Exon Skipping Research Articles

The methyltransferase SETD3 regulates mRNA alternative splicing through interacting with hnRNPK

The methyltransferase SETD3 is an enzyme essential for catalyzing histidine-73 methylation on β-Actin, thereby promoting its polymerization and regulating muscle contraction. Although increasing evidence suggests that SETD3 is involved in multiple physiological or pathological events, its biological functions remain incompletely understood. In this study, we utilize in situ proximity labeling combined with mass spectrometry analysis to detect potential interacting partners of SETD3. Unexpectedly, we find that many splicing factors are associated with SETD3. Genome-wide RNA sequencing reveals that SETD3 regulates pre-mRNA splicing events, predominantly influencing exon skipping. Biochemical and bioinformatic analyses suggest that SETD3 interacts with hnRNPK, and they collaboratively regulate exon skipping in a common subset of genes. Functionally, we demonstrate that SETD3 and hnRNPK are required for retention of exon 7 skipping in the FNIP1 gene. This promotes FNIP1-mediated nuclear translocation of the transcription factor TFEB and the subsequent induction of lysosomal and mitochondrial biogenesis. Overall, this study uncovers a novel function of SETD3 in modulating mRNA exon splicing.

Characterization of pathogenic genetic variants in Russian patients with primary ciliary dyskinesia using gene panel sequencing and transcript analysis

BackgroundPrimary ciliary dyskinesia (PCD) is a group of rare genetically heterogeneous disorders caused by defective cilia and flagella motility. The clinical phenotype of PCD patients commonly includes chronic oto-sino-pulmonary disease, infertility, and, in about half of cases, laterality defects due to randomization of left–right body asymmetry. To date, pathogenic variants in more than 50 genes responsible for motile cilia structure and assembly have been reported in such patients. While multiple population-specific mutations have been described in PCD cohorts from different countries, the data on genetic spectrum of PCD in Russian population are still extremely limited.ResultsThe present study provides a comprehensive clinical and genetic characterization of 21 Russian families with PCD living in various country regions. Anomalies of ciliary beating in patients` respiratory epithelial cells were confirmed by high-speed video microscopy. In the most cases, custom-designed panel sequencing allowed to uncover causative variants in well-known or rarely mentioned PCD-related genes, including DNAH5, DNAH11, CFAP300, LRRC6, ZMYND10, CCDC103, HYDIN, ODAD4, DNAL1, and OFD1. The variations comprised common mutations, as well as novel genetic variants, some of which probably specific for Russian patients. Additional targeted analysis of mRNA transcripts from ciliated cells enabled us to specify functional effects of newly identified genetic variants in DNAH5 (c.2052+3G>T, c.3599-2A>G), HYDIN (c.10949-2A>G, c.1797C>G), and ZMYND10 (c.510+1G>C) on splicing process. In particular, the splice site variant c.2052+3G>T, detected in four unrelated families, resulted in skipping of exon 14 in DNAH5 transcripts and, according to haplotype analysis of affected probands, was proposed as an ancestral founder mutation in Udmurt population.ConclusionsThe reported data provide a vital insight into genetic background of primary ciliary dyskinesia in the Russian population. The findings clearly illustrate the utility of gene panel sequencing coupled with transcriptional analysis in identification and clinical interpretation of novel genetic variants.

Disruption of nuclear speckle integrity dysregulates RNA splicing in C9ORF72-FTD/ALS

Expansion of an intronic (GGGGCC)n repeat within the C9ORF72 gene is the most common genetic cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) (C9-FTD/ALS), characterized with aberrant repeat RNA foci and noncanonical translation-produced dipeptide repeat (DPR) protein inclusions. Here, we elucidate that the (GGGGCC)n repeat RNA co-localizes with nuclear speckles and alters their phase separation properties and granule dynamics. Moreover, the essential nuclear speckle scaffold protein SRRM2 is sequestered into the poly-GR cytoplasmic inclusions in the C9-FTD/ALS mouse model and patient postmortem tissues, exacerbating the nuclear speckle dysfunction. Impaired nuclear speckle integrity induces global exon skipping and intron retention in human iPSC-derived neurons and causes neuronal toxicity. Similar alternative splicing changes can be found in C9-FTD/ALS patient postmortem tissues. This work identified novel molecular mechanisms of global RNA splicing defects caused by impaired nuclear speckle function in C9-FTD/ALS and revealed novel potential biomarkers or therapeutic targets.

