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Species-specific interferon responses are shaped by the virus-host arms race. The human interferon-induced transmembrane protein (IFITM) family consists of three antiviral IFITM genes that arose by gene duplication. These genes restrict virus entry and are key players in antiviral interferon responses. The unique IFITM repertoires in different species influence their resistance to viral infections, but the role of IFITMs in shaping the enhanced antiviral immunity of reservoir bat species is unclear. Here, we identified an IFITM gene in Chinese rufous horseshoe bat, a natural host of severe acute respiratory syndrome (SARS)-related coronaviruses, that is alternatively spliced to produce two IFITM isoforms in native cells as shown by transcriptomics. These bat IFITMs have conserved structures in vitro as demonstrated by circular dichroism spectroscopy, yet they exhibit distinct antiviral specificities against influenza A virus, Nipah virus and coronaviruses including SARS-CoV, SARS-CoV-2 and MERS-CoV. In parallel with human IFITM1-3, bat IFITM isoforms localize to distinct sites of virus entry which influences their antiviral potency. Further bioinformatic analysis of IFITM repertoires in 206 mammals reveals that alternative splicing is a recurring strategy for IFITM diversification, albeit less widely adopted than gene duplication. These findings demonstrate that alternative splicing is a key strategy for evolutionary diversification in the IFITM family. Our study also highlights an example of convergent evolution where species-specific selection pressures led to expansion of the IFITM family through multiple means, underscoring the importance of IFITM diversity as a component of innate immunity. Author summary: Zoonotic transmission occurs when viruses 'jump' from animals into human. This may lead to viral outbreaks such as the COVID-19 pandemic, posing a significant threat to public health. Bats are the origin of many zoonotic viruses as their unique immunity may allow them to carry viruses without developing disease. Interferon-induced transmembrane proteins (IFITMs) are important antiviral proteins that have been shown to influence the pathogenesis of viral infections. It is currently unclear whether IFITMs also contribute to the high viral tolerance of bats, so characterization of bat IFITMs is needed to identify factors that predispose species to act as viral reservoirs. Here, we find that the Chinese rufous horseshoe bat, a natural host of SARS-related coronaviruses, adopts a distinct strategy known as alternative splicing to functionally diversify their IFITM family. We also demonstrate that alternative splicing is a recurring strategy in the evolution of IFITMs and is evident in at least 75 mammalian species. Our study therefore provides novel insights into how epidemiologically significant species could take advantage of different evolutionary strategies to enhance their resistance to viruses. [ABSTRACT FROM AUTHOR]

Breast cancer (BCa) is a highly prevalent pathological condition (̴30% in women) with limited and subtype-dependent prognosis and therapeutic options. Therefore, BCa management might benefit from the identification of novel molecular elements with clinical potential. Since splicing process is gaining a great relevance in cancer, this work analysed the expression of multiple Spliceosome Components (SCs = 17) and Splicing Factors (SFs = 26) and found a drastic dysregulation in BCa (n = 69) vs. control (negative biopsies; n = 50) samples. Among all the components analysed, we highlight the upregulation of ESRP1 and down-regulation of PRPF8 and NOVA1 in BCa vs. control samples. Indeed, ESRP1 was specially overexpressed in triple-negative BCa (TNBCa) and associated with worse prognosis (i.e., higher BCa grade and lower overall survival), suggesting an association of ESRP1 with BCa aggressiveness. On the other hand, PRPF8 expression was generally downregulated in BCa with no associations to clinical characteristics, while NOVA1 expression was lower in TNBCa patients and highly aggressive tumours. Consistently, NOVA1 overexpression in vitro reduced functional parameters of aggressiveness in ER-/PR- cell lines (MDA-MB-231 and BT-549) but not in ER+/PR+ cells (MCF7), suggesting a critical role of NOVA1 in subtype-specific BCa. Finally, the in vitro pharmacological inhibition of splicing machinery using pladienolide B decreased aggressiveness features in all the BCa cell lines, showing a subtype-independent inhibitory potential, but being relatively innocuous in normal-like breast cells. These results demonstrate the profound dysregulation of the splicing machinery in BCa and their potential as source of promising diagnosis/prognosis markers, as well as valuable therapeutic targets for BCa. [ABSTRACT FROM AUTHOR]

Mutations that affect RNA splicing significantly impact human diversity and disease. Here we present a method using transformers, a type of machine learning model, to detect splicing from raw 45,000-nucleotide sequences. We generate embeddings with residual neural networks and apply hard attention to select splice site candidates, enabling efficient training on long sequences. Our method surpasses the leading tool, SpliceAI, in detecting splice sites in GENCODE and ENSEMBL annotations. Using extensive RNA sequencing data from an Icelandic cohort of 17,848 individuals and the Genotype-Tissue Expression (GTEx) project, our method demonstrates superior performance in detecting splice junctions compared to SpliceAI-10k (PR-AUC = 0.834 vs. PR-AUC = 0.820) and is more effective at identifying disease-related splice variants in ClinVar (PR-AUC = 0.997 vs. PR-AUC = 0.996). These advancements hold promise for improving genetic research and clinical diagnostics, potentially leading to better understanding and treatment of splicing-related diseases. A transformer-based method efficiently detects RNA splicing from 45,000-nucleotide sequences by applying hard attention to select splice site candidates, outperforming SpliceAI in identifying splice sites and disease-related variants. [ABSTRACT FROM AUTHOR]

Introduction: RNA splicing is a crucial posttranscriptional process that governs gene expression, and defects in alternative splicing contribute to various diseases, including cancer. Tumor suppressing subtransferable candidate 4 (TSSC4) is a known tumor suppressor and has been identified as part of the U5 small nuclear ribonucleoprotein (snRNP), which is involved in tri-snRNP biogenesis. However, the precise role of TSSC4 in regulating alternative splicing and its impact on tumor growth remain unclear. Methods: To explore the link between splicing modulation and tumor suppression driven by TSSC4, we conducted transcriptome sequencing (RNA-seq) on TSSC4-knockout and wild-type HeLa cells. Additionally, we analyzed alternative splicing and gene expression in various cancer cell lines, including TSSC4-knockout A549 cells and TSSC4-knockdown PANC-1, MDA-MB-231, and MCF-7 cells. Splicing patterns and gene expression profiles were compared between TSSC4-deficient and control cells. Results: Our RNA-seq analysis revealed that TSSC4 deficiency in HeLa cells results in widespread alterations in splicing patterns and gene expression. Specifically, the loss of TSSC4 led to abnormal alternative splicing events and dysregulation of tumor-associated genes, including several oncogenes. This effect was confirmed across multiple cancer cell lines, highlighting a consistent role of TSSC4 in splicing regulation. Discussion: These findings demonstrate that TSSC4 plays a crucial role in regulating RNA splicing, particularly in controlling the splicing of many oncogenes. Our results reveal a novel mechanism by which TSSC4 mediates tumor suppression through the modulation of alternative splicing, which could provide implications for understanding TSSC4's role in cancer biology. [ABSTRACT FROM AUTHOR]

Regulated precursor messenger RNA (pre‐mRNA) splicing modulates gene expression and promotes alternative splicing. The process is regulated by modifications of spliceosomal proteins and small nuclear RNAs (snRNAs). Here, we show that the protein phosphatase Psr1, known for its plasma membrane localisation and function in general stress response in Saccharomyces cerevisiae, also plays a regulatory role in pre‐mRNA splicing. Independently of its presence at the plasma membrane, Psr1 binds and dephosphorylates the core splicing factor Snu66. The enzyme is not an integral component of the spliceosome. Psr1 deletion in yeast, or tethering of its catalytic mutant to Snu66, results in splicing defects of introns with non‐canonical 5′ splice sites (ss). While the Psr1 binding site on Snu66 is distinct from the Hub1 interaction domains (HIND), Hub1 displaces Psr1 from Snu66. Thus, Psr1 phosphatase plays a regulatory role in pre‐mRNA splicing by modulating Snu66 functions. [ABSTRACT FROM AUTHOR]

