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The Epstein-Barr virus (EBV) infects over 95% of adults, establishing lifelong latency and contributing to the development of various malignancies, including Burkitt lymphoma and nasopharyngeal carcinoma. However, the RNA structures regulating the splicing of the critical EBV gene, latent membrane protein 1 (LMP1), remain uncharacterized. To identify these regulatory elements, we applied spliceosome inhibition with RNA probing and sequencing (SIRP-seq) to the BJAB-B1 cell line. By utilizing the spliceosome inhibitor pladienolide B, we enriched pre-mRNA species, enabling the detection of structural features within both the full-length pre-mRNA (LMP1-FL) and an alternatively spliced isoform retaining intron 2 (LMP1-AS). The resulting chemical probing datasets informed the RNA folding algorithms RNAfold and ScanFold to generate the first high-resolution secondary structure models for the LMP1 pre-mRNA, encompassing both exonic and intronic regions. Our results identify 11 novel, thermodynamically stable RNA structures, with several key elements positioned near splice junctions. Notably, three structures (Structures 8, 9, and 10) were identified near the 3' splice site of intron 2, appearing in alternative conformations that may influence splicing accessibility. Furthermore, these structures map to regions containing disease-relevant mutations associated with patient survival in Burkitt lymphoma. This structural framework provides new insights into how LMP1 splicing may be regulated by RNA structure and identifies potential novel therapeutic targets for mitigating EBV-associated diseases.

Pre-mRNA splicing is carried out by the spliceosome, a large and dynamic ribonucleoprotein complex. The spliceosome is known to be stored in nuclear speckles (NS), which are recognized as active subnuclear organelles for splicing. However, it remains poorly understood how spliceosomal protein-protein interactions are functionally coupled to NS organization to maintain splicing robustness in plants. Here, we report the functional significance of a specific interaction between two U4/U6·U5 tri-snRNP components of the spliceosome, STA1 and DOT2, in regulating NS organization, pre-mRNA splicing, and heat stress responses in Arabidopsis. We identified a missense mutation in DOT2 (a Snu66/SART1 homolog) from a genetic suppressor of the PRP6 homolog mutant sta1-1 (named S307). This mutation restored the weakened interaction between STA1 and DOT2 in the sta1-1 mutant background. Genetic, biochemical, and cell biological analyses showed that variation in the strength of the STA1-DOT2 interaction was closely associated with changes in NS formation, splicing efficiency, transcript abundance, as well as growth and heat tolerance. Pharmacological inhibition of STA1-associated NS formation by tubercidin recapitulated sta1-1-like phenotypes and splicing defects, supporting a functional link between NS organization and splicing outcomes. In addition, heat-induced weakening of the STA1-DOT2 interaction was accompanied by reduced NS formation and increased intron retention at the transcriptome-wide level including key heat-responsive transcripts. Based on these observations, we propose that the STA1-DOT2 interaction, likely reflecting the assembly state of the U4/U6·U5 tri-snRNP, may represent one of the heat-sensitive interaction nodes that couple spliceosome assembly to NS organization and splicing robustness under stress conditions.

RNA splicing is a fundamental feature of eukaryotic gene expression that is co-opted by many nuclear-replicating viruses to produce viral transcripts. Recent clinical development of spliceosome-targeting therapeutics has demonstrated that splicing can be safely modulated in vivo, raising the possibility that viral dependence on host RNA processing may represent an exploitable antiviral vulnerability. Here, we show that pharmacologic inhibition of cellular RNA splicing preferentially suppresses the expression of spliced viral transcripts and profoundly impairs replication of multiple splicing-dependent viruses. In human adenovirus, low-dose inhibition of the spliceosome using mechanistically distinct chemical inhibitors, or genetic depletion of SF3B1, disrupted splicing of complex late viral transcripts while largely sparing simple early transcripts. This resulted in impaired viral DNA replication, reduced late protein accumulation, and multi-log decreases in infectious progeny production. Splicing inhibitors similarly impaired RNA splicing and viral replication of DNA virus Herpes Simplex virus 1 and RNA virus influenza A, which both rely on host-dependent RNA processing strategies. In contrast, cytoplasmic RNA viruses lacking spliceosome dependence were unaffected, supporting an on-target mechanism. Together, these findings identify viral RNA processing complexity as a determinant of antiviral sensitivity and establish host spliceosome dependence as a shared and targetable vulnerability across diverse viral families.