Identification of a novel alternative splicing isoform of the Hippo kinase STK3/MST2 with impaired kinase and cell growth suppressing activities.

Mammalian Ste-20-like Kinases 1 and 2 (MST1/2) are core serine-threonine kinases of the Hippo pathway regulating several cellular processes, including cell cycle arrest and cell death. Here, we discovered a novel alternative splicing variant of the MST2 encoding gene, STK3, in malignant cells and tumor datasets. This variant, named STK3∆7 or MST2∆7 (for mRNA or protein, respectively), resulted from the skipping of exon 7. MST2∆7 exhibited increased ubiquitylation and interaction with the E3 ubiquitin-protein ligase CHIP compared to the full-length protein (MST2FL). Exon 7 in STK3 encodes a segment within the kinase domain, and its exclusion compromised MST2 interaction with and phosphorylation of MOB, a major MST1/2 substrate. Nevertheless, MST2∆7 was capable of interacting with MST1 and MST2FL. Unlike MST2FL, overexpression of MST2∆7 did not lead to increased cell death and growth arrest. Strikingly, we observed the exclusion of STK3 exon 7 in 3.2-15% of tumor samples from patients of several types of cancer, while STK3∆7 was seldomly found in healthy tissues. Our study identified a novel STK3 splicing variant with loss of function and the potential to disturb tissue homeostasis by impacting on MST2 activities in the regulation of cell death and quiescence.

Splice modulation strategy applied to deep intronic variants in COL7A1 causing recessive dystrophic epidermolysis bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare and most often severe genetic disease characterized by recurrent blistering and erosions of the skin and mucous membranes after minor trauma, leading to major local and systemic complications. The disease is caused by loss-of-function variants in COL7A1 encoding type VII collagen (C7), the main component of anchoring fibrils, which form attachment structures stabilizing the cutaneous basement membrane zone. Alterations in C7 protein structure and/or expression lead to abnormal, rare or absent anchoring fibrils resulting in loss of dermal-epidermal adherence and skin blistering. To date, more than 1,200 distinct COL7A1 deleterious variants have been reported and 19% are splice variants. Here, we describe two RDEB patients for whom we identified two pathogenic deep intronic pathogenic variants in COL7A1. One of these variants (c.7795-97C > G) promotes the inclusion of a pseudoexon between exons 104 and 105 in the COL7A1 transcript, while the other causes partial or complete retention of intron 51. We used antisense oligonucleotide (ASO) mediated exon skipping to correct these aberrant splicing events in vitro. This led to increased normal mRNA splicing above 94% and restoration of C7 protein expression at a level (up to 56%) that should be sufficient to reverse the phenotype. This first report of exon skipping applied to counteract deep intronic variants in COL7A1 represents a promising therapeutic strategy for personalized medicine directed at patients with intronic variants at a distance of consensus splice sites.

High mobility group box1 (HMGB1) is a potential disease biomarker in cell and mouse models of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder affecting 1:3500 male births and is associated with myofiber degeneration, regeneration, and inflammation. Glucocorticoid treatments have been the standard of care due to immunomodulatory/immunosuppressive properties but novel genetic approaches, including exon skipping and gene replacement therapy, are currently being developed. The identification of additional biomarkers to assess DMD-related inflammatory responses and the potential efficacy of these therapeutic approaches are thus of critical importance. The current study uses RNA sequencing of skeletal muscle from two mdx mouse models to identify high mobility group box1 (HMGB1) as a candidate biomarker potentially contributing to DMD-related inflammation. HMGB1 protein content was increased in a human iPSC-derived skeletal myocyte model of DMD and microdystrophin treatment decreased HMGB1 back to control levels. In vivo, HMGB1 protein levels were increased in vehicle treated B10-mdx skeletal muscle compared to B10-WT and significantly decreased in B10-mdx animals treated with adeno-associated virus (AAV)-microdystrophin. However, HMGB1 protein levels were not increased in D2-mdx skeletal muscle compared to D2-WT, demonstrating a strain-specific difference in DMD-related immunopathology.