Taybi-Linder syndrome (TALS) is a rare autosomal recessive disorder characterized by severe microcephaly with abnormal gyral pattern, severe growth retardation and bone abnormalities. It is caused by pathogenic variants in the RNU4ATAC gene. Its transcript, the small nuclear RNA U4atac, is involved in the excision of ~850 minor introns. Here, we report a patient presenting with TALS features but no pathogenic variants were found in RNU4ATAC, instead the homozygous RTTN c.2953A>G variant was detected by whole-exome sequencing. After deciphering the impact of the variant on the RTTN protein function at centrosome in engineered RTTN-depleted RPE1 cells and patient fibroblasts, we analysed neuronal stem cells (NSC) derived from CRISPR/Cas9-edited induced pluripotent stem cells and revealed major cell cycle and mitotic abnormalities, leading to aneuploidy, cell cycle arrest and cell death. In cortical organoids, we discovered an additional function of RTTN in the self-organisation of NSC into neural rosettes, by observing delayed apico-basal polarization of NSC. Altogether, these defects resulted to a marked delay of rosette formation in RTTN-mutated organoids, thus impeding their overall growth and shedding light on mechanisms leading to microcephaly. Author summary: Primary microcephaly is defined as a severe reduction of the brain size that occurs prenatally. Variants in about 50 genes have been associated to primary microcephaly, and most of them encode proteins that regulate cell cycle, notably by participating to centrosome biogenesis. Intriguingly, some other genes involved in the process of minor splicing, such as RNU4ATAC, are also related to primary microcephaly without clear understanding of the underlying pathophysiological mechanisms. In our previous work, we discovered that alterations of minor splicing result into dysfunction of the centrosome/cilium complex. Here, we further feed this link between minor splicing and centrosome/primary cilium by reporting the particular case of a patient who presents with all features of the rare RNU4ATAC-associated syndrome, called the Taybi-Linder syndrome, and yet, is homozygous for the only recurrent pathogenic variant in the centrosomal RTTN gene. Hence, to decipher the underlying cellular mechanisms, we generated unique human neuronal cellular models–iPSC-derived neuronal stem cells (NSC) and cortical organoids–and unveiled the combination of events that contribute to the depletion of the NSC pool and explain RTTN-associated microcephaly. Our work gives thus precious hints for the understanding of the Taybi-Linder syndrome physiopathology. [ABSTRACT FROM AUTHOR]

AbstractObjectiveDatasourceStudy SelectionsResultsConclusionTo synthesize the current understanding of RNA-based regulatory mechanisms, focusing on how RNA splicing and non-coding RNAs shape immune responses and airway remodeling in asthma, with the aim of exploring their potential as therapeutic targets for asthma treatment.Recent advances and emerging research in molecular biology and immunology related to RNA splicing, non-coding RNAs (lncRNAs, circRNAs), and N6-methyladenosine (m6A) RNA methylation in asthma pathogenesis.The review incorporates studies highlighting the roles of alternative RNA splicing, non-coding RNAs (lncRNAs and circRNAs), and RNA methylation (m6A) in regulating immune and inflammatory pathways involved in asthma.RNA splicing events, non-coding RNAs, and m6A RNA methylation are critical in modulating immune dysregulation, airway remodeling, and inflammation in asthma. These mechanisms influence key inflammatory pathways, mRNA stability, and the overall immune landscape of the disease.RNA splicing and non-coding RNAs represent promising areas of research for understanding asthma’s immune pathology and hold potential as novel therapeutic targets for more effective treatment strategies. [ABSTRACT FROM AUTHOR]

SUMMARY: In plants, pre‐mRNA alternative splicing has been demonstrated to be a crucial tier that regulates gene expression in response to salt stress. However, the underlying mechanisms remain elusive. Here, we studied the roles of DIGEORGE‐SYNDROME CRITICAL REGION 14‐like (AtDGCR14L) in regulating pre‐mRNA splicing and salt stress tolerance. We discovered that Arabidopsis AtDGCR14L is required for maintaining plant salt stress tolerance and the constitutively spliced and active isoforms of important stress‐ and/or abscisic acid (ABA)‐responsive genes. We also identified the interaction between AtDGCR14L and splicing factor U1‐70k, which needs a highly conserved three amino acid (TWG) motif in DGCR14. Different from wild‐type AtDGCR14L, the overexpression of the TWG‐substituted AtDGCR14L mutant did not change salt stress tolerance or pre‐mRNA splicing of stress/ABA‐responsive genes. Additionally, SWITCH3A (SWI3A) is a core subunit of the SWI/SUCROSE NONFERMENTING (SWI/SNF) chromatin‐remodeling complexes. We found that SWI3A, whose splicing depends on AtDGCR14L, actively enhances salt stress tolerance. These results revealed that AtDGCR14L may play an essential role in crosstalk between plant salt‐stress response and pre‐mRNA splicing mechanisms. We also unveiled the potential role of SWI3A in controlling salt stress tolerance. The TWG motif in the intrinsically disordered region of AtDGCR14L is highly conserved and crucial for DGCR14 functions. [ABSTRACT FROM AUTHOR]

Polycystic ovary syndrome (PCOS) is a prevalent yet complex reproductive endocrine disorder affecting 11–13% of women worldwide. Its main symptoms include elevated androgen levels, irregular menstrual cycles, and long-term metabolic and offspring health implications. Despite the disease's multifaceted nature involving genetic, epigenetic, and environmental factors, the role of alternative splicing in ovarian granulosa cells remains relatively unexplored. This study aims to investigate the transcriptional and alternative splicing characteristics of granulosa cells in PCOS patients and to elucidate the potential functional consequences of these changes. Analysis of previous published transcriptome sequencing data identified 491 upregulated and 401 downregulated genes in granulosa cells of PCOS patients, significantly involved in immune-related processes. Additionally, 1250 differential splicing events, predominantly involving exon skipping and affecting 947 genes, were detected. These genes with alternative splicing patterns were found to be enriched in endoplasmic reticulum stress and protein post-translational modification processes, suggesting their role in PCOS pathology. Moreover, the study highlighted that the utilization of different splice isoforms of the YAP1 gene may impact its interaction in the Hippo signaling pathway, influencing the pathogenesis of PCOS. These findings underscore substantial alterations in alternative splicing in granulosa cells of PCOS patients, providing a novel viewpoint for comprehending the molecular underpinnings of PCOS and suggesting potential avenues for therapeutic intervention. [ABSTRACT FROM AUTHOR]

RNA-modifying enzymes have recently garnered considerable attention due to their relevance in cancer biology, identifying them as potential targets for novel therapeutic intervention. THUMPD3 was recently identified as an RNA methyltransferase catalysing N2-methylguanosine (m2G) within certain tRNAs. In this study, we unveil a novel role for THUMPD3 in lung cancer cells. Depletion of the enzyme from lung cancer cells significantly impairs their fitness, negatively impacting key cellular processes such as proliferation and migration. Notably, exogenous expression of THUMPD3 in normal lung fibroblasts stimulates their proliferation rate. Additionally, transcriptome-wide analyses reveal that depletion of THUMPD3 from lung cancer cells induces substantial changes in the expression of cell surface proteins, including those comprising the extracellular matrix (ECM). We further demonstrate that THUMPD3 maintains expression of an extra-domain B (EDB) containing pro-tumour isoform of Fibronectin-1 mRNA, encoding FN1, an important ECM protein. Crucially, depletion of THUMPD3 promotes an alternative splicing event that removes the EDB-encoding exon from Fibronectin-1. This is consistent with THUMPD3 depletion reducing cellular proliferation and migration. Moreover, depletion of THUMPD3 selectively and preferentially affects the alternative splicing of ECM and cell adhesion molecule encoding transcripts, as well as those encoding neurodevelopmental proteins. Overall, these findings highlight THUMPD3 as an important player in regulating cancer-relevant alternative splicing and they provide a rationale for further investigations into THUMPD3 as a candidate target in anti-cancer therapy. [ABSTRACT FROM AUTHOR]

Introduction: The main obstacle to achieving an HIV-1 cure is the proviral reservoir. To promote equity in HIV cure strategies, it is crucial to study the viral reservoir of the predominant HIV-1 subtype C in both women and men. Therefore, we investigated the dynamics of the (intact) viral reservoir in relation to plasma viral load (VL), CD4+ T cell count, and immune activation before and during 96 weeks of successful antiretroviral therapy (ART). Methods: Eighty-two participants (62% female) newly initiating ART in a rural clinic in South Africa were included in the study. Blood samples were collected at baseline, week 48, and week 96, and CD4 count was determined. Plasma was used for VL and immune marker analyses, while isolated peripheral blood mononuclear cells (PBMCs) were used for the quantification of cellular multiple spliced HIV-1 RNA (msRNA) and the intact proviral DNA assay. For the longitudinal analyses on ART, we selected only those participants who durably suppressed their VL to <200 copies/mL during 48 (n=65) and/or 96 (n=60) weeks of treatment. Results: At ART initiation, the median CD4 count was 234 cells/mm3 and VL was 68,897 copies/mL. Interestingly, at baseline the number of defective proviruses was significantly correlated with VL (p<0.0001), msRNA (p<0.0001), CD4 count (p=0.0008), CXCL10 (p=0.0003) and TNF-α (p=0.0394). During successful ART, a significant decrease of both the intact and defective proviral reservoir was observed (p<0.0001). The decrease of the intact proviral reservoir was more profound compared to the defective fraction after 96 weeks of therapy. In addition, a significant decrease in cellular msRNA and IL-6, IL-7, TNF-α, sCD14, sCD163, CCL2, CXCL10, and CRP was detected. Discussion: This study underscores the significant relationship observed prior to therapy initiation between the number of defective proviruses, viral transcription/production and their association with immune response indicators such as CD4 count, CXCL10, and TNF-α. Furthermore, the observation of a less pronounced decrease of the defective proviral DNA highlights the importance of addressing both intact and defective proviruses in therapeutic strategies to enhance clinical outcomes for people with HIV-1. Together, these findings suggest a significant role of the defective proviruses in HIV-related disease progression. [ABSTRACT FROM AUTHOR]

