Regulation Of Splicing Research Articles

Spatially exploring RNA biology in archival formalin-fixed paraffin-embedded tissues.

The capability to spatially explore RNA biology in formalin-fixed paraffin-embedded (FFPE) tissues holds transformative potential for histopathology research. Here, we present pathology-compatible deterministic barcoding in tissue (Patho-DBiT) by combining in situ polyadenylation and computational innovation for spatial whole transcriptome sequencing, tailored to probe the diverse RNA species in clinically archived FFPE samples. It permits spatial co-profiling of gene expression and RNA processing, unveiling region-specific splicing isoforms, and high-sensitivity transcriptomic mapping of clinical tumor FFPE tissues stored for 5 years. Furthermore, genome-wide single-nucleotide RNA variants can be captured to distinguish malignant subclones from non-malignant cells in human lymphomas. Patho-DBiT also maps microRNA regulatory networks and RNA splicing dynamics, decoding their roles in spatial tumorigenesis. Single-cell level Patho-DBiT dissects the spatiotemporal cellular dynamics driving tumor clonal architecture and progression. Patho-DBiT stands poised as a valuable platform to unravel rich RNA biology in FFPE tissues to aid in clinical pathology evaluation.

Regulation of Ptbp1-controlled alternative splicing of pyruvate kinase muscle by Liver kinase b1 governs vascular smooth muscle cell plasticity in vivo.

Vascular smooth muscle cell (VSMC) plasticity is a state in which VSMCs undergo phenotypic switching from a quiescent contractile phenotype into other functionally distinct phenotypes. Although emerging evidence suggest that VSMC plasticity plays critical roles in the development of vascular diseases, little is known about the key determinant for controlling VSMC plasticity and fate. We found that smooth muscle cell-specific deletion of Lkb1 in tamoxifen-inducible Lkb1flox/flox; Myh11-Cre/ERT2 mice spontaneously and progressively induced aortic/arterial dilation, aneurysm, rupture, and premature death. Single-cell RNA sequencing and imaging-based lineage tracing showed that Lkb1-deficient VSMCs transdifferentiated gradually from early modulated VSMCs to fibroblast-like and chondrocyte-like cells, leading to ossification and blood-vessel rupture. Mechanistically, Lkb1 regulates polypyrimidine tract binding protein 1 (Ptbp1) expression and controls alternative splicing of pyruvate kinase muscle (PKM) isoforms 1 and 2. Lkb1 loss in VSMC results in an increased PKM2/PKM1 ratio and alters the metabolic profile by promoting aerobic glycolysis. Treatment with PKM2 activator TEPP-46 rescues VSMC transformation and aortic dilation in Lkb1flox/flox; Myh11-Cre/ERT2 mice. Furthermore, we found that Lkb1 expression decreased in human aortic aneurysm tissue compared to control tissue, along with changes in markers of VSMC fate. Lkb1, via its regulation of Ptbp1-dependent alterative splicing of PKM, maintains VSMC in contractile states by suppressing VSMC plasticity.

The Biological Mechanisms and Clinical Roles of RNA-Binding Proteins in Cardiovascular Diseases.

RNA-binding proteins (RBPs) have pivotal roles in cardiovascular biology, influencing various molecular mechanisms underlying cardiovascular diseases (CVDs). This review explores the significant roles of RBPs, focusing on their regulation of RNA alternative splicing, polyadenylation, and RNA editing, and their impact on CVD pathogenesis. For instance, RBPs are crucial in myocardial injury, contributing to disease progression and repair mechanisms. This review systematically analyzes the roles of RBPs in myocardial injury, arrhythmias, myocardial infarction, and heart failure, revealing intricate interactions that influence disease outcomes. Furthermore, the potential of RBPs as therapeutic targets for cardiovascular dysfunction is explored, highlighting the advances in drug development and clinical research. This review also discusses the emerging role of RBPs as biomarkers for cardiovascular diseases, offering insights into their diagnostic and prognostic potential. Despite significant progress, current research faces several limitations, which are critically examined. Finally, this review identifies the major challenges and outlines future research directions to advance the understanding and application of RBPs in cardiovascular medicine.

A plant-specific clade of serine/arginine-rich proteins regulates RNA splicing homeostasis and thermotolerance in tomato.

