Isoform Switching Research Articles

Dynamic transcriptional and chromatin accessibility landscape of medaka embryogenesis.

Medaka (Oryzias latipes) has become an important vertebrate model widely used in genetics, developmental biology, environmental sciences, and many other fields. A high-quality genome sequence and a variety of genetic tools are available for this model organism. However, existing genome annotation is still rudimentary, as it was mainly based on computational prediction and short-read RNA-seq data. Here we report a dynamic transcriptome landscape of medaka embryogenesis profiled by long-read RNA-seq, short-read RNA-seq, and ATAC-seq. By integrating these data sets, we constructed a much-improved gene model set including about 17,000 novel isoforms and identified 1600 transcription factors, 1100 long noncoding RNAs, and 150,000 potential cis-regulatory elements as well. Time-series data sets provided another dimension of information. With the expression dynamics of genes and accessibility dynamics of cis-regulatory elements, we investigated isoform switching, as well as regulatory logic between accessible elements and genes, during embryogenesis. We built a user-friendly medaka omics data portal to present these data sets. This resource provides the first comprehensive omics data sets of medaka embryogenesis. Ultimately, we term these three assays as the minimum ENCODE toolbox and propose the use of it as the initial and essential profiling genomic assays for model organisms that have limited data available. This work will be of great value for the research community using medaka as the model organism and many others as well.

A Glutaminase Isoform Switch Drives Therapeutic Resistance and Disease Progression of Prostate Cancer

Cellular metabolism in cancer is significantly altered to support the uncontrolled tumor growth. How metabolic alterations contribute to the hormonal therapy resistance and disease progression of prostate cancer (PCa) remains poorly understood. Here we report a glutaminase isoform switch mechanism that mediates the initial therapeutic effect but eventual failure of hormonal therapy of PCa. Androgen deprivation therapy inhibits the expression of kidney-type glutaminase (KGA), a splicing isoform of glutaminase 1 (GLS1) upregulated by androgen receptor (AR), to achieve therapeutic effect by suppressing glutaminolysis. Eventually the tumor cells switch to the expression of glutaminase C (GAC), a shorter splicing isoform of GLS1that is upregulated by MYC (c-MYC and N-Myc) with more potent in glutaminolysis, under the androgen-deprived condition. This switch leads to increased glutamine utilization, hyper-proliferation and aggressive behavior of tumor cells. Pharmacological inhibition or RNA interference of GLS1 isoforms differentially suppresses the growth of hormone sensitive prostate cancer and castration resistant prostate cancer in vitro and in vivo. Therefore, we have identified a metabolic function of AR action in PCa and discovered that the GLS1 isoform switch is a key mechanism in therapeutic resistance and disease progression.

Role of PKC\u03b5 in the epithelial-mesenchymal transition induced by FGFR2 isoform switch

BackgroundThe epithelial isoform of the fibroblast growth factor receptor 2 (FGFR2b) controls the entire program of keratinocyte differentiation via the sequential involvement of protein kinase C (PKC) δ and PKCα. In contrast, the FGFR2 isoform switch and the aberrant expression of the mesenchymal FGFR2c isoform leads to impairment of differentiation, epithelial-mesenchymal transition (EMT) and tumorigenic features. Aim of our present study was to contribute in clarifying the complex network of signaling pathways involved in the FGFR2c-mediated oncogenic outcomes focusing on PKCε, which appears to be involved in the induction of EMT and tumorigenesis in several epithelial contexts.MethodsBiochemical and molecular analysis, as well as in vitro invasion assays, combined with the use of specific small interfering RNA (siRNA), were performed in human keratinocytes stably expressing FGFR2c or FGFR2b isoforms.ResultsOur results showed that aberrant expression and signaling of FGFR2c, but not those of FGFR2b, in human keratinocytes induced a strong phosphorylation/activation of PKCε. The use of siRNA approach showed that PKCε is the hub signaling downstream FGFR2c responsible for the modulation of EMT markers and for the induction of the EMT-related transcription factors STAT3, Snail1 and FRA1, as well as for the acquisition of the invasive behavior. Moreover, experiments of depletion of ESRP1, responsible for FGFR2 splicing in epithelial cells, indicated that the activation of PKCε is the key molecular event triggered by FGFR2 isoform switch and underlying EMT induction.ConclusionsOverall, our results point to the identification of the downstream PKC isoform responsible for the FGFR signaling deregulation occurring in epithelial tissues from the physiological oncosoppressive to the pathological oncogenic profile.DoUzGs4sbo_jjVe6wgq-stVideo Graphical abstract

DHA Supplementation Attenuates MI-Induced LV Matrix Remodeling and Dysfunction in Mice.