The KEAP1-NRF2 system plays a crucial role in responding to oxidative and electrophilic stress. Its dysregulation can cause the overexpression of downstream genes, a known cancer hallmark. Understanding and detecting abnormal KEAP1-NRF2 activity is essential for understanding disease mechanisms and identifying therapeutic targets. This study presents an approach that analyzes splicing patterns by a naive Bayes-based classifier to identify constitutive activation of the KEAP1-NRF2 system, focusing on the higher presence of abnormal splicing junctions as a subproduct of overexpression of downstream genes. Our splicing-based classifier demonstrated robust performance, reliably identifying activation of the KEAP1-NRF2 pathway across extensive datasets, including The Cancer Genome Atlas and the Sequence Read Archive. This shows the classifier's potential to analyze hundreds of thousands of transcriptomes, highlighting its utility in broad-scale genomic studies and provides a new perspective on utilizing splicing aberrations caused by overexpression as diagnostic markers, offering potential improvements in diagnosis and treatment strategies. [ABSTRACT FROM AUTHOR]

Cellular growth and organismal development are remarkably complex processes that require the nutrient-responsive kinase mechanistic target of rapamycin complex 1 (mTORC1). Anticipating that important mTORC1 functions remained to be identified, we employed genetic and bioinformatic screening in C. elegans to uncover mechanisms of mTORC1 action. Here, we show that during larval growth, nutrients induce an extensive reprogramming of gene expression and alternative mRNA splicing by acting through mTORC1. mTORC1 regulates mRNA splicing and the production of protein-coding mRNA isoforms largely independently of its target p70 S6 kinase (S6K) by increasing the activity of the serine/arginine-rich (SR) protein RSP-6 (SRSF3/7) and other splicing factors. mTORC1-mediated mRNA splicing regulation is critical for growth; mediates nutrient control of mechanisms that include energy, nucleotide, amino acid, and other metabolic pathways; and may be conserved in humans. Although mTORC1 inhibition delays aging, mTORC1-induced mRNA splicing promotes longevity, suggesting that when mTORC1 is inhibited, enhancement of this splicing might provide additional anti-aging benefits. [Display omitted] • During C. elegans growth, mTORC1 broadly controls mRNA splicing and expression • mTORC1 increases the expression and activity of SR proteins and other splicing factors • mTORC1 controls growth-related metabolic pathways by regulating mRNA splicing • Regulating metabolic genes through mRNA splicing is a conserved mTORC1 function Ogawa et al. show in Caenorhabditis elegans that the essential growth regulator mTORC1 extensively reprograms gene expression and alternative mRNA splicing. This splicing regulation is critical for growth, orchestrates growth-associated metabolism, and appears to be conserved. Maintaining mTORC1-induced splicing may be beneficial for longevity under conditions of mTORC1 inhibition. [ABSTRACT FROM AUTHOR]

Background: Alternative splicing is a fundamental mechanism in the post‐transcriptional regulation of genes. The multifunctional transmembrane glycoprotein receptor carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1) undergoes extensive alternative splicing to allow for tunable functions in cell signalling, adhesion and modulation of immune and metabolic responses. Splice isoforms that differ in their ectodomain and short or long cytoplasmic tail (CEACAM1‐S/CEACAM1‐L) have distinct functional roles. The mechanisms that regulate CEACAM1 RNA splicing remain elusive. Methods: This narrative review summarizes the current knowledge of the mechanism and function of CEACAM1 splice isoforms. Historical perspectives address the biological significance of the glycosylated Ig domains, the variable exon 7, and phosphorylation events that dictate its signal transduction pathways. The use of small antisense molecules to target mis‐spliced variable exon 7 is discussed. Results: The Ig variable‐like N domain mediates cell adhesion and immune checkpoint inhibitory functions. Gly and Tyr residues in the transmembrane (TM) domain are essential for dimerization. Calmodulin, Calcium/Calmodulin‐dependent protein kinase II delta (CamK2D), Actin and Annexin A2 are binding partners of CEACAM1‐S. Homology studies of the muCEACAM1‐S and huCEACAM1‐S TM predict differences in their signal transduction pathways. Hypoxia‐inducible factor 1‐α (HIF‐1‐α) induces alternative splicing to produce CEACAM1‐S under limited oxygen conditions. Antisense small molecules directed to exon 7 may correct faulty expression of the short and long cytoplasmic tail splicing isoforms. Conclusion: More pre‐clinical and clinical studies are needed to elucidate the precise mechanisms by which CEACAM1 RNA splicing may be exploited to develop targeted interventions towards novel therapeutic strategies. [ABSTRACT FROM AUTHOR]

Signaling through the platelet-derived growth factor receptor alpha (PDGFRα) plays a critical role in craniofacial development. Phosphatidylinositol 3-kinase (PI3K)/Akt is the primary effector of PDGFRα signaling during mouse skeletal development. We previously demonstrated that Akt phosphorylates the RNA-binding protein serine/arginine-rich splicing factor 3 (Srsf3) downstream of PI3K-mediated PDGFRα signaling in mouse embryonic palatal mesenchyme (MEPM) cells, leading to its nuclear translocation. We further showed that ablation of Srsf3 in the murine neural crest lineage results in severe midline facial clefting and widespread alternative RNA splicing (AS) changes. Here, we demonstrated via enhanced UV-crosslinking and immunoprecipitation of MEPM cells that PDGF-AA stimulation leads to preferential binding of Srsf3 to exons and loss of binding to canonical Srsf3 CA-rich motifs. Through the analysis of complementary RNA-seq data, we showed that Srsf3 activity results in the preferential inclusion of exons with increased GC content and lower intron to exon length ratio. We found that Srsf3 activity downstream of PDGFRα signaling leads to retention of the receptor in early endosomes and increases in downstream PI3K-mediated Akt signaling. Taken together, our findings reveal that growth factor-mediated phosphorylation of an RNA-binding protein underlies gene expression regulation necessary for mammalian craniofacial development. [ABSTRACT FROM AUTHOR]

Background: Ductal carcinoma in-situ (DCIS) is a pre-invasive form of invasive breast cancer (IBC). Due to improved breast cancer screening, it now accounts for ~ 25% of all breast cancers. While the treatment success rates are over 90%, this comes at the cost of considerable morbidity, considering that the majority of DCIS never become invasive and our understanding of the molecular changes occurring in DCIS that predispose to invasive disease is limited. The aim of this study is to characterize molecular changes that occur in DCIS, with the goal of improving DCIS risk stratification. Methods: We identified and obtained a total of 197 breast tissue samples from 5 institutions (93 DCIS progressors, 93 DCIS non-progressors, and 11 adjacent normal breast tissues) that had at least 10-year follow-up. We isolated DNA and RNA from archival tissue blocks and characterized genome-wide mRNA expression, DNA methylation, DNA copy number variation, and RNA splicing variation. Results: We obtained all four genomic data sets in 122 of the 197 samples. Our intrinsic expression subtype-stratified analyses identified multiple molecular differences both between DCIS subtypes and between DCIS and IBC. While there was heterogeneity in molecular signatures and outcomes within intrinsic subtypes, several gene sets that differed significantly between progressing and non-progressing DCIS were identified by Gene Set Enrichment Analysis. Conclusion: DCIS is a molecularly highly heterogenous disease with variable outcomes, and the molecular events determining DCIS disease progression remain poorly defined. Our genome-wide multi-omic survey documents DCIS-associated alterations and reveals molecular heterogeneity within the intrinsic DCIS subtypes. Further studies investigating intrinsic subtype-stratified characteristics and molecular signatures are needed to determine if these may be exploitable for risk assessment and mitigation of DCIS progression. The highly significant associations of specific gene sets with IBC progression revealed by our Gene Set Enrichment Analysis may lend themselves to the development of a prognostic molecular score, to be validated on independent DCIS cohorts. [ABSTRACT FROM AUTHOR]