The spectrum of novel ABCB11 gene variations in children with progressive familial intrahepatic cholestasis type 2 in Pakistani cohorts

Progressive familial intrahepatic cholestasis (PFIC) is a rare childhood manifested disease associated with impaired bile secretion with severe pruritus yellow stool, and sometimes hepatosplenomegaly. PFIC is caused by mutations in ATP8B1, ABCB11, ABCB4, TJP2, NR1H4, SLC51A, USP53, KIF12, ZFYVE19, and MYO5B genes depending on its type. ABCB11 mutations lead to PFIC2 that encodes the bile salt export pump (BSEP). Different mutations of ABCB11 have been reported in different population groups but no data available in Pakistani population being a consanguineous one. We sequenced coding exons of the ABCB11 gene along with its flanking regions in 66 unrelated Pakistani children along with parents with PFIC2 phenotype. We identified 20 variations of ABCB11: 12 in homozygous form, one compound heterozygous, and seven heterozygous. These variants include 11 missenses, two frameshifts, two nonsense mutations, and five splicing variants. Seven variants are novel candidate variants and are not detected in any of the 120 chromosomes from normal ethnically matched individuals. Insilico analysis revealed that four homozygous missense variations have high pathogenic scores. Minigene analysis of splicing variants showed exon skipping and the addition of exon. This data is a useful addition to the disease variants genomic database and would be used in the future to build a diagnostic algorithm.

Is Exon Skipping a Viable Therapeutic Approach for Vascular Ehlers-Danlos Syndrome with Mutations in COL3A1 Exon 10 or 15?

Vascular Ehlers-Danlos syndrome or Ehlers-Danlos syndrome type IV (vEDS) is a connective tissue disorder characterised by skin hyperextensibility, joint hypermobility and fatal vascular rupture caused by COL3A1 mutations that affect collagen III expression, homo-trimer assembly and secretion. Along with collagens I, II, V and XI, collagen III plays an important role in the extracellular matrix, particularly in the inner organs. To date, only symptomatic treatment for vEDS patients is available. Fibroblasts derived from vEDS patients carrying dominant negative and/or haploinsufficiency mutations in COL3A1 deposit reduced collagen III in the extracellular matrix. This study explored the potential of an antisense oligonucleotide (ASO)-mediated splice modulating strategy to bypass disease-causing COL3A1 mutations reported in the in-frame exons 10 and 15. Antisense oligonucleotides designed to redirect COL3A1 pre-mRNA processing and excise exons 10 or 15 were transfected into dermal fibroblasts derived from vEDS patients and a healthy control subject. Efficient exon 10 or 15 excision from the mature COL3A1 mRNA was achieved and intracellular collagen III expression was increased after treatment with ASOs; however, collagen III deposition into the extracellular matrix was reduced in patient cells. The region encoded by exon 10 includes a glycosylation site, and exon 15 encodes hydroxyproline and hydroxylysine-containing triplet repeats, predicted to be crucial for collagen III assembly. These results emphasize the importance of post-translational modification for collagen III homo-trimer assembly. In conclusion, while efficient skipping of target COL3A1 exons was achieved, the induced collagen III isoforms generated showed defects in extracellular matrix formation. While therapeutic ASO-mediated exon skipping is not indicated for the patients in this study, the observations are restricted to exons 10 and 15 and may not be applicable to other collagen III in-frame exons.

Outcomes of EGFR, ALK, ROS1, BRAF, MET, and RET mutated non-small cell lung cancer with brain metastases (NSCLC BM).