Objective Myocardial ischemia and reperfusion (I/R) injury is associated with oxidative stress and inflammation, leading to scar development and malfunction. The marine omega-3 fatty acids (ω-3 FA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are mediating cardioprotection and improving clinical outcomes in patients with heart disease. Therefore, we tested the hypothesis that docosahexaenoic acid (DHA) supplementation prior to LAD occlusion-induced myocardial injury (MI) confers cardioprotection in mice. Methods C57BL/6N mice were placed on DHA or control diets (CD) beginning 7 d prior to 60 min LAD occlusion-induced MI or sham surgery. The expression of inflammatory mediators was measured via RT-qPCR. Besides FACS analysis for macrophage quantification and subtype evaluation, macrophage accumulation as well as collagen deposition was quantified in histological sections. Cardiac function was assessed using a pressure-volume catheter for up to 14 d. Results DHA supplementation significantly attenuated the induction of peroxisome proliferator-activated receptor-α (PPAR-α) (2.3 ± 0.4 CD vs. 1.4 ± 0.3 DHA) after LAD occlusion. Furthermore, TNF-α (4.0 ± 0.6 CD vs. 1.5 ± 0.2 DHA), IL-1β (60.7 ± 7.0 CD vs. 11.6 ± 1.9 DHA), and IL-10 (223.8 ± 62.1 CD vs. 135.5 ± 38.5 DHA) mRNA expression increase was diminished in DHA-supplemented mice after 72 h reperfusion. These changes were accompanied by a less prominent switch in α/β myosin heavy chain isoforms. Chemokine mRNA expression was stronger initiated (CCL2 6 h: 32.8 ± 11.5 CD vs. 78.8 ± 13.6 DHA) but terminated earlier (CCL2 72 h: 39.5 ± 7.8 CD vs. 8.2 ± 1.9 DHA; CCL3 72 h: 794.3 ± 270.9 CD vs. 258.2 ± 57.8 DHA) in DHA supplementation compared to CD mice after LAD occlusion. Correspondingly, DHA supplementation was associated with a stronger increase of predominantly alternatively activated Ly6C-positive macrophage phenotype, being associated with less collagen deposition and better LV function (EF 14 d: 17.6 ± 2.6 CD vs. 31.4 ± 1.5 DHA). Conclusion Our data indicate that DHA supplementation mediates cardioprotection from MI via modulation of the inflammatory response with timely and attenuated remodeling. DHA seems to attenuate MI-induced cardiomyocyte injury partly by transient PPAR-α downregulation, diminishing the need for antioxidant mechanisms including mitochondrial function, or α- to β-MHC isoform switch.

The Regulation of Heme Metabolism in Myocytes by Hypoxia and Ischemia: Substrate‐Enzyme Dyssynchrony

