To explore the clinical phenotypes and genetic etiology of a child with MRXS34 syndrome due to a variant of NONO gene. A child patient who presented at Shanxi Provincial Maternity and Child Care Hospital on September 28, 2020 was selected as study subject. Clinical data of the child were retrospectively collected. Peripheral blood samples were collected from the child and his parents. Following extraction of genomic DNA, whole exome sequencing (WES) was carried out. Candidate variant was verified by Sanger sequencing of the family members. Pathogenicity of the variant was assessed based on guidelines from the American College of Medical Genetics and Genomics (ACMG). Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to detect the effect of the NONO gene variant on messenger RNA (mRNA) expression level in the proband, and complementary DNA (cDNA) sequencing was performed to validate the splicing patterns of the NONO gene variant in the proband. Using the keywords "NONO gene" "MRXS34" "developmental delay" "intellectual disability" and "congenital heart disease", a literature search was conducted in databases including the China National Knowledge Infrastructure(CNKI), Wanfang Data and PubMed databases to identify studies on the clinical and genotypic characteristics of children with MRXS34 caused by NONO gene variants. The search period was set from the inception of the databases to April 2025, and a comprehensive analysis of the findings from the identified studies was performed. This study was approved by the Medical Ethics Committee of the hospital (Ethics No.: IRB-KYHZ-2019-006). The proband, a 3-year-old male, exhibited global developmental delay, intellectual disability, facial dysmorphism, macrocephaly, corpus callosum dysgenesis, cavum septum pellucidum, atrial septal defect, tricuspid valve insufficiency with regurgitation, cryptorchidism, inguinal hernia, and anal cutaneous fistula. The results of WES and Sanger sequencing validation showed that the proband carried a heterozygous variant of the NONO gene c.577_581del (p.Val193fs), while both parents were wild-type, indicating that this variant was a de novo variant. According to the ACMG guidelines, this variant was classified as pathogenic (PVS1+PS2_Moderate+PM2_Supporting). RT-qPCR results showed that the relative mRNA expression level of the NONO gene in the proband was significantly lower than that in the control group of normal children, and cDNA sequencing results verified that the frameshift variant due to base deletion led to nonsense-mediated mRNA decay (NMD), causing premature termination of transcription without exon skipping at the splice site. Using the literature search strategy established in this study, a total of 15 studies on the clinical and genotypic characteristics of children with MRXS34 caused by NONO gene variants were identified, involving a total of 32 patients. Together with the proband of this study, a total of 33 patients were included in the comprehensive analysis. The results revealed a total of 23 types of variants involving the NONO gene. The main clinical manifestations of MRXS34 included developmental delay/intellectual disability (23/24, 95.8%), cardiovascular abnormalities (23/30, 76.7%), craniofacial/somatic malformations (21/24, 87.5%), and corpus callosum dysgenesis (16/21, 76.2%). The NONO gene variant probably underlay the pathogenesis of MRXS34 in this proband. The findings of this study has expanded of the variant and clinical spectra associated with the NONO gene.

Aberrant alternative polyadenylation (APA) and alternative splicing (AS) contribute to numerous diseases, including cancer; however, their coordinated roles in ovarian cancer remain poorly understood. Here, we investigated CPSF7, an APA factor markedly upregulated in ovarian cancer and associated with poor prognosis. Silencing CPSF7 suppressed proliferation, migration, and invasion of ovarian cancer cells, while antisense oligonucleotides (ASOs) targeting CPSF7 reduced tumor growth in a patient‑derived xenograft (PDX) model. Mechanistically, knockdown of the splicing factor SNRPD2 induced exon 4 skipping in CPSF7 pre‑mRNA. Loss of exon 4 disrupted the RNA recognition motif (RRM) domain essential for CPSF7‑mediated pre‑mRNA cleavage and polyadenylation, and introduced premature termination codons (PTCs) that generated noncoding transcripts subject to nonsense‑mediated decay (NMD), thereby reducing CPSF7 expression. Thus, efficient splicing mediated by SNRPD2 is crucial for sustaining high CPSF7 levels in ovarian cancer cells. Functional assays showed that CPSF7 knockdown reduced proliferation and metastatic potential in cells with elevated SNRPD2, suggesting that CPSF7 is a key mediator of SNRPD2-driven oncogenesis. Moreover, CPSF7 governed specific APA events to maintain transcript stability, with UBE2K identified as a critical downstream target. CPSF7 preferentially bound distal polyadenylation signals (PASs) within the predominant UBE2K transcript (UBE2K-201), thereby increasing its mRNA stability and maintaining high functional UBE2K expression. Collectively, these findings reveal that AS and APA are interconnected in ovarian cancer via the SNRPD2-CPSF7-UBE2K axis, which drives disease progression and represents a promising target for therapeutic intervention.

Antibody-secreting cells integrate efficient NMD with non‑canonical UPR signaling to maintain proteostasis and support massive immunoglobulin synthesis.

UPF3A and UPF3B shape the transcriptome cooperatively yet oppose cell function.

UPF1 at Work: Structural and Mechanistic Insights Into a Master Regulator of Nonsense-Mediated mRNA Decay.

Nonsense-Mediated Decay mRNA Quality Control System Is Essential for Root Development and Efficient Root Nodule Symbiosis in Medicago truncatula.

Targeting RNA quality-control defects in tauopathies: Pharmacodynamic biomarkers and therapeutic development.

De novo variants in LDB1 are linked to distinct neurodevelopmental phenotypes determined by variant location and differing pathomechanisms.