201 Background: Non-small cell lung cancer (NSCLC) is a leading cause of brain metastases. Advances in gene-directed therapies have shifted the focus away from traditional platinum-based chemotherapies. Key targeted mutations in NSCLC include EGFR, ALK, BRAF, MET exon skipping, RET fusions, and ROS1 rearrangements. This retrospective study aims to explore overall survival (OS) and progression-free survival (PFS) in NSCLC BM patients with these gene mutations. Methods: In this retrospective study, conducted by the Department of Medical Oncology at Beijing Tiantan Hospital and the First Medical Center, PLA General Hospital, we reviewed 1526 patients with NSCLC brain metastasis from 2010 to 2022. Data collected included molecular marker status, systemic therapies, and dates of progression. The analysis focused on determining OS and PFS from the time of brain metastasis diagnosis to the last follow-up or death. Statistical analysis used the Cox proportional hazards model. Results: The study reviewed 1526 patients, identifying the following mutation distribution: EGFR mutations in 628 patients, ALK rearrangements in 207 patients, ROS1 rearrangements in 64 patients, BRAF mutations in 31 patients, MET exon skipping in 55 patients, and RET fusions in 42 patients. Significant variations in median OS (mOS) and median PFS (mPFS) were observed across these mutation groups. Patients with EGFR and ALK mutations demonstrated longer mOS and mPFS. Patients with RET and ROS1 mutations also showed better outcomes compared to those with BRAF and MET mutations. Conclusions: This study highlights the importance of molecular mutations in NSCLC BM as prognostic indicators and therapeutic targets. EGFR and ALK mutations were associated with more favorable outcomes, emphasizing the need for personalized medicine in managing NSCLC BM. The study suggests further research focused on specific mutation types is crucial to refine treatment strategies and improve patient care.

Single-Base Substitution Causing Dual-Exon Skipping Event in PKD2 Gene: Unusual Molecular Finding from Exome Sequencing in a Patient with Autosomal Dominant Polycystic Kidney Disease.

Background: Pathogenic variants in the Polycystic Kidney Disease 2 (PKD2) gene are associated with Autosomal Dominant Polycystic Kidney Disease (ADPKD) in approximately 30% of cases. In recent years, the high-throughput sequencing techniques have significantly increased the number of variants identified in affected patients. Here, we described the peculiar effect of a PKD2 splicing variant, the c.1717-2A>G, identified in an Italian male patient with ADPKD. This variant led to the unusual and rare skipping of two consecutive exons, causing a large in-frame deletion. Methods: The genetic evaluation of the patient was performed using the Next-Generation Sequencing (NGS) assay Clinical Exome Solution® (SOPHiA Genetics). Bioinformatics analysis was performed using the SOPHiA DDM platform (SOPHiA Genetics). Prediction of pathogenicity was carried out by integrating several in silico tools. RNA evaluation was performed to test the effect of the variant on the PKD2 splicing using a Reverse-Transcription PCR coupled with cDNA sequencing. Results: NGS revealed the presence of the PKD2 c.1717-2A>G variant that lies in the canonical splice site of intron 7. This rare variant was predicted to have a significant impact on the splicing, proved by the RNA-based analysis. We identified the presence of a transcript characterised by the simultaneous skipping of exons 8 and 9, with a retained reading frame and the merging of exons 7-10. Conclusions: We described for the first time a dual-exon skip event related to the presence of a single-base substitution in the PKD2 gene in an ADPKD-affected patient. We assumed that the molecular basis of such a rare mechanism lies in the specific order of intron removal. The finding represents novel evidence of an alternative and unusual splicing mechanism in the PKD2 gene, adding insights to the pathogenesis of the ADPKD.

PTBP3 Mediates IL-18 Exon Skipping to Promote Immune Escape in Gallbladder Cancer.

Gallbladder cancer (GBC) is the most common malignant tumor of the biliary system, with poor response to current treatments. Abnormal alternative splicing has been associated with the development of a variety of tumors. Combining the GEO database and GBC mRNA-seq analysis, it is found high expression of the splicing factor polypyrimidine region- binding protein 3 (PTBP3) in GBC. Multi-omics analysis revealed that PTBP3 promoted exon skipping of interleukin-18 (IL-18), resulting in the expression of ΔIL-18, an isoform specifically expressed in tumors. That ΔIL-18 promotes GBC immune escape by down-regulating FBXO38 transcription levels in CD8+T cells to reduce PD-1 ubiquitin-mediated degradation is revealed. Using a HuPBMC mouse model, the role of PTBP3 and ΔIL-18 in promoting GBC growth is confirmed, and showed that an antisense oligonucleotide that blocked ΔIL-18 production displayed anti-tumor activity. Furthermore, that the H3K36me3 promotes exon skipping of IL-18 by recruiting PTBP3 via MRG15 is demonstrated, thereby coupling the processes of IL-18 transcription and alternative splicing. Interestingly, it is also found that the H3K36 methyltransferase SETD2 binds to hnRNPL, thereby interfering with PTBP3 binding to IL-18 pre-mRNA. Overall, this study provides new insights into how aberrant alternative splicing mechanisms affect immune escape, and provides potential new perspectives for improving GBC immunotherapy.