Current strategies for the treatment of acute myocardial infarction (AMI) are limited therefore better pharmacological interventions are needed to improve cardiac function post‐injury. Overexpression of the enzyme heme oxygenase‐1 (HMOX1) has been shown to exert cytoprotective effects in models of acute myocardial infarction and represents a highly attractive therapeutic target. HMOX1 is the stress‐inducible enzyme responsible for the catabolism of heme, the functional backbone by which oxygen is carried in proteins like hemoglobin and cytochromes in the electron transport chain. As a potent inducer of HMOX1 and an FDA‐approved heme‐surrogate for the treatment of porphyria, hemin represents a novel treatment strategy for AMI.Translation of HMOX1‐targeted therapeutics to the clinic has been stymied by a singular focus on HMOX1 and an incomplete understanding of fundamental heme metabolism regulation in response to hypoxia, ischemia or pharmacological stimulation with hemin. To date, studies investigating hemin pharmacology and its use in models of ischemic injury are limited. The objective of the present study was to provide a temporal and pharmacokinetic characterization of the changes in heme regulatory enzyme expression in response to hemin and ischemic cardiac injury (i.e. AMI).Echocardiographic, hemodynamic and protein quantification analyses were performed in male CD‐1 mice following a single intraperitoneal hemin injection, permanent ligation AMI, or daily hemin treatment pre‐ or post‐AMI with corresponding sham and vehicle controls. Temporal changes in left ventricular heme regulatory enzyme expression were measured in response to hemin and AMI. This is the first study to holistically characterize the effects of either AMI and/or hemin on endogenous heme regulation in the myocardium.Hemin markedly increased HMOX1 levels and instigated isoform switching between heme‐synthesizing enzymes in the left ventricle of healthy mice. This isoform switching was further observed with AMI and suggests a progressive increase in heme bioavailability with ischemic injury. Interestingly, HMOX1 — a heme and stress‐inducible enzyme — was induced at only 3 days post‐AMI and did not increase concomitantly with heme‐synthesizing enzymes.Echocardiographic analyses showed preserved left ventricular morphology and cardiac output with hemin administration both pre‐ and post‐AMI. Invasive hemodynamics revealed preserved left ventricular function in mice administered daily hemin when initiated 3h pre‐AMI but not when initiated 2h post‐AMI. Similarly, hemin administration significantly increased H9C2 cardiomyotubule viability when administered prior to H2O2‐mediated injury but not with simultaneous or 2h post‐injury hemin administration. This suggests that time‐dependent mechanisms are involved in conferring hemin‐mediated cardioprotection and could suggest differential mechanisms related to myocyte survival and cardiac remodelling.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada, Nova Scotia Health Research Foundation

MiR 208a Deficiency Promotes Mitochondrial Biogenesis in the metabolic heart

A high caloric regimen causes metabolic syndrome, which is a pre‐requisite for type 2 diabetes mellitus, and increases the risk for cardiovascular disease. Both metabolic syndrome and diabetes induce a common cardiac phenotype known as metabolic cardiomyopathy with an early onset as diastolic dysfunction that may progress to systolic dysfunction and congestive heart failure.While the normal heart is able to freely switch between glucose and fatty acid (FA) oxidation, the metabolic heart becomes almost completely reliant on FA oxidation for ATP production. This energetic dysregulation is supported by increased mitochondrial metabolism, which includes an increased mitochondrial mass and function. Based on the reported cardiac specificity of the microRNA (miR) 208a, we investigated its role in bioenergetic metabolism in the metabolic heart. Lewis rats exposed to a high fat diet (HFD) exhibit obesity, systemic insulin resistance, hyperglycemia, hyperlipidemia, cardiac diastolic dysfunction, and cardiac fibrosis. Microarray studies highlighted an altered miRNA expression profile in the heart upon HFD, including a decrease in miR 208a. CRISPR deletion of the miR 208a in cultured human SV40 cardiomyocytes exposed to diabetogenic conditions (high glucose, high albumin‐bound palmitate) led to a bioenergetics profile that favors mitochondrial FA metabolism including an increase in mitochondrial biogenesis signaling, electron transport chain complexes and critical enzymes for FA beta‐oxidation. In addition, miR 208‐a deficiency prevented the switch in the myosin heavy chain isoforms by increasing myosin heavy chain alpha, thus preserving cardiac contractility. We analyzed in silico for putative miR 208a targets focusing on mitochondrial outcomes using the TargetScanHuman database ( http://www.targetscan.org/cgi‐bin/targetscan/vert_71/targetscan.cgi). We found that mitochondrial ribosomal protein 28 (involved in mitochondrial translation and generation of mitochondrial DNA‐encoded electron transport chain subunits), MINOS1 (a conserved component of mitofilin complexes required for mitochondrial function and cristae organization), and MED7 (component of the Mediator Complex that regulates the transcription by facilitating the interaction between nuclear receptors, transcriptional co‐activator and co‐repressors, and chromatic modification factors with RNA Pol II) are rescued by miR 208a deficiency in cardiomyocytes exposed to diabetogenic conditions. In summary, miR 208a regulates nuclear and mitochondrial transcription, mitochondrial cristae organization and integrity in the diabetic heart, and supports the metabolic remodeling towards increased FA oxidation. Our data suggest that miR 208a may be a therapeutic target to promote mitochondrial biogenesis in chronic diseases associated with mitochondrial defects.Support or Funding InformationCentral Michigan University Early Career Grant, AIREA American Heart Association