Spliceosomal small nuclear RNA (snRNA) U1 and the U2AF heterodimer play critical functions by recognizing the highly conserved GT and AG dinucleotides, respectively, located at the start and at the end of introns. Here, we explore how changing these components contributed to maintaining splicing function in genomes where 95% of introns escape the GT/AG rule. By gaining access to new tunicate genomes, we could reveal that the emergence of non-canonical introns in the Fritillaria borealis lineage coincides with the duplication of U2AF subunits. Our findings indicate that paralogs U2AF1α and U2AF2α have preserved conserved functions, while divergent paralogs U2AF1β and U2AF2β form novel heterodimers that recognize introns with non-canonical 3' ends. The conserved m6A present on U6 snRNA has been considerably reduced in F. borealis, but its U1 snRNA retains a stable 5'-terminal m6A, which is typically suppressed in humans and other chordates. We propose that this unique m6A pattern stabilizes the binding of snRNA to non-canonical 5' splice sites. Although the core components of the spliceosome remain preserved, functional changes implemented through gene duplication and post-transcriptional modifications can significantly broaden the range of target splice sites.

Intellectual disability (ID) affects approximately 1%-3% of the population and spans diverse clinical presentations with marked genetic heterogeneity, especially in consanguineous populations where autosomal-recessive ID is common. Despite advances in diagnostic methods, ~50% of individuals with ID remain without a molecular diagnosis. Genome sequencing (GS) can detect variant classes poorly captured by other methodologies, including deep intronic splice changes and structural variants. We performed short-read GS on 38 Iranian autosomal recessive intellectual disability (ARID) families that remained unsolved after exome sequencing (ES) and two phases of reanalysis. Sequencing and variant calling were performed on DRAGEN Bio-IT Platform with GRCh38 and variants were annotated in Golden helix Varseq software and the AnnotSV tool. GS yielded diagnoses in 2 of 38 families (5.3%), identifying a homozygous deep-intronic ATP8A2 variant (c.1473 + 519C > T) that creates a cryptic donor site and a 57-bp pseudoexon, and a homozygous ~103-kb CNTNAP2 deletion removing the in-frame Exon 2. Additionally, GS uncovered a homozygous NCOR1 missense variant that represents a novel candidate gene for ARID, but further studies are required to confirm the gene-disease association. The ATP8A2 and CNTNAP2 variants were uniquely detectable by GS. In conclusion, in a challenging, ES-negative ARID cohort, GS provided an additional ~5.3% diagnostic yield by uncovering a noncoding splice alteration and a large intragenic deletion. These results underscore GS as a valuable, though still modest, tool over ES and highlight significant interpretation challenges in noncoding regions. Continued advances in functional assays and complementary long-read technologies will be essential to further reduce the diagnostic gap in unresolved ID.

Drug resistance, defined as the adaptive tolerance of human diseases and agricultural pests to therapeutic or chemical agents, threatens global health and food security. Alternative splicing (AS), a central posttranscriptional regulatory mechanism, is increasingly recognized as a conserved driver of resistance evolution. However, although AS-mediated resistance has been widely studied in cancer, its role in pesticide and fungicide resistance remains insufficiently explored, limiting cross-disciplinary reuse of mechanistic insights and intervention strategies. This review critically compares the spliceosome-mediated and AS-mediated mechanisms that underlie resistance to anticancer drugs, pesticides and fungicides within a unified framework, with the goal of clarifying how distinct selective pressures shape convergent adaptive resistance across biological kingdoms. We summarize how aberrant AS promotes resistance through target truncation, altered drug metabolism, apoptosis evasion, and rewiring of survival signaling. We further propose a customized Design-Build-Test-Learn (DBTL) roadmap for AS-targeted intervention, integrating long-read sequencing, AI-assisted prediction, splice-modulating therapeutics such as ASOs and CRISPR-based approaches, nanodelivery optimization, and dynamic resistance monitoring. By linking mechanistic comparison with translational implementation, this review provides an interdisciplinary framework for understanding, predicting, and reversing AS-mediated resistance in both clinical and agricultural settings.