Functional Characterization of Splice Variants in the Diagnosis of Albinism.

Albinism is a genetically heterogeneous disease in which 21 genes are known so far. Its inheritance mode is autosomal recessive except for one X-linked form. The molecular analysis of exonic sequences of these genes allows for about a 70% diagnostic rate. About half (15%) of the unsolved cases are heterozygous for one pathogenic or probably pathogenic variant. Assuming that the missing variant may be located in non-coding regions, we performed sequencing for 122 such heterozygous patients of either the whole genome (27 patients) or our NGS panel (95 patients) that includes, in addition to all exons of the 21 genes, the introns and flanking sequences of five genes, TYR, OCA2, SLC45A2, GPR143 and HPS1. Rare variants (MAF < 0.01) in trans to the first variant were tested by RT-PCR and/or minigene assay. Of the 14 variants tested, nine caused either exon skipping or the inclusion of a pseudoexon, allowing for the diagnosis of 11 patients. This represents 9.8% (12/122) supplementary diagnosis for formerly unsolved patients and 75% (12/16) of those in whom the candidate variant was in trans to the first variant. Of note, one missense variant was demonstrated to cause skipping of the exon in which it is located, thus shedding new light on its pathogenic mechanism. Searching for non-coding variants and testing them for an effect on RNA splicing is warranted in order to increase the diagnostic rate.

Microsatellite instability at U2AF-binding polypyrimidic tract sites perturbs alternative splicing during colorectal cancer initiation

BackgroundMicrosatellite instability (MSI) due to mismatch repair deficiency (dMMR) is common in colorectal cancer (CRC). These cancers are associated with somatic coding events, but the noncoding pathophysiological impact of this genomic instability is yet poorly understood. Here, we perform an analysis of coding and noncoding MSI events at the different steps of colorectal tumorigenesis using whole exome sequencing and search for associated splicing events via RNA sequencing at the bulk-tumor and single-cell levels.ResultsOur results demonstrate that MSI leads to hundreds of noncoding DNA mutations, notably at polypyrimidine U2AF RNA-binding sites which are endowed with cis-activity in splicing, while higher frequency of exon skipping events are observed in the mRNAs of MSI compared to non-MSI CRC. At the DNA level, these noncoding MSI mutations occur very early prior to cell transformation in the dMMR colonic crypt, accounting for only a fraction of the exon skipping in MSI CRC. At the RNA level, the aberrant exon skipping signature is likely to impair colonic cell differentiation in MSI CRC affecting the expression of alternative exons encoding protein isoforms governing cell fate, while also targeting constitutive exons, making dMMR cells immunogenic in early stage before the onset of coding mutations. This signature is characterized by its similarity to the oncogenic U2AF1-S34F splicing mutation observed in several other non-MSI cancer.ConclusionsOverall, these findings provide evidence that a very early RNA splicing signature partly driven by MSI impairs cell differentiation and promotes MSI CRC initiation, far before coding mutations which accumulate later during MSI tumorigenesis.

Tissue distribution of renadirsen sodium, a dystrophin exon-skipping antisense oligonucleotide, in heart and diaphragm after subcutaneous administration to cynomolgus monkeys