Differential Alternative Polyadenylation Landscapes Mediate Hematopoietic Stem Cell Activation and Regulate Glutamine Metabolism.

Alternative polyadenylation (APA) is emerging as an important regulatory mechanism of RNA and protein isoform expression by controlling 3' untranslated region (3'-UTR) composition. The relevance of APA in stem cell hierarchies remains elusive. Here, we first demonstrate the requirement of the APA regulator Pabpn1 for hematopoietic stem cell (HSC) function. We then determine the genome-wide APA landscape (APAome) of HSCs and progenitors by performing low-input 3' sequencing paired with bioinformatic pipelines. This reveals transcriptome-wide dynamic APA patterns and an overall shortening of 3'-UTRs during differentiation and upon homeostatic or stress-induced transition from quiescence to proliferation. Specifically, we show that APA regulates activation-induced Glutaminase (Gls) isoform switching by Nudt21. This adaptation of the glutamine metabolism by increasing the GAC:KGA isoform ratio fuels versatile metabolic pathways necessary for HSC self-renewal and proper stress response. Our study establishes APA as a critical regulatory layer orchestrating HSC self-renewal, behavior, and commitment.

Phosphorylation of GATA4 at serine 105 is required for left ventricular remodelling process in angiotensin II-induced hypertension in rats.

In this study, we investigated whether local intramyocardial GATA4 overexpression affects the left ventricular (LV) remodelling process and the importance of phosphorylation at serine 105 (S105) for the actions of GATA4 in an angiotensin II (AngII)‐induced hypertension rat model. Adenoviral constructs overexpressing wild‐type GATA4 or GATA4 mutated at S105 were delivered into the anterior LV free wall. AngII (33.3 µg/kg/h) was administered via subcutaneously implanted minipumps. Cardiac function and structure were examined by echocardiography, followed by histological immunostainings of LV sections and gene expression measurements by RT‐qPCR. The effects of GATA4 on cultured neonatal rat ventricular fibroblasts were evaluated. In AngII‐induced hypertension, GATA4 overexpression repressed fibrotic gene expression, reversed the hypertrophic adult‐to‐foetal isoform switch of myofibrillar genes and prevented apoptosis, whereas histological fibrosis was not affected. Overexpression of GATA4 mutated at S105 resulted in LV chamber dilatation, cardiac dysfunction and had minor effects on expression of myocardial remodelling genes. Fibrotic gene expression in cardiac fibroblasts was differently affected by overexpression of wild‐type or mutated GATA4. Our results indicate that GATA4 reduces AngII‐induced responses by interfering with pro‐fibrotic and hypertrophic gene expressions. GATA4 actions on LV remodelling and fibroblasts are dependent on phosphorylation site S105.

Edgetic perturbation signatures represent known and novel cancer biomarkers

Isoform switching is a recently characterized hallmark of cancer, and often translates to the loss or gain of domains mediating protein interactions and thus, the re-wiring of the interactome. Recent computational tools leverage domain-domain interaction data to resolve the condition-specific interaction networks from RNA-Seq data accounting for the domain content of the primary transcripts expressed. Here, we used The Cancer Genome Atlas RNA-Seq datasets to generate 642 patient-specific pairs of interactomes corresponding to both the tumor and the healthy tissues across 13 cancer types. The comparison of these interactomes provided a list of patient-specific edgetic perturbations of the interactomes associated with the cancerous state. We found that among the identified perturbations, select sets are robustly shared between patients at the multi-cancer, cancer-specific and cancer sub-type specific levels. Interestingly, the majority of the alterations do not directly involve significantly mutated genes, nevertheless, they strongly correlate with patient survival. The findings (available at EdgeExplorer: “http://webclu.bio.wzw.tum.de/EdgeExplorer”) are a new source of potential biomarkers for classifying cancer types and the proteins we identified are potential anti-cancer therapy targets.