Dysregulation of alternative pre‐mRNA splicing plays a critical role in the progression of cancers, yet the underlying molecular mechanisms remain largely unknown. It is reported that metastasis‐associated in colon cancer 1 (MACC1) is a novel prognostic and predictive marker in many types of cancers, including lung adenocarcinoma. Here, we reveal that the oncogene MACC1 specifically drives the progression of lung adenocarcinoma through its control over cancer‐related splicing events. MACC1 depletion inhibits lung adenocarcinoma progression through triggering IRAK1 from its long isoform, IRAK1‐L, to the shorter isoform, IRAK1‐S. Mechanistically, MACC1 interacts with splicing factor HNRNPH1 to prevent the production of the short isoform of IRAK1 mRNA. Specifically, the interaction between MACC1 and HNRNPH1 relies on the involvement of MACC1's SH3 domain and HNRNPH1's GYR domain. Further, HNRNPH1 can interact with the pre‐mRNA segment (comprising exon 11) of IRAK1, thereby bridging MACC1's regulation of IRAK1 splicing. Our research not only sheds light on the abnormal splicing regulation in cancer but also uncovers a hitherto unknown function of MACC1 in tumor progression, thereby presenting a novel potential therapeutic target for clinical treatment. [ABSTRACT FROM AUTHOR]

Variants in RNA binding motif protein 20 (RBM20) are causative in a severe form of dilated cardiomyopathy referred to as RBM20 cardiomyopathy, yet the mechanisms are unclear. Moreover, the reason(s) for phenotypic heterogeneity in carriers with different pathogenic variants are similarly opaque. To gain insight, we carried out multi-omics analysis, including the first analysis of gene expression changes at the protein level, of mice carrying two different pathogenic variants in the RBM20 nuclear localization signal (NLS). Direct comparison of the phenotypes confirmed greater premature morality in S639G variant carrying mice compared to mice with the S637A variant despite similar cardiac remodeling and dysfunction. Analysis of differentially spliced genes uncovered alterations in the splicing of both RBM20 target genes and non-target genes, including several genes previously implicated in arrhythmia. Global proteomics analysis found that a greater number of proteins were differentially expressed in the hearts of Rbm20 S639G mice relative to WT than in Rbm20 S637A versus WT. Gene ontology analysis suggested greater mitochondrial dysfunction in Rbm20 S639G mice, although direct comparison of protein expression in the hearts of Rbm20 S639G versus Rbm20 S637A mice failed to identify any significant differences. Similarly, few differences were found by direct comparison of gene expression at the transcript level in Rbm20 S639G and Rbm20 S637A despite greater coverage. Our data provide a comprehensive overview of gene splicing and expression differences associated with pathogenic variants in RBM20, as well as insights into the molecular underpinnings of phenotypic heterogeneity associated with different dilated cardiomyopathy-associated variants. [Display omitted] • Disparate RBM20 NLS variants causes distinct disease phenotypes. • S639G variant causes greater premature mortality compared to S637. • DSGs in Rbm20 S639G versus Rbm20 S637A included genes linked to arrhythmia. • Unique Rbm20 S639G DEPs implicate mitochondrial dysfunction in phenotype differences. • First protein-level and multi-omics characterization in RBM20 cardiomyopathy. [ABSTRACT FROM AUTHOR]

Background: An individual's genetic fingerprint is emerging as a pivotal predictor of numerous disease‐ and treatment‐related factors. Single nucleotide polymorphisms (SNPs) in drug‐metabolizing enzymes play key roles in an individual's exposure to a malignancy‐associated risk, such as Aflatoxin B1 (AFB1)‐induced hepatocellular carcinoma (HCC). Aim: This study aimed at reviewing literature on the polymorphisms that exist in CYP enzymes and their possible link with susceptibility to AFB1‐induced HCC. Materials & Methods: A set of keywords associated with the study subject of interest was used to search the Google Scholar and the PubMed database. The last ten years' worth of research projects were included in the results filter. The research involved HCC patients and any connection between polymorphic forms of CYP enzymes and their susceptibility to AFB1‐induced HCC, including older but significant data. Results: Variations in CYP1A2 and CYP3A4 were reported to impact the rate and magnitude of AFB1 bio‐activation, thus influencing an individual's vulnerability to develop HCC. In HCC patients, the activity of CYP isoforms varies, where increased activity has been reported with CYP2C9, CYP2D6, and CYP2E1, while CYP1A2, CYP2C8, and CYP2C19 exhibit decreased activity. CYP2D6*10 frequency has been discovered to differ considerably in HCC patients. Rs2740574 (an upstream polymorphism in CYP3A4 as detected in CYP3A4*1B) and rs776746 (which affects CYP3A5 RNA splicing), both of which influence CYP3A expression, thus impacting the variability of AFB1‐epoxide adducts in HCC patients. Discussion: CYP1A2 is the primary enzyme accountable for the formation of harmful AFBO globally. CYP3A4, CYP3A5, CYP3A7, CYP2B7, and CYP3A3 are also implicated in the bio‐activation of AFB1 to mutagenic metabolites. It is thought that CYP3A4 is the protein that interacts with AFB1 metabolism the most. Conclusion: Polymorphic variants of CYP enzymes have a functional impact on the susceptibility to AFB1‐induced HCC. Outlining such variation and their implications may provide deeper insights into approaching HCC in a more personalized manner for guiding future risk‐assessment, diagnosis, and treatment. [ABSTRACT FROM AUTHOR]

The document "CircRNA: Functions, Applications and Prospects" published in the journal Biomolecules discusses the functions, applications, and prospects of circular RNAs (circRNAs). It highlights the role of circRNAs in gene expression, their involvement in various diseases, and their potential as biomarkers and therapeutic targets. The document also explores the applications of circRNAs in clinical treatment, emphasizing their resistance to degradation and tissue-specific expression patterns. Additionally, it discusses the use of genome-editing tools, RNA delivery methods, and exosomes for circRNA research and therapy. [Extracted from the article]

The chloroplast and mitochondrion are semi-autonomous organelles that play essential roles in cell function. These two organelles are embellished with prokaryotic remnants and contain many new features emerging from the co-evolution of organelles and the nucleus. A typical plant chloroplast or mitochondrion genome encodes less than 100 genes, and the regulation of these genesˈ expression is remarkably complex. The regulation of chloroplast and mitochondrion gene expression can be achieved at multiple levels during development and in response to environmental cues, in which, RNA metabolism, including: RNA transcription, processing, translation, and degradation, plays an important role. RNA metabolism in plant chloroplasts and mitochondria combines bacterial-like traits with novel features evolved in the host cell and is regulated by a large number of nucleus-encoded proteins. Among these, pentatricopeptide repeat (PPR) proteins are deeply involved in multiple aspects of the RNA metabolism of organellar genes. Research over the past decades has revealed new insights into different RNA metabolic events in plant organelles, such as the composition of chloroplast and mitochondrion RNA editosomes. We summarize and discuss the most recent knowledge and biotechnological implications of various RNA metabolism processes in plant chloroplasts and mitochondria, with a focus on the nucleus-encoded factors supporting them, to gain a deeper understanding of the function and evolution of these two organelles in plant cells. Furthermore, a better understanding of the role of nucleus-encoded factors in chloroplast and mitochondrion RNA metabolism will motivate future studies on manipulating the plant gene expression machinery with engineered nucleus-encoded factors. [ABSTRACT FROM AUTHOR]

Purpose: To determine the genetic cause of infertility in two unrelated families of female patients suffering from oocyte degeneration and fertilization failure. Methods: Whole exome sequencing and Sanger sequencing were performed to identify the disease-causing genes of infertility in two unrelated female patients. Minigene experiments were conducted to confirm the effect of splice site mutations on mRNA splicing. Results: In two unrelated female infertility patients, a novel compound heterozygous splicing mutation (c.516-1G > T and c.877-1G > A) in PATL2 gene and a novel homozygous splicing mutation (c.1222-1G > A) in WEE2 gene were identified. Minigene splicing assays revealed that the c.516-1G > T mutation in PATL2 resulted in a deletion of 8 bases in mRNA that causes a frameshift (c.516-523delTCCCCCAG, p.P173Q fs*13). The c.877-1G > A mutation led to the skipping of exons 10 and 11 and retention of introns 8–9 in PATL2 mRNA. The c.1222-1G > A mutation resulted in the deletion of exon 9 in WEE2 mRNA, leading to an in-frame deletion of 57 amino acids in the WEE2 protein (p.408-464del). Conclusion: Our study discovered novel splicing mutations in PATL2 and WEE2, further expanding the mutation spectrum of these two genes and providing guidance for genetic counseling and diagnosis of female infertility. [ABSTRACT FROM AUTHOR]

DDX41 (DEAD‑box helicase 41) is a member of the largest family of RNA helicases. The DEAD-box RNA helicases share a highly conserved core structure and regulate all aspects of RNA metabolism. The functional role of DDX41 in innate immunity is also highly conserved. DDX41 acts as a sensor of viral DNA and activates the STING-TBK1-IRF3-type I IFN signaling pathway. Germline heterozygous variants in DDX41 have been reported in familial myelodysplasia syndrome (MDS)/acute myeloid leukemia (AML) patients; most patients also acquired a somatic variant in the second DDX41 allele. Here, we report a patient who inherited compound heterozygous DDX41 variants and presented with bone dysplasia, ichthyosis, and dysmorphic features. Functional analyses of the patient-derived dermal fibroblasts revealed a reduced abundance of DDX41 and abrogated activation of the IFN genes through the STING-type I interferon pathway. Genome-wide transcriptome analyses in the patient's fibroblasts revealed significant gene dysregulation and changes in the RNA splicing events. The patient's fibroblasts also displayed upregulation of periostin mRNA expression. Using an RNA binding protein assay, we identified DDX41 as a novel regulator of periostin expression. Our results suggest that functional impairment of DDX41, along with dysregulated periostin expression, likely contributes to this patient's multisystem disorder. [ABSTRACT FROM AUTHOR]