To explore the clinical phenotype and genetic etiology for a Chinese pedigree affected with Hereditary coagulation factor Ⅴ deficiency (FⅤD). A 47-year-old female who visited the First Affiliated Hospital of Wenzhou Medical University in July 2025 and her family members (husband, mother, younger brother and younger brother's wife, elder sister, second elder sister, daughter, son, elder niece, and second niece) were selected as study subjects. Clinical data and family history were retrospectively collected. Peripheral blood samples were collected from the proband and her family members. Prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), coagulation factor Ⅱ activity (FⅡ:C), coagulation factor Ⅶ activity (FⅦ:C), coagulation factor Ⅷ activity (FⅧ:C), coagulation factor Ⅹ activity (FⅩ:C), and FⅤ activity (FⅤ:C) were measured in all participants using the one-stage clotting assay. FⅤ antigen (FⅤ:Ag) levels in plasma were determined with enzyme-linked immunosorbent assay (ELISA). Genomic DNA was extracted from peripheral blood samples of all participants, and the coding regions of the F5 gene were amplified by PCR and subjected to Sanger sequencing. Candidate variants were verified with multiple online tools to predict their pathogenicity and impact on splicing. Based on guidelines formulated by the American College of Medical Genetics and Genomics (ACMG), the pathogenicity of candidate variants was evaluated. Coagulation function of all participants was evaluated using thrombin generation assay and thromboelastography. This study was approved by the Medical Ethics Committee of the hospital (Ethics No.: KY2022-R193). The proband's peripheral blood PT and APTT were 25.6 s and 58.3 s, respectively, while FⅤ:C and FⅤ:Ag were 7% and 6%, respectively, which was in keeping with a phenotype of type I FⅤD. Her brother also showed significantly decreased FⅤ:C and FⅤ:Ag levels. The proband's mother, eldest sister, second sister, daughter, son, eldest niece, second niece had FⅤ:C and FⅤ:Ag approximately 50% of those of normal controls, whereas her husband and sister-in-law showed no obvious abnormality in such indices. Genetic testing revealed that the proband and her brother both harbored compound heterozygous variants of the F5 gene, namely c.6528+3A>T (IVS24+3A>T) and c.6665A>G (p.Asp2222Gly), for which other family members with reduced FⅤ:C and FⅤ:Ag were heterozygous carriers, and those with normal coagulation indices were of the wild-type. Bioinformatics analyses using multiple software indicated that the IVS24+3A>T variant is located at a splice donor site and can significantly weaken the strength of the splice site, potentially leading to exon skipping. Based on the ACMG guidelines, the novel variant is classified as likely pathogenic (PM2_Moderate+PM3_Moderate+PP1_Supporting+PP3_Supporting+PP4_Supporting). Results of thrombin generation assay indicated reduced thrombin generation capacity in the proband, whilst thromboelastography results showed abnormal coagulation characteristics manifested as prolonged coagulation initiation time. Ultimately, both the proband and her younger brother were diagnosed with hereditary type I FⅤD. The IVS24+3A>T and p.Asp2222Gly compound heterozygous variants of the F5 gene probably underlay the pathogenesis of FⅤD in this pedigree. Above finding has enriched the mutational spectrum of the F5 gene and provided a basis for genetic counseling and molecular diagnosis.

Hereditary cancers arise from germline pathogenic variants that confer increased lifetime cancer risk. In Palestine, the genetic basis of hereditary cancer predisposition remains limited. The objective of this study is to investigate germline variants associated with hereditary cancer susceptibility in Palestinian families with strong cancer history. Families with suspected hereditary breast cancer were recruited, and germline DNA extracted from peripheral blood was first analyzed using a BRCA1/2 panel. Whole-exome sequencing (WES) was subsequently performed in BRCA-negative probands to identify additional candidate hereditary variants. A total of 34 individuals from three unrelated families were included in the study, comprising 8 affected and 26 unaffected individuals. Segregation analysis and in silico functional assessment were conducted to evaluate variant pathogenicity. We identified a splice-site variant in RAD50 (c.2524+3A>G) in Family I, a truncating MSH4 variant (c.328C>T; p.R110X) and a missense STAT6 variant (c.1216C>G; p.L406V) in Family II, and a splice-region PRKAR1A variant (c.973+6T>C) in Family III. These variants are segregated with disease in the respective families and are predicted to affect protein function. Our findings highlight the importance of germline genomic analysis in characterizing hereditary cancer predisposition in underrepresented populations and provide preliminary data for future risk assessment and genetic counseling strategies in Palestine.

