the shortened circadian period of the xct mutant. Therefore, our results demonstrated that mutations in XCT shortened the circadian period partly by alternative splicing of LHY and TIC particularly in 3′ splice site recognition, which provides new insight into the link between alternative splicing and the circadian clock.