Dynamic Alternative Splicing During Mouse Preimplantation Embryo Development.

The mechanism of alternative pre-mRNA splicing (AS) during preimplantation development is largely unknown. In order to capture the dynamic changes of AS occurring during embryogenesis, we carried out bioinformatics analysis based on scRNA-seq data over the time-course preimplantation development in mouse. We detected numerous previously-unreported differentially expressed genes at specific developmental stages and investigated the nature of AS at both minor and major zygotic genome activation (ZGA). The AS and differential AS atlas over preimplantation development were established. The differentially alternatively spliced genes (DASGs) are likely to be key splicing factors (SFs) during preimplantation development. We also demonstrated that there is a regulatory cascade of AS events in which some key SFs are regulated by differentially AS of their own gene transcripts. Moreover, 212 isoform switches (ISs) during preimplantation development were detected, which may be critical for decoding the mechanism of early embryogenesis. Importantly, we uncovered that zygotic AS activation (ZASA) is in conformity with ZGA and revealed that AS is coupled with transcription during preimplantation development. Our results may provide a deeper insight into the regulation of early embryogenesis.

Sex biased expression and co-expression networks in development, using the hymenopteran Nasonia vitripennis.

Sexual dimorphism requires regulation of gene expression in developing organisms. These developmental differences are caused by differential expression of genes and isoforms. The effect of expressing a gene is also influenced by which other genes are simultaneously expressed (functional interactions). However, few studies have described how these processes change across development. We compare the dynamics of differential expression, isoform switching and functional interactions in the sexual development of the model parasitoid wasp Nasonia vitripennis, a system that permits genome wide analysis of sex bias from early embryos to adults. We find relatively little sex-bias in embryos and larvae at the gene level, but several sub-networks show sex-biased functional interactions in early developmental stages. These networks provide new candidates for hymenopteran sex determination, including histone modification. In contrast, sex-bias in pupae and adults is driven by the differential expression of genes. We observe sex-biased isoform switching consistently across development, but mostly in genes that are already differentially expressed. Finally, we discover that sex-biased networks are enriched by genes specific to the Nasonia clade, and that those genes possess the topological properties of key regulators. These findings suggest that regulators in sex-biased networks evolve more rapidly than regulators of other developmental networks.

The RNA-binding protein AKAP8 suppresses tumor metastasis by antagonizing EMT-associated alternative splicing

Alternative splicing has been shown to causally contribute to the epithelial–mesenchymal transition (EMT) and tumor metastasis. However, the scope of splicing factors that govern alternative splicing in these processes remains largely unexplored. Here we report the identification of A-Kinase Anchor Protein (AKAP8) as a splicing regulatory factor that impedes EMT and breast cancer metastasis. AKAP8 not only is capable of inhibiting splicing activity of the EMT-promoting splicing regulator hnRNPM through protein–protein interaction, it also directly binds to RNA and alters splicing outcomes. Genome-wide analysis shows that AKAP8 promotes an epithelial cell state splicing program. Experimental manipulation of an AKAP8 splicing target CLSTN1 revealed that splice isoform switching of CLSTN1 is crucial for EMT. Moreover, AKAP8 expression and the alternative splicing of CLSTN1 predict breast cancer patient survival. Together, our work demonstrates the essentiality of RNA metabolism that impinges on metastatic breast cancer.

Genetic basis of oxygen sensing in the carotid body: HIF2α and an isoform switch in cytochrome c oxidase subunit 4.

The mechanistic basis of the marked oxygen sensitivity of glomus cells in the carotid body has long puzzled physiologists. In this issue of Science Signaling, Moreno-Domínguez et al. show the critical importance of high levels of hypoxia-inducible factor, HIF2α/EPAS1, and the nuclear-encoded mitochondrial cytochrome c oxidase subunit, COX4I2, in glomus cell sensitivity to hypoxia.