The pharmacokinetics and tissue distribution of renadirsen sodium, a dystrophin exon-skipping phosphorothioate-modified antisense oligonucleotide with 2’-O,4’-C-ethylene-bridged nucleic acid (ENA), after subcutaneous or intravenous administration to cynomolgus monkeys were investigated. The plasma concentration of renadirsen after subcutaneous administration at 1, 3, and 10 mg/kg increased with the dose. The absolute bioavailability at 3 mg/kg after subcutaneous administration was calculated as 88.6%, and the time to reach maximum plasma concentration of renadirsen was within 4 h, indicating the efficient and rapid absorption following subcutaneous administration. The exposure of muscle tissues to renadirsen was found to increase with repeated dosing at 6 mg/kg, and higher exposure was observed in the diaphragm and heart than in the quadriceps femoris and anterior tibialis muscles. Renadirsen achieved more exon 45-skipped dystrophin mRNA in the diaphragm and heart than in the quadriceps femoris and anterior tibialis muscles. Renadirsen also showed a cumulative skipping effect in a repeated-dose study. The findings on exon 45-skipped dystrophin mRNA in these muscle tissues were consistent with the concentration of renadirsen in these tissues. Because it is not feasible to directly evaluate drug concentration and exon skipping in the heart and diaphragm in humans, the pharmacokinetics and pharmacodynamics of renadirsen in these tissues in monkeys are crucial for the design and interpretation of clinical settings.

In Vitro Studies to Evaluate the Intestinal Permeation of an Ursodeoxycholic Acid-Conjugated Oligonucleotide for Duchenne Muscular Dystrophy Treatment.

Delivery represents a major hurdle to the clinical advancement of oligonucleotide therapeutics for the treatment of disorders such as Duchenne muscular dystrophy (DMD). In this preliminary study, we explored the ability of 2'-O-methyl-phosphorothioate antisense oligonucleotides (ASOs) conjugated with lipophilic ursodeoxycholic acid (UDCA) to permeate across intestinal barriers in vitro by a co-culture system of non-contacting IEC-6 cells and DMD myotubes, either alone or encapsulated in exosomes. UDCA was used to enhance the lipophilicity and membrane permeability of ASOs, potentially improving oral bioavailability. Exosomes were employed due to their biocompatibility and ability to deliver therapeutic cargo across biological barriers. Exon skipping was evaluated in the DMD myotubes to reveal the targeting efficiency. Exosomes extracted from milk and wild-type myotubes loaded with 5'-UDC-3'Cy3-ASO and seeded directly on DMD myotubes appear able to fuse to myotubes and induce exon skipping, up to ~20%. Permeation studies using the co-culture system were performed with 5'-UDC-3'Cy3-ASO 51 alone or loaded in milk-derived exosomes. In this setting, only gymnotic delivery induced significant levels of exon skipping (almost 30%) implying a possible role of the intestinal cells in enhancing delivery of ASOs. These results warrant further investigations to elucidate the delivery of ASOs by gymnosis or exosomes.

Decoding RNA Metabolism by RNA-linked CRISPR Screening in Human Cells.

RNAs undergo a complex choreography of metabolic processes in human cells that are regulated by thousands of RNA-associated proteins. While the effects of individual RNA-associated proteins on RNA metabolism have been extensively characterized, the full complement of regulators for most RNA metabolic events remain unknown. Here we present a massively parallel RNA-linked CRISPR (ReLiC) screening approach to measure the responses of diverse RNA metabolic events to knockout of 2,092 human genes encoding all known RNA-associated proteins. ReLiC screens highlight modular interactions between gene networks regulating splicing, translation, and decay of mRNAs. When combined with biochemical fractionation of polysomes, ReLiC reveals striking pathway-specific coupling between growth fitness and mRNA translation. Perturbing different components of the translation and proteostasis machineries have distinct effects on ribosome occupancy, while perturbing mRNA transcription leaves ribosome occupancy largely intact. Isoform-selective ReLiC screens capture differential regulation of intron retention and exon skipping by SF3b complex subunits. Chemogenomic screens using ReLiC decipher translational regulators upstream of mRNA decay and uncover a role for the ribosome collision sensor GCN1 during treatment with the anti-leukemic drug homoharringtonine. Our work demonstrates ReLiC as a versatile platform for discovering and dissecting regulatory principles of human RNA metabolism.

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.

Local and systemic transcriptome and spliceome reprogramming induced by the root-knot nematode Meloidogyne incognita in tomato.