Global warming poses a threat for crops, therefore, the identification of thermotolerance mechanisms is a priority. In plants, the core factors that regulate transcription under heat stress (HS) are well described and include several HS transcription factors (HSFs). Despite the relevance of alternative splicing in HS response and thermotolerance, the core regulators of HS-sensitive alternative splicing have not been identified. In tomato, alternative splicing of HSFA2 is important for acclimation to HS. Here, we show that several members of the serine/arginine-rich family of splicing factors (SRSFs) suppress HSFA2 intron splicing. Individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) combined with RNA-Seq revealed that RS2Z35 and RS2Z36, which make up a plant-specific clade of SR proteins, not only regulate HSFA2 but approximately 50% of RNAs that undergo HS-sensitive alternative splicing, with preferential binding to purine-rich RNA motifs. Single and double CRISPR rs2z mutant lines show a dysregulation of splicing and exhibit lower basal and acquired thermotolerance compared to wild type plants. Our results suggest that RS2Z35 and RS2Z36 have a central role in mitigation of the negative effects of HS on RNA splicing homeostasis, and their emergence might have contributed to the increased capacity of plants to acclimate to high temperatures.

Expression levels of core spliceosomal proteins modulate the MBNL-mediated spliceopathy in DM1.

Myotonic dystrophy type 1 (DM1) is a heterogeneous multisystemic disease caused by a CTG repeat expansion in DMPK. Transcription of the expanded allele produces toxic CUG repeat RNA that sequesters the MBNL family of alternative splicing (AS) regulators into ribonuclear foci, leading to pathogenic mis-splicing. To identify genetic modifiers of toxic CUG RNA levels and the spliceopathy, we performed a genome-scale siRNA screen using an established HeLa DM1 repeat-selective screening platform. We unexpectedly identified core spliceosomal proteins as a new class of modifiers that rescue the spliceopathy in DM1. Modest knockdown of one of our top hits, SNRPD2, in DM1 fibroblasts and myoblasts, significantly reduces DMPK expression and partially rescues MBNL-regulated AS dysfunction. While the focus on the DM1 spliceopathy has centered around the MBNL proteins, our work reveals an unappreciated role for MBNL:spliceosomal protein stoichiometry in modulating the spliceopathy, revealing new biological and therapeutic avenues for DM1.

Functional analysis of ESRP1/2 gene variants and CTNND1 isoforms in orofacial cleft pathogenesis

Orofacial cleft (OFC) is a common human congenital anomaly. Epithelial-specific RNA splicing regulators ESRP1 and ESRP2 regulate craniofacial morphogenesis and their disruption result in OFC in zebrafish, mouse and humans. Using esrp1/2 mutant zebrafish and murine Py2T cell line models, we functionally tested the pathogenicity of human ESRP1/2 gene variants. We found that many variants predicted by in silico methods to be pathogenic were functionally benign. Esrp1 also regulates the alternative splicing of Ctnnd1 and these genes are co-expressed in the embryonic and oral epithelium. In fact, over-expression of ctnnd1 is sufficient to rescue morphogenesis of epithelial-derived structures in esrp1/2 zebrafish mutants. Additionally, we identified 13 CTNND1 variants from genome sequencing of OFC cohorts, confirming CTNND1 as a key gene in human OFC. This work highlights the importance of functional assessment of human gene variants and demonstrates the critical requirement of Esrp-Ctnnd1 acting in the embryonic epithelium to regulate palatogenesis.

Interdependent regulation of alternative splicing by SR and hnRNP proteins.