Accurate quantification of circular RNAs identifies extensive circular isoform switching events

Detection and quantification of circular RNAs (circRNAs) face several significant challenges, including high false discovery rate, uneven rRNA depletion and RNase R treatment efficiency, and underestimation of back-spliced junction reads. Here, we propose a novel algorithm, CIRIquant, for accurate circRNA quantification and differential expression analysis. By constructing pseudo-circular reference for re-alignment of RNA-seq reads and employing sophisticated statistical models to correct RNase R treatment biases, CIRIquant can provide more accurate expression values for circRNAs with significantly reduced false discovery rate. We further develop a one-stop differential expression analysis pipeline implementing two independent measures, which helps unveil the regulation of competitive splicing between circRNAs and their linear counterparts. We apply CIRIquant to RNA-seq datasets of hepatocellular carcinoma, and characterize two important groups of linear-circular switching and circular transcript usage switching events, which demonstrate the promising ability to explore extensive transcriptomic changes in liver tumorigenesis.

Retinoic acid promotes metabolic maturation of human Embryonic Stem Cell-derived Cardiomyocytes.

Cardiomyocytes differentiated from human embryonic stem cells (hESCs) represent a promising cell source for heart repair, disease modeling and drug testing. However, improving the differentiation efficiency and maturation of hESC-derived cardiomyocytes (hESC-CMs) is still a major concern. Retinoic acid (RA) signaling plays multiple roles in heart development. However, the effects of RA on cardiomyocyte differentiation efficiency and maturation are still unknown.Methods: RA was added at different time intervals to identify the best treatment windows for cardiomyocyte differentiation and maturation. The efficiency of cardiomyocyte differentiation was detected by quantitative real-time PCR and flow cytometry. Cardiomyocytes maturation was detected by immunofluorescence staining, metabolic assays and patch clamp to verify structural, metabolic and electrophysiological maturation, respectively. RNA sequencing was used for splicing analysis.Results: We found that RA treatment at the lateral mesoderm stage (days 2-4) significantly improved cardiomyocyte differentiation, as evidenced by the upregulation of TNNT2, NKX2.5 and MYH6 on day 10 of differentiation. In addition, flow cytometry showed that the proportion of differentiated cardiomyocytes in the RA-treated group was significantly higher than that in control group. RA treatment on days 15-20 increased cardiomyocyte area, sarcomere length, multinucleation and mitochondrial copy number. RNA sequencing revealed RA promoted RNA isoform switch to the maturation-related form. Meanwhile, RA promoted electrophysiological maturation and calcium handling of hESC-CMs. Importantly, RA-treated cardiomyocytes showed decreased glycolysis and enhanced mitochondrial oxidative phosphorylation, with the increased utilization of fatty acid and exogenous pyruvate but not glutamine.Conclusion: Our data indicated that RA treatment at an early time window (days 2-4) promotes the efficiency of cardiomyocyte differentiation and that RA treatment post beating (days 15-20) promotes cardiomyocyte maturation. The biphasic effects of RA provide new insights for improving cardiomyocyte differentiation and quality.

Forced isoform switching of Neat1_1 to Neat1_2 leads to the loss of Neat1_1 and the hyperformation of paraspeckles but does not affect the development and growth of mice

Neat1 is a long noncoding RNA (lncRNA) that serves as an architectural component of the nuclear bodies known as paraspeckles. Two isoforms of Neat1, the short isoform Neat1_1 and the long isoform Neat1_2, are generated from the same gene locus by alternative 3′ processing. Neat1_1 is the most abundant and the best conserved isoform expressed in various cell types, whereas Neat1_2 is expressed in a small population of particular cell types, including the tip cells of the intestinal epithelium. To investigate the physiological significance of isoform switching, we created mutant mice that solely expressed Neat1_2 by deleting the upstream polyadenylation (poly-A) signal (PAS) required for the production of Neat1_1. We observed the loss of Neat1_1 and strong up-regulation of Neat1_2 in various tissues and cells and the subsequent hyperformation of paraspeckles, especially in cells that normally express Neat1_2. However, the mutant mice were born at the expected Mendelian ratios and did not exhibit obvious external and histological abnormalities. These observations suggested that the hyperformation of paraspeckles does not interfere with the development and growth of these animals under normal laboratory conditions.