Rice lesion mimic mutants are important materials for studying the mechanisms of cell death. In-depth research on these mutants can provide insights into the molecular mechanisms underlying rice growth and development, offering a theoretical basis for crop improvement. In this study, rice variety Wuyunjing 21 (WYJ21) was mutagenized with ethyl methanesulfonate to obtain a lesion mimic mutant, lmm28. Unlike wild-type (WT) plants, the lmm28 mutant exhibits brown lesions on the leaves starting from the early tillering stage. The size of the lesions increases as the plant grows. Additionally, the lmm28 mutant shows significantly reduced plant height, tiller number, number of effective panicles, seed setting rate, and 1000-grain weight compared to the WT. Leaf staining of the mutant revealed an accumulation of reactive oxygen species and cell death in the lesion leaves. Transmission electron microscopy images showed that, in the lmm28 mutant, the nuclear boundaries in leaf cells became indistinct and damage to the chloroplast membrane structures was observed, with thylakoid disorganization occurring in some chloroplasts. Genetic analysis and map-based cloning localized the candidate gene of the mutant to a 167.79 kb region on chromosome 5. After analyzing the annotated genes within this region, the candidate gene was preliminarily identified as OsBON3. Sequencing analysis revealed that, in lmm28, a base change from GT to GC occurred at the 5′ splice junction of the 15th intron of OsBON3. Further analysis, using cDNA amplification of exons 14–16 followed by sequencing, showed that the mutation at the splice recognition site caused the incorrect splicing of OsBON3 pre-mRNA, leading to an increased number of transcripts in lmm28. The transcript containing an inserted intron is present at much higher levels than the normal transcript, which may lead to a reduction in the protein levels containing the functional vWA domain. Therefore, the vWA domain of OsBON3 is likely crucial for maintaining ROS homeostasis in rice and plays a key role in regulating its growth and development. [ABSTRACT FROM AUTHOR]

Alternative splicing refers to the process that genes produce RNA isoforms under different splicing modes, and it is an important mechanism for post-transcriptional expression regulation of genes. Alternative splicing has been proved to play an important role in the occurrence and development of cancer, and abnormal splicing is considered as an important symbol of tumor occurrence. Long noncoding RNA (lncRNA) is an important participant and regulator in the process of gene alternative splicing, and many evidences show that lncRNA can affect the occurrence and development of cancer by regulating alternative splicing. In this paper, the research on alternative splicing regulated by lncRNA in cancer is reviewed, and the related bioinformatics tools and databases are summarized. [ABSTRACT FROM AUTHOR]

Huntington's disease (HD) is a fatal neurodegenerative disease caused by a trinucleotide repeat expansion in exon 1 of the huntingtin gene (HTT) that results in toxic gain of function and cell death. Despite its monogenic cause, the pathogenesis of HD is highly complex, and increasing evidence indicates that, in addition to the full-length (FL) mutant HTT protein, the expanded exon 1 HTT (HTTexon1) protein that is translated from the HTT1a transcript generated by aberrant splicing is prone to aggregate and might contribute to HD pathology. This finding suggests that reducing the expression of HTT1a might achieve a greater therapeutic benefit than targeting only FL mutant HTT. Conversely, strategies that exclusively target FL HTT might not completely prevent the pathogenesis of HD. We have developed an engineered microRNA targeting the HTT exon 1 sequence (miHTT), delivered via adeno-associated virus serotype 5 (AAV5). The target sequence of miHTT is present in both FL HTT and HTT1a transcripts. Preclinical studies with AAV5-miHTT have demonstrated efficacy in several rodent and large animal models by reducing FL HTT mRNA and protein and rescuing HD-like phenotypes and have been the rationale for phase I/II clinical studies now ongoing in the USA and Europe. In the present study, we evaluated the ability of AAV5-miHTT to reduce the levels of aberrantly spliced HTT1a mRNA and the HTTexon1 protein in the brain of two mouse models of HD (heterozygous zQ175 knock-in mice and humanized Hu128/21 mice). Polyadenylated HTT1a mRNA and HTTexon1 protein were detected in the striatum and cortex of heterozygous zQ175 knock-in mice, but not in wild-type littermate control mice. Intrastriatal administration of AAV5-miHTT resulted in dose-dependent expression of mature miHTT microRNA in cortical brain regions, accompanied by significant lowering of both FL HTT and HTT1a mRNA expression at 2 months postinjection. Mutant HTT and HTTexon1 protein levels were also significantly reduced in the striatum and cortex of heterozygous zQ175 knock-in mice at 2 months after AAV5-miHTT treatment and in humanized Hu128/21 mice 7 months post-treatment. The effects were confirmed in primary Hu128/21 neuronal cultures. These results demonstrate that AAV5-miHTT gene therapy is an effective approach to lower both FL HTT and the pathogenic HTTexon1 levels, which could potentially have an additive therapeutic benefit in comparison to other HTT-targeting modalities. [ABSTRACT FROM AUTHOR]

Leukemia represents the most prevalent malignancy in children, constituting 30% of childhood cancer cases, with acute lymphoblastic leukemia (ALL) being particularly heterogeneous. This paper explores the role of alternative splicing in leukemia, highlighting its significance in cancer development and progression. Aberrant splicing is often driven by mutations in splicing-factor genes, which can lead to the production of variant proteins that contribute to oncogenesis. The spliceosome, a complex of small nuclear RNAs and proteins, facilitates RNA splicing, a process critical for generating diverse mRNA and protein products from single genes. Mutations in splicing factors, such as U2AF1, SF3B1, SRSF2, ZRSR2, and HNRNPH1, are frequently observed across various hematological malignancies and are associated with poor prognosis and treatment resistance. This research underscores the necessity of understanding the mechanisms of RNA splicing dysregulation in order to develop targeted therapies to correct these aberrant processes, thereby improving outcomes for patients with leukemia and related disorders. [ABSTRACT FROM AUTHOR]

Mitochondria play a crucial role in eukaryotic organisms, housing their own genome with genes vital for oxidative phosphorylation. Coordination between nuclear and mitochondrial genomes is pivotal for organelle gene expression. Splicing, editing and processing of mitochondrial transcripts are regulated by nuclear-encoded factors. Splicing efficiency (SEf) of the many group II introns present in plant mitochondrial genes is critical for mitochondrial function since a splicing defect or splicing deficiency can severely impact plant growth and development. This study investigates SEf in free-living and holoparasitic plants, focusing on 25 group II introns from 15 angiosperm species. Our comparative analyses reveal distinctive splicing patterns with holoparasites exhibiting significantly lower SEf, potentially linked to their unique evolutionary trajectory. Given the preponderance of horizontal gene transfer (HGT) in parasitic plants, we investigated the effect of HGT on SEf, such as the presence of foreign introns or foreign nuclear-encoded splicing factors. Contrary to expectations, the SEf reductions do not correlate with HGT events, suggesting that other factors are at play, such as the loss of photosynthesis or the transition to a holoparasitic lifestyle. The findings of this study broaden our understanding of the molecular evolution in parasitic plants and shed light on the multifaceted factors influencing organelle gene expression. [ABSTRACT FROM AUTHOR]

In Arabidopsis thaliana, the transcription factors WRKY7, WRKY11 and WRKY17 act as negative defence regulators against Pseudomonas syringae pv. tomato (Pst) DC3000. However, their coordinated regulation of gene expression has yet to be fully explored. In this study, we conducted a transcriptomic analysis on the triple mutant wrky7/11/17 in response to Pst DC3000 at 0, 3 and 24 h post‐inoculation (hpi). Our results suggest that at early infection stages (0 and 3 hpi), WRKY7, WRKY11 and WRKY17 significantly repress a group of genes involved in signal perception and transduction, including receptor‐like kinases. Furthermore, at later stages of interaction (24 hpi), these transcription factors induce genes related to the biosynthesis and signalling of the jasmonic acid (JA) pathway. Further infection experiments with Pst DC3000 in plants treated with methyl jasmonate (a JA analogue) and infections with Botrytis cinerea, a pathogen against which JA‐mediated responses are crucial for effective defence, support this proposal. Moreover, we analysed the role of WRKY7, WRKY11 and WRKY17 in alternative splicing regulation. A comparison between differentially expressed (DEG) and spliced (DAS) genes revealed that over 80% of DAS events do not occur in conjunction with overall changes in gene expression. Alternative splicing events were found in genes with functions in splicing and the JA pathway, such as ALY4, PRP40A, JAZ3 and JAZ10. These results suggest that WRKY7, WRKY11 and WRKY17 can also participate in this layer of gene expression regulation to modulate immunity negatively. [ABSTRACT FROM AUTHOR]