Prostate cancer (PCa) represents a hormone-dependent malignancy where androgen receptor (AR) signaling plays a central role in disease initiation, progression, and therapeutic resistance. Recent advances have revealed that long non-coding RNAs (lncRNAs) constitute a critical regulatory layer in prostate cancer, with implications for endocrine signaling pathways. lncRNAs orchestrate complex gene regulatory networks through diverse molecular mechanisms including chromatin remodeling, AR splice variant regulation, competitive endogenous RNA networks, translational control, and metabolic reprogramming. In castration-resistant prostate cancer, dysregulated lncRNAs contribute to resistance against androgen deprivation therapy and next-generation AR antagonists such as enzalutamide. This review synthesizes current knowledge on lncRNA biology in PCa, emphasizing lncRNA relationships with AR signaling and endocrine resistance mechanisms. We discuss key lncRNAs that modulate AR activity, metabolic adaptation, and lineage plasticity. Additionally, we examine structure-function relationships that enable rational therapeutic design, lncRNA roles in bone metastasis and neuroendocrine differentiation, and lncRNA clinical utility as biomarkers for disease progression and treatment stratification. Therapeutic strategies include antisense oligonucleotides, small-molecule inhibitors of lncRNA-protein interaction disruptors, and combination approaches with DNA-damaging agents and AR inhibitors. Understanding lncRNA-mediated endocrine regulation provides insights into prostate cancer biology and offers avenues for overcoming therapeutic resistance in advanced disease.

Circular RNAs (circRNAs) were first identified approximately 50 years ago in pathogenic viroids as single-stranded, covalently closed RNA molecules. Initially considered by-products of splicing, circRNAs are now recognised as an important class of regulatory RNAs involved in microRNA sponging, RNA-protein interactions, and cellular pathways. Their closed-loop structure, generated through backsplicing, confers resistance to exonucleolytic degradation and contributes to their stability. Owing to their tissue- and disease-specific expression, circRNAs have emerged as promising biomarkers for cancer, neurodegenerative disorders, and cardiovascular disease. Over the past decade, numerous bioinformatics tools utilising RNA-sequencing (RNA-seq) data have been developed for circRNA detection and analysis. Detection methods have evolved from manual split-read inspection to automated identification of the back spliced junction, while annotation pipelines now resolve the genomic origins and structural characteristics of circRNAs. Because individual circRNA callers vary considerably in sensitivity and specificity, a combined usage of tools in circRNA detection has become the preferred strategy for generating high-confidence datasets. Beyond their non-coding functions, increasing evidence suggests that some circRNAs possess protein-coding potential through open reading frames, cap-independent translation mechanisms, internal ribosome entry sites (IRESs), and N6-methyladenosine modifications. A new generation of bioinformatic tools can now assess the protein-coding potential of circRNAs, integrating the above features, as well as machine learning and deep learning approaches refining these predictions. This review summarises recently developed short-read RNA-seq bioinformatics tools for circRNA detection, consensus calling, annotation, and protein-coding potential prediction, with a particular focus on advances from the past five years that facilitate the identification of translatable circRNAs.

TRPV4 is a non-selective cation channel of the TRPV family and plays a key role in fibrosis, but its pathological mechanisms in genetically susceptible individuals remain unclear. This study aimed to investigate the potential role of the Trpv4 c.1491+1G>A splice-site mutation in pulmonary fibrosis using a gene-edited mouse model. The mutation was identified in a family with autosomal dominant familial digital arthropathy-brachydactyly (FDAB). A corresponding gene-edited mouse model was generated using CRISPR/Cas9 technology. Histopathological analysis, single-cell RNA sequencing (scRNA-seq), qPCR, Western blot, and immunofluorescence co-staining were employed to assess phenotypic, transcriptomic, and molecular changes in the lungs. The model mice exhibited skeletal abnormalities and multi-organ damage, with pronounced pulmonary fibrosis. In the lung tissues of homozygous mutant (Trpv4-Hom) mice, wild-type Trpv4 expression was significantly reduced, accompanied by thickened alveolar septa, pulmonary congestion, and increased collagen deposition. scRNA-seq revealed a decrease in the proportions of alveolar macrophages and NK cells, while Clara cells, fibroblasts, mesothelial cells, and alveolar type II cells increased. The ALCAM-CD6 and MIF-CD74 signaling axes were significantly upregulated. qPCR confirmed the transcriptional upregulation of Alcam, Cd6, Cd74, and Mif; Western blot further validated the increased protein levels of ALCAM, CD6, and CD74; and immunofluorescence confirmed the enhanced co-localization of MIF and CD74 in lung tissue. The Trpv4 c.1491+1G>A mutation is associated with exacerbated pulmonary fibrosis, altered lung cellular composition, disrupted ALCAM-CD6 immune regulatory pathways and MIF signaling homeostasis, and enhanced pro-fibrotic cell communication. These findings provide novel molecular targets for the development of anti-fibrotic therapies targeting this pathway.