Alternative pre-mRNA splicing is a combinatorial process involving SR and hnRNP splicing factors. These proteins can silence or enhance splicing based on their expression levels and binding positions. To better understand their combinatorial and interdependent regulation, computational analyses were performed using HepG2 and K562 cell knockdown and binding datasets from the ENCODE Project. Analyses of diMerential splicing for 6 SR proteins and 13 hnRNP knockdowns revealed statistically significant exon overlap among most RBP combinations, albeit at diMerent levels. Neither SR proteins nor hnRNPs showed strong preferences for collaborating with specific RBP classes in mediating exon inclusion. While SRSF1, hnRNPK, and hnRNPC stand out as major influencers of alternative splicing, they do so predominantly independent of other RBPs. Meanwhile, minor influencers of alternative splicing such as hnRNPAB and hnRNPA0 predominantly regulate exon inclusion in concert with other RBPs, indicating that inclusion can be mediated by both single and multiple RBPs. Interestingly, the higher the number of RBPs that regulate the inclusion of an exon, the more variable exon inclusion preferences become. Interdependently regulated exons are more modular and have diMerent physical characteristics such as reduced exon length compared to their independent counterparts. A comparison of RBP interdependence between HepG2 and K562 cells provides the framework that explains cell-type-specific alternative splicing. Our study highlights the importance of the interdependent regulation of alternative exons and identifies characteristics of interdependently regulated exons that diMer from independently regulated exons.

Alternative splicing regulation in plants by SP7-like effectors from symbiotic arbuscular mycorrhizal fungi

Most plants in natural ecosystems associate with arbuscular mycorrhizal (AM) fungi to survive soil nutrient limitations. To engage in symbiosis, AM fungi secrete effector molecules that, similar to pathogenic effectors, reprogram plant cells. Here we show that the Glomeromycotina-specific SP7 effector family impacts on the alternative splicing program of their hosts. SP7-like effectors localize at nuclear condensates and interact with the plant mRNA processing machinery, most prominently with the splicing factor SR45 and the core splicing proteins U1-70K and U2AF35. Ectopic expression of these effectors in the crop plant potato and in Arabidopsis induced developmental changes that paralleled to the alternative splicing modulation of a specific subset of genes. We propose that SP7-like proteins act as negative regulators of SR45 to modulate the fate of specific mRNAs in arbuscule-containing cells. Unraveling the communication mechanisms between symbiotic fungi and their host plants will help to identify targets to improve plant nutrition.

Ribosomal RNA transcription governs splicing through ribosomal protein RPL22.

Ribosome biosynthesis is a cancer vulnerability executed by targeting RNA polymerase I (Pol I) transcription. We developed advanced, specific Pol I inhibitors to identify drivers of this sensitivity. By integrating multi-omics features and drug sensitivity data from a large cancer cell panel, we discovered that RPL22 frameshift mutation conferred Pol I inhibitor sensitivity in microsatellite instable cancers. Mechanistically, RPL22 directly interacts with 28S rRNA and mRNA splice junctions, functioning as a splicing regulator. RPL22 deficiency, intensified by 28S rRNA sequestration, promoted the splicing of its paralog RPL22L1 and p53 negative regulator MDM4. Chemical and genetic inhibition of rRNA synthesis broadly remodeled mRNA splicing controlling hundreds of targets. Strikingly, RPL22-dependent alternative splicing was reversed by Pol I inhibition revealing a ribotoxic stress-initiated tumor suppressive pathway. We identify a mechanism that robustly connects rRNA synthesis activity to splicing and reveals their coordination by ribosomal protein RPL22.

Role of U11/U12 minor spliceosome gene ZCRB1 in Ciliogenesis and WNT Signaling.

Despite the fact that 0.5% of human introns are processed by the U11/U12 minor spliceosome, the latter influences gene expression across multiple cellular processes. The ZCRB1 protein is a recently described core component of the U12 mono-snRNP minor spliceosome, but its functional significance to minor splicing, gene regulation, and biological signaling cascades is poorly understood. Using CRISPR-Cas9 and siRNA targeted knockout and knockdown strategies, we show that human cell lines with a partial reduction in ZCRB1 expression exhibit significant dysregulation of the splicing and expression of U12-type genes, primarily due to dysregulation of U12 mono-snRNA. RNA-Seq and targeted analyses of minor intron-containing genes indicate a downregulation in the expression of genes involved in ciliogenesis, and consequentially an upregulation in WNT signaling. Additionally, zcrb1 CRISPR-Cas12a knockdown in zebrafish embryos led to gross developmental and body axis abnormalities, disrupted ciliogenesis, and upregulated WNT signaling, complementing our human cell studies. This work highlights a conserved and essential biological role of the minor spliceosome in general, and the ZCRB1 protein specifically in cellular and developmental processes across species, shedding light on the multifaceted relationship between splicing regulation, ciliogenesis, and WNT signaling.