The core spliceosome Sm proteins are gaining attention as potential targets for cancer treatment. Here, we evaluate this, with focus on SmD2. A pan-cancer analysis including 26 solid tumor types revealed that the SmD2-encoding SNRPD2 gene was overexpressed in almost all cancers. In several cancers, high SNRPD2 expression was associated with a poor prognosis. To investigate the vulnerability of human cells to the loss of SmD2 expression, we silenced SNRPD2 using a short hairpin-expressing lentiviral vector in established cancer cell lines; in short-term cultured melanoma cells; and in several normal cell cultures, including cancer-associated fibroblasts cultured from non-small cell lung cancer resections. Additionally, we analyzed publicly available cell viability datasets for the dependency of cancer cell lines to SmD2 expression. Together, these studies clearly established SmD2 as a cancer-selective lethal target. Delving into genes with similar essentiality profiles to SNRPD2, we uncovered the intersected lethal stress between the loss of SmD2 and the loss of gene products participating in not only different mRNA processing steps including mRNA splicing, but also processes for coordinated protein production, as well as mitosis. Furthermore, we could correlate SNRPD2 expression to the responses of cancer cells to several FDA-approved anti-tumor drugs, especially to drugs inhibiting the cell cycle. Overall, our study confirms the anticipated role for targeting SmD2 in cancer treatment and reveals non-canonical SmD2 functions beyond mRNA splicing that could contribute to the dependency of cancer cells to high SNRPD2 expression. [ABSTRACT FROM AUTHOR]

Alternative splicing allows a single gene to produce a variety of protein isoforms. Changes in splicing isoform usage characterize virtually every stage of the differentiation process and define the physiological differences between cardiomyocytes with different function, at different stages of development, and pathological function. Recent identification of cardiac splicing factors provided insights into the mechanisms underlying alternative splicing and revealed how these splicing factors impact functional properties of the heart. Alterations of the splicing of sarcomeric genes, cell signaling proteins, and ion channels have been associated with the development of pathological conditions such as cardiomyopathy and arrhythmia. RBM20, RBM24, PTBP1, RBFOX, and QKI play key roles in cardiac development and pathology. A better understanding of their regulation will yield insights into healthy cardiac development and inform the development of molecular therapeutics. [ABSTRACT FROM AUTHOR]

Chikungunya virus (CHIKV) is an emerging, mosquito-borne arthritic alphavirus increasingly associated with severe neurological sequelae and long-term morbidity. However, there is limited understanding of the crucial host components involved in CHIKV replicase assembly complex formation, and thus virus replication and virulence-determining factors, within the central nervous system (CNS). Furthermore, the majority of CHIKV CNS studies focus on neuronal infection, even though astrocytes represent the main cerebral target. Heterogeneous ribonucleoprotein K (hnRNP K), an RNA-binding protein involved in RNA splicing, trafficking, and translation, is a regulatory component of alphavirus replicase assembly complexes, but has yet to be thoroughly studied in the context of CHIKV infection. We identified the hnRNP K CHIKV viral RNA (vRNA) binding site via sequence alignment and performed site-directed mutagenesis to generate a mutant, ΔhnRNPK-BS1, with disrupted hnRNPK–vRNA binding, as verified through RNA coimmunoprecipitation and RT-qPCR. CHIKV ΔhnRNPK-BS1 demonstrated hampered replication in both NSC-34 neuronal and C8-D1A astrocytic cultures. In astrocytes, disruption of the hnRNPK–vRNA interaction curtailed viral RNA transcription and shut down subgenomic RNA translation. Our study demonstrates that hnRNP K serves as a crucial RNA-binding host factor that regulates CHIKV replication through the modulation of subgenomic RNA translation. [ABSTRACT FROM AUTHOR]

Mutations in SF3B1 are common in many types of cancer, promoting cancer progression through aberrant RNA splicing. Recently, mRNA nuclear export has been reported to be defective in cells with the SF3B1 K700E mutation. However, the mechanism remains unclear. Our study reveals that the K700E mutation in SF3B1 attenuates its interaction with THOC5, an essential component of the mRNA nuclear export complex THO. Furthermore, the SF3B1 mutation caused reduced binding of THOC5 with some mRNA and inhibited the nuclear export of these mRNAs. Interestingly, overexpression of THOC5 restores the nuclear export of these mRNAs in cells with the SF3B1 K700E mutation. Importantly, other types of cancer-associated SF3B1 mutations also inhibited mRNA nuclear export similarly, suggesting that it is common for cancer-associated SF3B1 mutations to inhibit mRNA nuclear export. Our research highlights the critical role of the THOC5–SF3B1 interaction in the regulation of mRNA nuclear export and provides valuable insights into the impact of SF3B1 mutations on mRNA nuclear export. [ABSTRACT FROM AUTHOR]

• Provide an in-depth analysis of 27 different histone modifications in H1 cell. • Causal relationships among histone modifications in alternative splicing event were inferred by using Bayesian network. • Distinct histone modification patterns associated with alternative splicing were reported. Alternative splicing is a crucial process of gene expression. Over 90% multi-exonic genes in human genome undergo alternative splicing. Although the splicing code has been proposed, it still couldn't satisfactorily explain the tissue-specific alternative splicing. Results of co-transcriptional RNA processing analysis demonstrated that, except for trans- and cis-acting elements, histone modifications also play a role in alternative splicing. In the present work, we analyzed the associations among 27 kinds of histone modifications in H1 human embryonic stem cell. In order to illustrate the casual relationships between histone modification and alternative splicing, we built the Bayesian network and validated its robustness by using cross validation test. In addition to the combinatorial patterns, distinct histone modification patterns were also observed in the alternative spliced exons and surrounding intron regions, indicating that histone modifications could substantially mark alternative splicing. [ABSTRACT FROM AUTHOR]

Protein arginine methyltransferases (PRMTs) are important posttranslational modifying enzymes in eukaryotic proteins and regulate diverse pathways from gene transcription, RNA splicing, and signal transduction to metabolism. Increasing evidence supports that PRMTs exhibit the capacity to form higher-order oligomeric structures, but the structural basis of PRMT oligomerization and its functional consequence are elusive. Herein, we revealed for the first time different oligomeric structural forms of the predominant arginine methyltransferase PRMT1 using cryo-EM, which included tetramer (dimer of dimers), hexamer (trimer of dimers), octamer (tetramer of dimers), decamer (pentamer of dimers), and also helical filaments. Through a host of biochemical assays, we showed that PRMT1 methyltransferase activity was substantially enhanced as a result of the high-ordered oligomerization. High-ordered oligomerization increased the catalytic turnover and the multimethylation processivity of PRMT1. Presence of a catalytically dead PRMT1 mutant also enhanced the activity of WT PRMT1, pointing out a noncatalytic role of oligomerization. Structural modeling demonstrates that oligomerization enhances substrate retention at the PRMT1 surface through electrostatic force. Our studies offered key insights into PRMT1 oligomerization and established that oligomerization constitutes a novel molecular mechanism that positively regulates the enzymatic activity of PRMTs in biology. [ABSTRACT FROM AUTHOR]

Effective cancer immunotherapy relies on enhancing the host’s immune response, particularly by boosting T cell-mediated cytotoxicity against tumor cells. In this study, we identify CWF19-like cell cycle control factor 1 (CWF19L1) as a novel splicing regulator that enhances T cell-mediated cytotoxicity. CWF19L1 interacts prominently with key splicing factors within the nucleus, including components of the U5 small nuclear ribonucleoprotein and the pre-mRNA processing factor 19 (PRPF19) complex. Deficiency of CWF19L1 disrupts alternative splicing of immune-related genes, resulting in diminished expression of cytotoxic molecules. Furthermore, CWF19L1 plays a critical role in promoting T cell-mediated antitumor responses by upregulating the expression of effector cytokines. Our findings unveil previously undocumented functions of CWF19L1 in alternative splicing and its involvement in the regulation of antitumor immunity, highlighting its potential as a therapeutic target for novel cancer immunotherapies. [ABSTRACT FROM AUTHOR]