This study investigated genetic determinants of the pharmacokinetics of the CYP2C8 index drugs repaglinide and gemfibrozil, and their interaction in healthy participants. Sequencing data from a study with montelukast revealed a novel functional CYP2C8 allele (rs2071426, CYP2C8*19), predicted to create an intronic splice donor site. In human liver samples, CYP2C8*19 associated with transcript-specific changes in CYP2C8 mRNA expression, reduced CYP2C8 protein expression, and decreased enzyme activity. Consistently, participants with the CYP2C8*19/*19 genotype had 45% greater area under the plasma repaglinide concentration-time curve from time zero to infinity (AUC0-&#x221e;) than participants with CYP2C8*1/*1 (P&#x2009;=&#x2009;1.6&#x2009;&#xd7;&#x2009;10-4). Participants with CYP2C8*1/*3 had 26% smaller AUC0-&#x221e; (P&#x2009;=&#x2009;0.0033) and those with CYP2C8*1/*4 had 51% greater AUC0-&#x221e; (P&#x2009;=&#x2009;8.2&#x2009;&#xd7;&#x2009;10-4). The fold increase in repaglinide AUC0-&#x221e; caused by gemfibrozil was 36% (P&#x2009;=&#x2009;1.3&#x2009;&#xd7;&#x2009;10-4) smaller in CYP2C8*19/*19 participants than in CYP2C8*1/*1 participants. In a genome-wide association study (GWAS), SLCO1B1 c.521&#x2009;T>C (rs4149056) associated with increased repaglinide AUC0-&#x221e; (P&#x2009;=&#x2009;4.5&#x2009;&#xd7;&#x2009;10-15; n&#x2009;=&#x2009;172) and SLCO1A2 variants associated with decreased AUC0-&#x221e; (P&#x2009;<&#x2009;10-8). In a GWAS of repaglinide after gemfibrozil pretreatment, SLCO1C1 variants associated with decreased AUC0-&#x221e; (P&#x2009;<&#x2009;1.6&#x2009;&#xd7;&#x2009;10-8; n&#x2009;=&#x2009;66). Participants with the poor function SLCO1B1 genotype showed a 32% smaller fold increase in repaglinide AUC0-&#x221e; following gemfibrozil than participants with the normal function SLCO1B1 genotype (P&#x2009;=&#x2009;0.0045). This study characterizes CYP2C8*19 as a novel decreased function allele and shows that CYP2C8 and SLCO1B1 genotypes affect the gemfibrozil-repaglinide interaction.

Normal-tension glaucoma (NTG) is an age-related cause of irreversible vision loss, yet the contribution of genetic variants to susceptibility across the lifespan remains unclear. The splice-site variant METTL23 c.84+60delAT (delAT), which induces aberrant exon skipping, has been implicated in NTG. We evaluated the age-dependent genetic risk associated with delAT by integrating age-stratified carrier frequency analyses in population datasets with age-specific case control association analyses in 3843 individuals. In the gnomAD database, the frequency of delAT carriers declined sharply with age, decreasing approximately sixfold in individuals aged &#x2265;40 years. Consistent with this finding, case-control analyses demonstrated increasing effect sizes with advancing age, with odds ratios of 1.84 in individuals aged &#x2265;40 years and 2.59 in those &#x2265;50 years (P&#x2009;=&#x2009;0.039). These complementary analyses provide convergent evidence that delAT confers an age-dependent increase in NTG risk, underscoring the importance of incorporating age into genetic analyses of late-onset disease.

With the rise of checkpoint blockade therapies and neoantigen-based vaccines reaching later-stage trials, there is a growing need for computational tools to identify and prioritize neoantigens. pVACtools, initially introduced in 2016, is an open-source informatic suite designed to support basic and translational neoantigen research. pVACtools assists prediction, prioritization, and visualization of neoantigens, as well as design of neoantigen-based therapies. We describe several major advances to pVACtools since the last update: (1) expanded neoantigen quality and safety assessment features, including support for peptide presentation scoring, immunogenicity prediction, anchor residue analysis, reference proteome similarity, percentile score calculation; (2) addition of pVACsplice, a new tool for predicting neoantigens from tumor-specific cis-splicing mutations; (3) addition of pVACbind, a flexible tool that supports noncanonical neoantigen sources; (4) improvement in neoantigen selection strategies; (5) a substantially improved pVACvector algorithm that achieves higher DNA/mRNA vector vaccine design success rates with shorter runtimes; (6) new utilities to support synthetic long peptide vaccine design; (7) extended prediction support for many non-human species; and (8) addition of pVACcompare, a tool to support comparison between two pVACseq results. Together, these updates reinforce pVACtools as the field's most comprehensive toolkit for neoantigen research, from basic discovery to the design and execution of personalized cancer vaccine clinical trials.