A defective splicing machinery promotes senescence through MDM4 alternative splicing.

Defects in the splicing machinery are implicated in various diseases, including cancer. We observed a general reduction in the expression of spliceosome components and splicing regulators in human cell lines undergoing replicative, stress-induced, and telomere uncapping-induced senescence. Supporting the view that defective splicing contributes to senescence, splicing inhibitors herboxidiene, and pladienolide B induced senescence in normal and cancer cell lines. Furthermore, depleting individual spliceosome components also promoted senescence. All senescence types were associated with an alternative splicing transition from the MDM4-FL variant to MDM4-S. The MDM4 splicing shift was reproduced when splicing was inhibited, and spliceosome components were depleted. While decreasing the level of endogenous MDM4 promoted senescence and cell survival independently of the MDM4-S expression status, cell survival was also improved by increasing MDM4-S. Overall, our work establishes that splicing defects modulate the alternative splicing of MDM4 to promote senescence and cell survival.

Abstract We130: Novel Role of RBFox1 in Myocardial Infarction Induced Heart Failure

Introduction: Alternative mRNA splicing affects a broad spectrum of cardiac genes during pathological manifestation of heart diseases. Earlier studies from our lab have identified a muscle-specific isoform of RBFox1 to be a key RNA splicing regulator in pressure overload induced heart failure through regulating cardiac transcription factor alternative splicing. However, the physiological impact of RBFox1 in myocardial infarction (MI), and the RBFox1 downstream mRNA alternative splicing events during MI induced cardiac remodeling remains unknown. Goals: To investigate the functional impact of RBFox1 in MI induced cardiac remodeling. Method and Results: At both mRNA and protein levels, we found RBFox1 to be significantly decreased in Sprague-Dawley Rat hearts post MI. To investigate the functional impact of RBFox1 in myocardial infarction, we utilized AAV9 to achieve cardiac specific expression of RBFox1 in rats. Interestingly, expression of RBFox1 prevented cardiac dysfunction post MI characterized by improved cardiac function based on echocardiography. Expression of RBFox1 further reduced hypertrophy, cell death and fibrotic remodeling at both molecular and histological levels post MI. In vitro, expression of RBFox1 is sufficient to prevent hypoxia induced cardiac cell death based on TUNEL staining and cleaved caspase 3 level, while inactivation of RBFox1 promoted cardiac cell death. To further understand the molecular mechanism underlying RBFox1 mediated cardiac protection, we performed RNA-seq analysis to determine the transcriptome changes in vivo. RNA-seq showed insulin secretion, cAMP signaling and muscle contraction pathways to be top significantly changed pathways in RBFox1 expressed rat hearts post MI. Lastly, we have identified Mbnl1 mRNA alternative splicing to be regulated by RBFox1 through promoting a 36bp exon exclusion at the C-terminal of Mbnl1 protein, potentially affecting the function of Mbnl1. Conclusion: In summary, we have identified a previously uncharacterized role of RBFox1 in myocardial infarction injury. RBFox1 expression is critical to preserve cardiac function post MI, potentially through regulating downstream targets alternative splicing, including Mbnl1.

Abstract We084: Deciphering the Molecular Code Underlining Postnatal Cardiomyocyte Maturation by an RNA Splicing Factor RBFox1