Splicing Busts a Move: Isoform Switching Regulates Migration.

Cell migration is essential for normal development, neural patterning, pathogen eradication, and cancer metastasis. Pre-mRNA processing events such as alternative splicing and alternative polyadenylation result in greater transcript and protein diversity as well as function and activity. A critical role for alternative pre-mRNA processing in cell migration has emerged in axon outgrowth during neuronal development, immune cell migration, and cancer metastasis. These findings suggest that migratory signals result in expression changes of post-translational modifications of splicing or polyadenylation factors, leading to splicing events that generate promigratory isoforms. We summarize this recent progress and suggest emerging technologies that may facilitate a deeper understanding of the role of alternative splicing and polyadenylation in cell migration.

SAM68-Specific Splicing Is Required for Proper Selection of Alternative 3′ UTR Isoforms in the Nervous System

SummaryNeuronal alternative splicing is a core mechanism for functional diversification. We previously found that STAR family proteins (SAM68, SLM1, SLM2) regulate spatiotemporal alternative splicing in the nervous system. However, the whole aspect of alternative splicing programs by STARs remains unclear. Here, we performed a transcriptomic analysis using SAM68 knockout and SAM68/SLM1 double-knockout midbrains. We revealed different alternative splicing activity between SAM68 and SLM1; SAM68 preferentially targets alternative 3′ UTR exons. SAM68 knockout causes a long-to-short isoform switch of a number of neuronal targets through the alteration in alternative last exon (ALE) selection or alternative polyadenylation. The altered ALE usage of a novel target, interleukin 1 receptor accessory protein (Il1rap), results in remarkable conversion from a membrane-bound type to a secreted type in Sam68KO brains. Proper ALE selection is necessary for IL1RAP neuronal function. Thus the SAM68-specific splicing program provides a mechanism for neuronal selection of alternative 3′ UTR isoforms.

N6-Methyladenosine Modulates Nonsense-Mediated mRNA Decay in Human Glioblastoma.

The N6-methyladenosine (m6A) modification influences various mRNA metabolic events and tumorigenesis, however, its functions in nonsense-mediated mRNA decay (NMD) and whether NMD detects induced carcinogenesis pathways remain undefined. Here, we showed that the m6A methyltransferase METTL3 sustained its oncogenic role by modulating NMD of splicing factors and alternative splicing isoform switches in glioblastoma (GBM). Methylated RNA immunoprecipitation-seq (MeRIP-seq) analyses showed that m6A modification peaks were enriched at metabolic pathway-related transcripts in glioma stem cells (GSC) compared with neural progenitor cells. In addition, the clinical aggressiveness of malignant gliomas was associated with elevated expression of METTL3. Furthermore, silencing METTL3 or overexpressing dominant-negative mutant METTL3 suppressed the growth and self-renewal of GSCs. Integrated transcriptome and MeRIP-seq analyses revealed that downregulating the expression of METTL3 decreased m6A modification levels of serine- and arginine-rich splicing factors (SRSF), which led to YTHDC1-dependent NMD of SRSF transcripts and decreased SRSF protein expression. Reduced expression of SRSFs led to larger changes in alternative splicing isoform switches. Importantly, the phenotypes mediated by METTL3 deficiency could be rescued by downregulating BCL-X or NCOR2 isoforms. Overall, these results establish a novel function of m6A in modulating NMD and uncover the mechanism by which METTL3 promotes GBM tumor growth and progression. SIGNIFICANCE: These findings establish the oncogenic role of m6A writer METTL3 in glioblastoma stem cells.

Complex Polyclonal Resistance Mechanisms to Ivosidenib Monotherapy in IDH1-Mutant Relapsed or Refractory Acute Myeloid Leukemia Revealed By Single Cell Sequencing Analyses

Complex Polyclonal Resistance Mechanisms to Ivosidenib Monotherapy in IDH1-Mutant Relapsed or Refractory Acute Myeloid Leukemia Revealed By Single Cell Sequencing Analyses