Background: It has been demonstrated that nintedanib can inhibit the proliferation of gastric cancer cells, but the specific mechanism of action is unclear. Objective: Investigating the changes of key factors involved in gene transcription and post-transcriptional regulation during the process of treating gastric cancer with nintedanib. Methods: In this study, we performed transcriptome sequencing on gastric cancer cell groups treated with nintedanib and control groups. The SUVA (Splice sites Usage Variation Analysis) software was used to identify differential alternative splicing (AS) events between the nintedanib-treated group and the control group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted to assess the functional differences and pathways associated with these events. Finally, a co-expression regulatory network of differentially expressed RNA-binding proteins (RBPs) and differentially spliced genes was established. Results: A total of 915 differential AS events were identified between the two groups, and these differential genes were closely related to the apoptosis pathway. Further analysis revealed that differential RBPs (TAGLN2, TAGLN, SRSF6, PKM, SRSF2, NOC2L, IPO4, C1QBP, DHX9) may affect the anti-proliferative effect of nintedanib on gastric cancer cells by regulating downstream genes involved in cell proliferation and angiogenesis (NR4A1, BBC3, IFI27) through alternative splicing. Conclusion: This study systematically identified important changes in alternative splicing and RNA-binding proteins during the process of nintedanib-induced apoptosis in gastric cancer cells. It innovatively revealed the mechanisms of action of nintedanib in gastric cancer cells and expanded the selection of new targets for gastric cancer treatment. [ABSTRACT FROM AUTHOR]

NAC (NAM, ATAF, and CUC) transcription factors are essential in regulating plant stress response and senescence, with their functions being modulated by alternative splicing. The molecular mechanisms of stress-induced premature flowering and drought tolerance in Phyllostachys edulis (moso bamboo) are not yet fully understood. In this study, a novel NAC variant derived from PheNAC23, named PheNAC23ES, was isolated. PheNAC23ES exhibited distinct expression patterns compared to PheNAC23 during leaf senescence and drought stress response. Overexpression of PheNAC23 promoted flowering and reduced its tolerance to drought stress in Arabidopsis thaliana (A. thaliana). However, overexpression of PheNAC23ES exhibited the opposite functions. PheNAC23 was localized in the nucleus and had transactivation activity, while PheNAC23ES had a similar localization to the control green fluorescent protein and no transactivation activity. Further functional analysis revealed that PheNAC23ES could interact with PheNAC23, suggesting that PheNAC23ES might serve as a small interfering peptide that affects the function of PheNAC23 by binding to it. [ABSTRACT FROM AUTHOR]

Thousands of genes are activated in late two-cell embryos, which means that numerous pre-mRNAs are generated during this time. These pre-mRNAs must be accurately spliced to ensure that the mature mRNAs are translated into functional proteins. However, little is known about the roles of pre-mRNA splicing and the cellular factors modulating pre-mRNA splicing during early embryonic development. Here, we report that downregulation of SON, a large Ser/Arg (SR)-related protein, reduced embryonic development and caused deficient blastomere cleavage. These embryonic developmental defects result from dysregulated nuclear speckle organization and premRNA splicing of a set of cell cycle-related genes. Furthermore, SON downregulation disrupted the transcriptome (2128 upregulated and 1399 downregulated) in four-cell embryos. Increased H3K4me3, H3K9me3, and H3K27me3 levels were detected in four-cell embryos after SON downregulation. Taken together, these results demonstrate that accurate pre-mRNA splicing is essential for early embryonic development and that SON plays important roles in nuclear speckle organization, pre-mRNA splicing, transcriptome establishment, and histone methylation reprogramming during early embryonic development. [ABSTRACT FROM AUTHOR]

Jusvinza is an immunomodulatory drug composed of an altered peptide ligand (APL) designed from a novel CD4+ T cell epitope of human heat shock protein 60 (HSP60), an autoantigen involved in the pathogenesis of rheumatoid arthritis (RA). The peptide induces regulatory T cells and decreases levels of TNF-α and IL-17; pre-clinical and phase I clinical studies support its use for the treatment of RA. This peptide was repositioned for the treatment of COVID-19 patients with signs of hyperinflammation. Neutrophils play a pathogenic role in both RA and severe forms of COVID-19. To add novel evidence about the mechanism of action of Jusvinza, the proteomic profile regulated by this peptide of neutrophils isolated from four RA patients was investigated using LC-MS/MS and bioinformatics analysis. A total of 149 proteins were found to be differentially modulated in neutrophils treated with Jusvinza. The proteomic profile regulated by Jusvinza is characterized by the presence of proteins related to RNA splicing, phagocytosis, endocytosis, and immune functions. In response to Jusvinza treatment, several proteins that regulate the NF-κB signaling pathway were differentially modulated, supporting the peptide's anti-inflammatory effect. Proteins related to metabolic pathways that supply ATP for cellular functions or lipid metabolites with immunoregulatory properties were also identified. Additionally, several structural components of neutrophil extracellular traps (NETs) were decreased in Jusvinza-treated cells, supporting its impairment of this biological process. Of note, these findings were validated by in vitro experiments which confirmed that Jusvinza decreased NET formation. Such results provide evidence of the molecular mechanism of action and support the therapeutic potentialities of Jusvinza to treat other diseases characterized by hyperinflammation besides RA and COVID-19. [ABSTRACT FROM AUTHOR]

In recent years, advances in biomedicine have revealed an important role for post-transcriptional mechanisms of gene expression regulation in pathologic conditions. In cancer in general and leukaemia specifically, RNA binding proteins have emerged as important regulator of RNA homoeostasis that are often dysregulated in the disease state. Having established the importance of these pathogenetic mechanisms, there have been a number of efforts to target RNA binding proteins using oligonucleotide-based strategies, as well as with small organic molecules. The field is at an exciting inflection point with the convergence of biomedical knowledge, small molecule screening strategies and improved chemical methods for synthesis and construction of sophisticated small molecules. Here, we review the mechanisms of post-transcriptional gene regulation, specifically in leukaemia, current small-molecule based efforts to target RNA binding proteins, and future prospects.

Nuclear clearance and cytoplasmic accumulations of the RNA-binding protein TDP-43 are pathological hallmarks in almost all patients with amyotrophic lateral sclerosis (ALS) and up to 50% of patients with frontotemporal dementia (FTD) and Alzheimers disease. In Alzheimers disease, TDP-43 pathology is predominantly observed in the limbic system and correlates with cognitive decline and reduced hippocampal volume. Disruption of nuclear TDP-43 function leads to abnormal RNA splicing and incorporation of erroneous cryptic exons in numerous transcripts including Stathmin-2 (STMN2, also known as SCG10) and UNC13A, recently reported in tissues from patients with ALS and FTD. Here, we identify both STMN2 and UNC13A cryptic exons in Alzheimers disease patients, that correlate with TDP-43 pathology burden, but not with amyloid-β or tau deposits. We also demonstrate that processing of the STMN2 pre-mRNA is more sensitive to TDP-43 loss of function than UNC13A. In addition, full-length RNAs encoding STMN2 and UNC13A are suppressed in large RNA-seq datasets generated from Alzheimers disease post-mortem brain tissue. Collectively, these results open exciting new avenues to use STMN2 and UNC13A as potential therapeutic targets in a broad range of neurodegenerative conditions with TDP-43 proteinopathy including Alzheimers disease.

BackgroundRNA-seq has brought forth significant discoveries regarding aberrations in RNA processing, implicating these RNA variants in a variety of diseases. Aberrant splicing and single nucleotide variants (SNVs) in RNA have been demonstrated to alter transcript stability, localization, and function. In particular, the upregulation of ADAR, an enzyme that mediates adenosine-to-inosine editing, has been previously linked to an increase in the invasiveness of lung adenocarcinoma cells and associated with splicing regulation. Despite the functional importance of studying splicing and SNVs, the use of short-read RNA-seq has limited the community's ability to interrogate both forms of RNA variation simultaneously.ResultsWe employ long-read sequencing technology to obtain full-length transcript sequences, elucidating cis-effects of variants on splicing changes at a single molecule level. We develop a computational workflow that augments FLAIR, a tool that calls isoform models expressed in long-read data, to integrate RNA variant calls with the associated isoforms that bear them. We generate nanopore data with high sequence accuracy from H1975 lung adenocarcinoma cells with and without knockdown of ADAR. We apply our workflow to identify key inosine isoform associations to help clarify the prominence of ADAR in tumorigenesis.ConclusionsUltimately, we find that a long-read approach provides valuable insight toward characterizing the relationship between RNA variants and splicing patterns.

Branching is a critical step in RNA splicing that is essential for 5 splice site selection. Recent spliceosome structures have led to competing models for the recognition of the invariant adenosine at the branch point. However, there are no structures of any splicing complex with the adenosine nucleophile docked in the active site and positioned to attack the 5 splice site. Thus we lack a mechanistic understanding of adenosine selection and splice site recognition during RNA splicing. Here we present a cryo-electron microscopy structure of a group II intron that reveals that active site dynamics are coupled to the formation of a base triple within the branch-site helix that positions the 2-OH of the adenosine for nucleophilic attack on the 5 scissile phosphate. This structure, complemented with biochemistry and comparative analyses to splicing complexes, supports a base triple model of adenosine recognition for branching within group II introns and the evolutionarily related spliceosome.