Root-knot nematodes (Meloidogyne spp.) are widely spread root parasites that infect thousands of vascular plant species. These highly polyphagous nematodes engage in sophisticated interactions with host plants that results in the formation of knot-like structures known as galls whose ontogeny remains largely unknown. Here, we determined transcriptome changes and alternative splicing variants induced by Megalaima incognita in galls and neighboring root cells at two distinct infective stages. M. incognita induced substantial transcriptome changes in tomato roots both locally in galls and systemically in neighboring cells. A considerable parallel regulation of gene expression in galls and neighboring cells were detected, indicative of effective intercellular communications exemplified by suppression of basal defense responses particularly during the early stage of infection. The transcriptome analysis also revealed that M. incognita exerts a tight control over the cell cycle process as a whole that results in an increase of ploidy levels in the feeding sites and accelerated mitotic activity of the gall cells. Alternative splicing analysis indicated that M. incognita significantly modulates pre-mRNA splicing as a total of 9064 differentially spliced events from 2898 genes were identified where intron retention and exon skipping events were largely suppressed. Furthermore, a number of differentially spliced events were functionally validated using transgenic hairy root system and found to impact gall formation and nematode egg mass production. Together, our data provide unprecedented insights into the transcriptome and spliceome reprogramming induced by M. incognita in tomato with respect to gall ontogeny and nematode parasitism.

Transcriptomic analysis reveals regulation of adipogenesis via long non-coding RNA, alternative splicing, and alternative polyadenylation

Obesity is characterized by dysregulated adipogenesis that leads to increased number and/or size of adipocytes. Understanding the molecular mechanisms governing adipogenesis is therefore key to designing therapeutic interventions against obesity. In our study, we analyzed 3’-end sequencing data that we generated from human preadipocytes and adipocytes, as well as previously published RNA-seq datasets, to elucidate mechanisms of regulation via long non-coding RNA (lncRNA), alternative splicing (AS) and alternative polyadenylation (APA). We discovered lncRNAs that have not been previously characterized but may be key regulators of white adipogenesis. We also detected 100 AS events and, using motif enrichment analysis, identified RNA binding proteins (RBPs) that could mediate exon skipping—the most prevalent AS event. In addition, we show that usage of alternative poly(A) sites in introns or 3’-UTRs of key adipogenesis genes leads to isoform diversity, which can have significant biological consequences on differentiation efficiency. We also identified RBPs that may modulate APA and defined how 3’-UTR APA can regulate gene expression through gain or loss of specific microRNA binding sites. Taken together, our bioinformatics-based analysis reveals potential therapeutic avenues for obesity through manipulation of lncRNA levels and the profile of mRNA isoforms via alternative splicing and polyadenylation.

An Alternatively Spliced Gain-of-Function NT5C2 Isoform Contributes to Chemoresistance in Acute Lymphoblastic Leukemia.

Relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL) is a major cause of pediatric cancer-related deaths. Relapse-specific mutations do not account for all chemotherapy failures in B- ALL patients, suggesting additional mechanisms of resistance. By mining RNA-seq datasets of paired diagnostic/relapse pediatric B-ALL samples, we discovered pervasive alternative splicing (AS) patterns linked to relapse and affecting drivers of resistance to glucocorticoids, anti-folates, and thiopurines. Most splicing variations represented cassette exon skipping, "poison" exon inclusion, and intron retention, phenocopying well-documented loss-of-function mutations. In contrast, relapse-associated AS of NT5C2 mRNA yielded an isoform with the functionally uncharacterized in-frame exon 6a. Incorporation of the 8-amino acid sequence SQVAVQKR into this enzyme created a putative phosphorylation site and resulted in elevated nucleosidase activity, which is a known consequence of gain-of-function mutations in NT5C2 and a common determinant of 6-mercaptopurine (6-MP) resistance. Consistent with this finding, NT5C2ex6a and the R238W hotspot variant conferred comparable levels of resistance to 6-MP in B-ALL cells both in vitro and in vivo. Furthermore, both the NT5C2ex6a and R238W variants induced collateral sensitivity to the inosine monophosphate dehydrogenase (IMPDH) inhibitor mizoribine. These results ascribe an important role for splicing perturbations in chemotherapy resistance in relapsed B-ALL and suggest that IMPDH inhibitors, including the commonly used immunosuppressive agent mycophenolate mofetil, could be a valuable therapeutic option for treating thiopurine-resistant leukemias.