Mammalian hearts undergo major changes after birth during the perinatal period. While several extrinsic factors such as mechanical load, electrical stimulation, hormones, and nutrients have been implicated in this process, the intrinsic regulatory circuit governing cardiomyocyte postnatal maturation is largely unknown. Importantly, stem-cell derived cardiomyocytes commonly exhibit immaturity which is a major limiting factor for their application as disease models or cell therapy agents. Therefore, uncovering the intrinsic regulatory mechanism of mammalian cardiomyocyte maturation has significant implications in both basic cardiac biology and cell-based therapy and disease modeling. In our recent analysis of the global transcriptome transition from neonatal to adult rat hearts, we found RNA splicing among the top changed pathways. Additionally, we found that expression of RBFox1, a cardiac enriched RNA splicing regulator, was significantly increased during the postnatal transition to adolescence. We further demonstrate that RBFox1 mediated post-transcriptional regulation has a potent effect to promote neonatal and stem-cell derived cardiomyocyte maturation. RNA-seq followed by Gene Ontology enrichment analysis showed that Rbfox1 led to differentially expressed genes enriched in cardiac contraction and conduction, sarcomere organization, K + ion import and muscle filament sliding, as well as differential isoform switches enriched in cardiac contraction and sarcomere structure. An evolutionarily conserved, temporal specific super enhancer exists upstream of cardiac Rbfox1 , which is occupied by active epigenetic markers in the postnatal period. We have identified the tissue and temporal transcriptional activity of this super enhancer in vitro and in vivo . Several binding motifs of transcription factors are predicted within it and a key DNA fragment was identified to be essential for enhancer activity. A transgenic mouse strain has been established to facilitate the tracing of myocyte maturation in vivo by constructing a reporter system driven by a 4xSuperEnhancer-Rbfox1Promoter-tdTomato construct. In conclusion, we propose a mechanism that RBFox1 is an intracellular regulator with tissue- and temporal-specific expression patterns that regulates cardiac maturation by promoting alternative splicing that fine tunes the genetic code.

Analysis of host factor networks during hepatitis B virus infection in primary human hepatocytes

BackgroundChronic hepatitis B virus (HBV) infection affects around 250 million people worldwide, causing approximately 887,000 deaths annually, primarily owing to cirrhosis and hepatocellular carcinoma (HCC). The current approved treatments for chronic HBV infection, such as interferon and nucleos(t)ide analogs, have certain limitations as they cannot completely eradicate covalently closed circular DNA (cccDNA). Considering that HBV replication relies on host transcription factors, focusing on host factors in the HBV genome may provide insights into new therapeutic targets against HBV. Therefore, understanding the mechanisms underlying viral persistence and hepatocyte pathogenesis, along with the associated host factors, is crucial. In this study, we investigated novel therapeutic targets for HBV infection by identifying gene and pathway networks involved in HBV replication in primary human hepatocytes (PHHs). Importantly, our study utilized cultured primary hepatocytes, allowing transcriptomic profiling in a biologically relevant context and enabling the investigation of early HBV-mediated effects.MethodsPHHs were infected with HBV virion particles derived from HepAD38 cells at 80 HBV genome equivalents per cell (Geq/cell). For transcriptomic sequencing, PHHs were harvested 1, 2-, 3-, 5-, and 7 days post-infection (dpi). After preparing the libraries, clustering and sequencing were conducted to generate RNA-sequencing data. This data was processed using Bioinformatics tools and software to analyze DEGs and obtain statistically significant results. Furthermore, qRT-PCR was performed to validate the RNA-sequencing results, ensuring consistent findings.ResultsWe observed significant alterations in the expression patterns of 149 genes from days 1 to 7 following HBV infection (R2 > 0.7, q < 0.05). Functional analysis of these genes identified RNA-binding proteins involved in mRNA metabolism and the regulation of alternative splicing during HBV infection. Results from qRT-PCR experiments and the analysis of two validation datasets suggest that RBM14 and RPL28 may serve as potential biomarkers for HBV-associated HCC.ConclusionsTranscriptome analysis of gene expression changes during HBV infection in PHHs provided valuable insights into chronic HBV infection. Additionally, understanding the functional involvement of host factor networks in the molecular mechanisms of HBV replication and transcription may facilitate the development of novel strategies for HBV treatment.

RPL22 is a tumor suppressor in MSI-high cancers and a splicing regulator of MDM4

Microsatellite instability-high (MSI-H) tumors are malignant tumors that, despite harboring a high mutational burden, often have intact TP53. One of the most frequent mutations in MSI-H tumors is a frameshift mutation in RPL22, a ribosomal protein. Here, we identified RPL22 as a modulator of MDM4 splicing through an alternative splicing switch in exon 6. RPL22 loss increases MDM4 exon 6 inclusion and cell proliferation and augments resistance to the MDM inhibitor Nutlin-3a. RPL22 represses the expression of its paralog, RPL22L1, by mediating the splicing of a cryptic exon corresponding to a truncated transcript. Therefore, damaging mutations in RPL22 drive oncogenic MDM4 induction and reveal a common splicing circuit in MSI-H tumors that may inform therapeutic targeting of the MDM4-p53 axis and oncogenic RPL22L1 induction.