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disease characterized by loss of motor neurons. Human genetic studies have linked mutations in RNA-binding proteins as causative for this disease. The hnRNPA1 protein, a known pre-mRNA splicing factor, is mutated in some ALS patients. Here, two human cell models were generated to investigate how a mutation in the C-terminal low-complexity domain (LCD) of hnRNPA1 can cause splicing changes of thousands of transcripts that collectively are linked to the DNA damage response, cilium organization, and translation. We show that the hnRNPA1 D262V mutant protein binds to new binding sites on differentially spliced transcripts from genes that are linked to ALS. We demonstrate that this ALS-linked hnRNPA1 mutation alters normal RNA-dependent protein-protein interactions. Furthermore, cells expressing this hnRNPA1 mutant exhibit a cell aggregation phenotype, markedly reduced growth rates, changes in stress granule kinetics, and aberrant growth of neuronal processes. This study provides insight into how a single amino acid mutation in a splicing factor can alter RNA splicing networks of genes linked to ALS.

In recent years, advances in biomedicine have revealed an important role for post-transcriptional mechanisms of gene expression regulation in pathologic conditions. In cancer in general and leukaemia specifically, RNA binding proteins have emerged as important regulator of RNA homoeostasis that are often dysregulated in the disease state. Having established the importance of these pathogenetic mechanisms, there have been a number of efforts to target RNA binding proteins using oligonucleotide-based strategies, as well as with small organic molecules. The field is at an exciting inflection point with the convergence of biomedical knowledge, small molecule screening strategies and improved chemical methods for synthesis and construction of sophisticated small molecules. Here, we review the mechanisms of post-transcriptional gene regulation, specifically in leukaemia, current small-molecule based efforts to target RNA binding proteins, and future prospects.

The fidelity of splice site selection is critical for proper gene expression. In particular, proper recognition of 3-splice site (3SS) sequences by the spliceosome is challenging considering the low complexity of the 3SS consensus sequence YAG. Here, we show that absence of the Prp18p splicing factor results in genome-wide activation of alternative 3SS in S. cerevisiae, including highly unusual non-YAG sequences. Usage of these non-canonical 3SS in the absence of Prp18p is enhanced by upstream poly(U) tracts and by their potential to interact with the first intronic nucleoside, allowing them to dock in the spliceosome active site instead of the normal 3SS. The role of Prp18p in 3SS fidelity is facilitated by interactions with Slu7p and Prp8p, but cannot be fulfilled by Slu7p, identifying a unique role for Prp18p in 3SS fidelity. This fidelity function is synergized by the downstream proofreading activity of the Prp22p helicase, but is independent from another late splicing helicase, Prp43p. Our results show that spliceosomes exhibit remarkably relaxed 3SS sequence usage in the absence of Prp18p and identify a network of spliceosomal interactions centered on Prp18p which are required to promote the fidelity of the recognition of consensus 3SS sequences.

BACKGROUND: Previous studies found that the proliferative scar-specific long non-coding RNA lncRNA HSFAS is a novel biomarker that can be used in the diagnosis of hypertrophic scar, but how it functions in hypertrophic scar is not clear. OBJECTIVE: To investigate the role and mechanism of lncRNA HSFAS in hypertrophic scar. METHODS: Fresh scar tissue and surrounding normal skin tissue samples from three patients with hypertrophic scar were collected, and tissue immunofluorescence was used to detect the expression of lncRNA HSFAS in frozen sections of two skin tissues. Primary fibroblasts were isolated from proliferative scarred skin tissue and normal skin tissue and cultured by enzyme digestion method. Quantitative real-time PCR was used to detect the mRNA expression of lncRNA HSFAS in cells. The proteins bound to lncRNA HSFAS were detected by RNA pull-down combined mass spectrometry. GO and KEGG were used to analyze the main functions and pathways of lncRNA HSFAS involved in hypertrophic scar progression. The targeted binding of lncRNA HSFAS to proteins was determined by catRAPID and RPISeq website analysis. RESULTS AND CONCLUSION: Compared with normal skin tissue and fibroblasts from normal skin tissue, the expression of lncRNA HSFAS in human hypertrophic scar tissue and primary fibroblasts from hypertrophic scar tissue was significantly increased (P < 0.05). There were 510 proteins clearly bound to lncRNA HSFAS by RNA pull-down combined mass spectrometry. The results of GO and KEGG analyses showed that these proteins were mainly involved in RNA splicing and processing, chromosome synthesis and separation, and cell cycle. Among them, the proteins involved in RNA splicing and processing included scaffold attachment factor B2 and DICER1, and the binding fraction with lncRNA HSFAS was higher. The results of bioinformatics analysis showed that lncRNA HSFAS was bound to scaffold attachment factor B2 and DICER1 proteins. To conclude, lncRNA HSFAS may affect gene expression by interacting with scaffold attachment factor B2 and DICER1 proteins to regulate RNA splicing and processing modification, thus promoting the occurrence and development of hypertrophic scar. [ABSTRACT FROM AUTHOR]

Abstract Lynch syndrome (LS) is one of the most common hereditary cancer syndrome in human populations, associated with germline variants in MLH1, MSH2/EPCAM, MSH6 and PMS2 genes. The advent of next generation sequencing has proven a significant impact in germline variant detection in the causative genes; however, a large proportion of patients with clinical criteria still receive uncertain or negative results. PMS2 is the least frequent reported gene, associated with up to 15% of LS cases with late-onset disease and low penetrance phenotype; however, the proportion of PMS2-LS cases is considered to be highly underestimated. In this context, our analysis aimed to improve the current diagnostic yield by focusing on missense and intronic PMS2 variants available in public clinical databases (ClinVar, LOVD). We performed an in silico assessment of the wild-type DNA sequence and the reported genetic variants, employing splicing bioinformatics tools known for their effectiveness in other genes. Splicing variants were predicted in silico and using GTEx short-read RNA expression data. Out of the 2384 missense variants discovered, 90% were classified with uncertain significance (VUS). 4.9% of missense variants were shown to have a potential splicing consequence (DS > 0.2) using SpliceAI. As described in the original publication, SpliceAI-visual was proven effective in annotation of short intronic variants ( 0.1). Exons 2, 3, 4, 5, 6, 7, 8, 11, 12 and 14 were consistently predicted in at least three out of eight software with weak canonical splice sites. Additionally, we noted that both Exonic Splicing Enhancers (ESEs) and Exonic Splicing Silencers (ESSs) contribute significantly to alternative splicing and exonic selection in PMS2 gene. Specifically, ESE motifs were consistently more abundant in highly expressed exons 5, 11 and 14, while ESS motifs played a fundamental role in exons 6, 7 and 10. Computational analysis performed in our study serves as a valuable filtering step for guiding further RNA experiments. Additional functional data is necessary to validate our findings.

Abstract Background Congenital heart disease (CHD) is the most common congenital anomaly. Almost 90% of isolated cases have an unexplained genetic etiology after clinical testing. Non-canonical splice variants that disrupt mRNA splicing through the loss or creation of exon boundaries are not routinely captured and/or evaluated by standard clinical genetic tests. Recent computational algorithms such as SpliceAI have shown an ability to predict such variants, but are not specific to cardiac-expressed genes and transcriptional isoforms. Methods We used genome sequencing (GS) (n = 1101 CHD probands) and myocardial RNA-Sequencing (RNA-Seq) (n = 154 CHD and n = 43 cardiomyopathy probands) to identify and validate splice disrupting variants, and to develop a heart-specific model for canonical and non-canonical splice variants that can be applied to patients with CHD and cardiomyopathy. Two thousand five hundred seventy GS samples from the Medical Genome Reference Bank were analyzed as healthy controls. Results Of 8583 rare DNA splice-disrupting variants initially identified using SpliceAI, 100 were associated with altered splice junctions in the corresponding patient myocardium affecting 95 genes. Using strength of myocardial gene expression and genome-wide DNA variant features that were confirmed to affect splicing in myocardial RNA, we trained a machine learning model for predicting cardiac-specific splice-disrupting variants (AUC 0.86 on internal validation). In a validation set of 48 CHD probands, the cardiac-specific model outperformed a SpliceAI model alone (AUC 0.94 vs 0.67 respectively). Application of this model to an additional 947 CHD probands with only GS data identified 1% patients with canonical and 11% patients with non-canonical splice-disrupting variants in CHD genes. Forty-nine percent of predicted splice-disrupting variants were intronic and > 10 bp from existing splice junctions. The burden of high-confidence splice-disrupting variants in CHD genes was 1.28-fold higher in CHD cases compared with healthy controls. Conclusions A new cardiac-specific in silico model was developed using complementary GS and RNA-Seq data that improved genetic yield by identifying a significant burden of non-canonical splice variants associated with CHD that would not be detectable through panel or exome sequencing.