SMARCA4 controls state plasticity in small cell lung cancer through regulation of neuroendocrine transcription factors and REST splicing

IntroductionSmall Cell Lung Cancer (SCLC) can be classified into transcriptional subtypes with distinct degrees of neuroendocrine (NE) differentiation. Recent evidence supports plasticity among subtypes with a bias toward adoption of low-NE states during disease progression or upon acquired chemotherapy resistance. Here, we identify a role for SMARCA4, the catalytic subunit of the SWI/SNF complex, as a regulator of subtype shift in SCLC.MethodsATACseq and RNAseq experiments were performed in SCLC cells after pharmacological inhibition of SMARCA4. DNA binding of SMARCA4 was characterized by ChIPseq in high-NE SCLC patient derived xenografts (PDXs). Enrichment analyses were applied to transcriptomic data. Combination of FHD-286 and afatinib was tested in vitro and in a set of chemo-resistant SCLC PDXs in vivo.ResultsSMARCA4 expression positively correlates with that of NE genes in both SCLC cell lines and patient tumors. Pharmacological inhibition of SMARCA4 with FHD-286 induces the loss of NE features and downregulates neuroendocrine and neuronal signaling pathways while activating non-NE factors. SMARCA4 binds to gene loci encoding NE-lineage transcription factors ASCL1 and NEUROD1 and alters chromatin accessibility, enhancing NE programs. Enrichment analysis applied to high-confidence SMARCA4 targets confirmed neuron related pathways as the top GO Biological processes regulated by SMARCA4 in SCLC. In parallel, SMARCA4 also controls REST, a known suppressor of the NE phenotype, by regulating SRRM4-dependent REST transcript splicing. Furthermore, SMARCA4 inhibition drives ERBB pathway activation in SCLC, rendering SCLC tumors sensitive to afatinib.ConclusionsThis study nominates SMARCA4 as a key regulator of the NE state plasticity and defines a novel therapeutic strategy for SCLC.

Identification of alternative splicing regulatory patterns and characteristic splicing factors in heart failure using RNA-seq data and machine learning

Heart failure (HF) represents the advanced stage of several cardiovascular disorders. This study aimed to build an alternative splicing regulatory network and identify potential splicing factors involved in HF utilizing RNA-seq data and machine learning algorithms. We performed bioinformatics analysis on RNA-seq datasets containing samples from HF patients and normal individuals to obtain gene expression matrices and identify differently regulated alternative splicing events in HF. By calculating percent spliced-in (PSI) value, we identified 4055 abnormal alternative splicing events of 3142 genes in HF. These genes were significantly enriched in PPAR signaling, regulation of actin cytoskeleton, and muscle contraction. Interestingly, based on abnormal alternative splicing events, two distinct clusters of HF patients with distinct molecular mechanisms and pathways were identified using unsupervised clustering. Additionally, we built a regulatory network consisting of heart failure-related alternative splicing and splicing factors. Subsequently, we identify 203 HF specific pairs between splicing factors and alternative splicing events. Four splicing factors (RBM5, ZRANB2, HnRNPF, and HnRNPA0) were found using LASSO and SVM-RFE algorithms, their expression patterns were confirmed in two other microarray datasets. Our study clarifies involvement of splicing factors and alternative splicing events in HF by thoroughly analyzing RNA-seq data with machine learning methods. The findings may advance our understanding of the regulatory systems underlying biological processes associated with heart failure by providing candidates for further investigation and markers for diagnostic and therapeutic purposes.

KPNA2 promotes the progression of gastric cancer by regulating the alternative splicing of related genes

RNA-binding proteins (RBPs) play critical roles in genome regulation. In this study, we explored the latent function of KPNA2, which is an essential member of the RBP family, in the regulation of alternative splicing (AS) in gastric cancer (GC). We analyzed the role of KPNA2 in regulating differential expression and AS via RNA sequencing (RNA-seq) and improved RNA immunoprecipitation sequencing (iRIP-seq). Clinical specimens were used to analyze the associations between KPNA2 expression and clinicopathological characteristics. CCK8 assays, transwell assays and wound healing assays were performed to explore the effect of KPNA2/WDR62 on GC cell progression. KPNA2 was shown to be highly expressed in GC cells and tissues and associated with lymph node metastases. KPNA2 promoted the proliferation, migration and invasion of GC cells and primarily regulated exon skipping, alternative 3's splice sites (A3SSs), alternative 5' splice sites (A5SSs), and cassette exons. We further revealed that KPNA2 participated in biological processes related to cell proliferation, and the immune response in GC via the regulation of transcription. In addition, KPNA2 preferentially bound to intron regions. Notably, KPNA2 regulated the A3SS AS mode of WDR62, and upregulation of WDR62 reversed the KPNA2 downregulation-induced inhibition of GC cell proliferation, migration and invasion. Finally, we discovered that the AS of immune-related molecules could be regulated by KPNA2. Overall, our results demonstrated for the first time that KPNA2 functions as an oncogenic splicing factor in GC that regulated the AS and differential expression of GC-related genes, and KPNA2 may be a potential target for GC treatment.

PARP4 interacts with hnRNPM to regulate splicing during lung cancer progression

BackgroundThe identification of cancer driver genes from sequencing data has been crucial in deepening our understanding of tumor biology and expanding targeted therapy options. However, apart from the most commonly altered genes, the mechanisms underlying the contribution of other mutations to cancer acquisition remain understudied. Leveraging on our whole-exome sequencing of the largest Asian lung adenocarcinoma (LUAD) cohort (n = 302), we now functionally assess the mechanistic role of a novel driver, PARP4.MethodsIn vitro and in vivo tumorigenicity assays were used to study the functional effects of PARP4 loss and mutation in multiple lung cancer cell lines. Interactomics analysis by quantitative mass spectrometry was conducted to identify PARP4’s interaction partners. Transcriptomic data from cell lines and patient tumors were used to investigate splicing alterations.ResultsPARP4 depletion or mutation (I1039T) promotes the tumorigenicity of KRAS- or EGFR-driven lung cancer cells. Disruption of the vault complex, with which PARP4 is commonly associated, did not alter tumorigenicity, indicating that PARP4’s tumor suppressive activity is mediated independently. The splicing regulator hnRNPM is a potentially novel PARP4 interaction partner, the loss of which likewise promotes tumor formation. hnRNPM loss results in splicing perturbations, with a propensity for dysregulated intronic splicing that was similarly observed in PARP4 knockdown cells and in LUAD cohort patients with PARP4 copy number loss.ConclusionsPARP4 is a novel modulator of lung adenocarcinoma, where its tumor suppressive activity is mediated not through the vault complex—unlike conventionally thought, but in association with its novel interaction partner hnRNPM, thus suggesting a role for splicing dysregulation in LUAD tumorigenesis.

N6-methyladenosine writer METTL16-mediated alternative splicing and translation control are essential for murine spermatogenesis

BackgroundThe mitosis-to-meiosis switch during spermatogenesis requires dynamic changes in gene expression. However, the regulation of meiotic transcriptional and post-transcriptional machinery during this transition remains elusive.ResultsWe report that methyltransferase-like protein 16 (METTL16), an N6-methyladenosine (m6A) writer, is required for mitosis-to-meiosis transition during spermatogenesis. Germline conditional knockout of Mettl16 in male mice impairs spermatogonial differentiation and meiosis initiation. Mechanistically, METTL16 interacts with splicing factors to regulate the alternative splicing of meiosis-related genes such as Stag3. Ribosome profiling reveals that the translation efficiency of many meiotic genes is dysregulated in METTL16-deficient testes. m6A-sequencing shows that ablation of METTL16 causes upregulation of the m6A-enriched transcripts and downregulation of the m6A-depleted transcripts, similar to Meioc and/or Ythdc2 mutants. Further in vivo and in vitro experiments demonstrate that the methyltransferase activity site (PP185-186AA) of METTL16 is necessary for spermatogenesis.ConclusionsOur findings support a molecular model wherein the m6A writer METTL16-mediated alternative splicing and translation efficiency regulation are required to control the mitosis-to-meiosis germ cell fate decision in mice, with implications for understanding meiosis-related male fertility disorders.