Somatic mutations in the gene encoding splicing factor 3B subunit 1 (SF3B1), a key component of the splicing machinery, have been described in patients with PRL-secreting pituitary neuroendocrine tumors (PRL-PitNET) and associated with aggressiveness and resistance to pharmacological therapy with dopamine agonists (DAs). Dopamine receptor type 2 (DRD2) represents the main target for PRL-PitNETs treatment with DAs, even if about 10% of patients is resistant. Aims of the study were to: 1) test the effects of SF3B1 inhibitor pladienolide B in tumoral lactotroph cells expressing wild-type or mutated SF3B1R625H; 2) investigate SF3B1 impact on tumoral cells responsiveness to DRD2 agonist cabergoline. Pladienolide B treatment reduced cell proliferation (-45.1(15.3)%,p<0.001), PRL secretion (-19.25(35.1)%,p<0.05) and increased apoptosis (+2.4(2.5)-fold,p<0.05) in rat tumoral MMQ cells. Pladienolide B antimitotic, proapoptotic and antisecretory effects were maintained in primary cultured cells from both resistant and responsive PRL-PitNETs. SF3B1R625H overexpression increased tumoral lactotrophs proliferation and migration. Moreover, the antimitotic efficacy of pladienolide B, but not of cabergoline, was maintained in MMQ cells transfected with SF3B1R625H. Cabergoline effects on cell proliferation, AKT activation, cyclin D3 and p27 were abolished in MMQ cells silenced for SF3B1. Accordingly, SF3B1R625H overexpression and SF3B1 silencing reduced DRD2 expression at both protein and transcript level, an effect reverted by nonsense-mediated decay inhibitor NMDI14. In conclusion, we demonstrated a relevant role of SF3B1 in PRL-PitNET. Indeed, SF3B1 inhibitor pladienolide B exerted antitumoral actions in PRL-PitNET cells bearing wild-type or mutated SF3B1. Moreover, both SF3B1R625H overexpression and SF3B1 genetic silencing reduced DRD2 expression and signaling.

RAPGEF6 is a member of the guanine nucleotide exchange factor (GEF) subfamily that acts on Rap small GTPases and contains a Ras/Rap-associating domain. Although deficiency of this gene has previously been linked to schizophrenia, no MIM phenotype entry currently associates RAPGEF6 with a defined clinical condition. In this study, trio-based whole-exome sequencing (WES) was performed in an individual presenting with psychiatric disorders and mild intellectual disability. WES revealed a de novo frameshift variant, c.272dup (p.Pro92Serfs*6), in the RAPGEF6 gene (NM_016340.6). This variant was classified as likely pathogenic according to ACMG criteria. Nonetheless, the contribution of additional genetic factors not detected by WES cannot be excluded. According to developmental transcriptomic data from the BrainSpan database, RAPGEF6 is expressed in the human brain across the entire lifespan and participates in neuron projection development, Rap-protein signal transduction, and regulation of GTPase activity. Structural variation data from DECIPHER further indicate that copy-number variants involving RAPGEF6 are primarily associated with intellectual disability and micrognathia. In addition, DECIPHER shows that RAPGEF6 is highly intolerant to loss-of-function (LoF) variants. Both NMD-Esc predictor and Mutation Taster suggest that the identified frameshift mutation is likely to trigger nonsense-mediated decay (NMD) of the RAPGEF6 transcript, resulting in loss of protein production. In addition, RAPGEF6 expression progressively increased during retinoic acid-induced neuronal differentiation of SK-N-BE neuroblastoma cells, supporting a potential role of this gene in neuronal maturation processes. Together, these data support a contributory role of RAPGEF6 haploinsufficiency in neurodevelopmental and psychiatric phenotypes, reinforcing its emerging relevance in neuropsychiatric disorders.

Joubert syndrome (JS) is a rare, predominantly autosomal recessive neurodevelopmental disorder characterized by hypotonia, motor delay, intellectual disability, oculomotor apraxia, and the hallmark "molar tooth sign" on axial view of MRI. JS is genetically heterogeneous, with pathogenic variants identified in more than 40 genes involved in primary cilia function. Among these, CEP290 is one of the most frequently mutated genes. In this study, we investigated two children-an 11-year-old boy (the proband) and his 5-year-old sister-both presenting with a similar phenotype consistent with JS. The parents, who self-identified as Chechen, reported distant consanguinity. The family also included a healthy 13-year-old daughter. The proband had previously been evaluated by a neurologist and underwent whole-genome sequencing (WGS); however, no causative variants were identified initially. After phenotype reassessment by a clinical geneticist, we performed a reanalysis of the raw WGS data and identified a novel homozygous intronic variant of uncertain significance (VUS), c.1910-15_1910-11delinsTTACA in CEP290 (NM_025114.4). Sanger sequencing confirmed that both the proband and his affected sister were homozygous for this variant, which they inherited from their heterozygous parents. Their healthy sister did not carry the variant. mRNA-sequencing and targeted cDNA sequencing (read depth ~ 100,000x) demonstrated that this intronic variant causes completely aberrant splicing of CEP290 pre-mRNA. Predominantly this variant causes the skipping of exon 20 in the main CEP290 transcript. Alternatively, the variant results in partial inclusion of intron 19 into the mRNA, elongation of exon 20 by 58 nucleotides, and a homozygous substitution chr12:88114573 (ACTGTGTA> TTACAGTA). No canonical mRNA isoform was detected when the variant was homozygous. Both the predicted severe truncation and the likely degradation of aberrant transcripts through nonsense-mediated decay (NMD) would correspond to complete loss of CEP290 function. Following the reclassification of this VUS to likely pathogenic, the family was able to pursue in vitro fertilization (IVF) with preimplantation genetic testing for monogenic disorders (PGT-M). Our study highlights the critical importance of proper phenotyping prior to referral for WES/WGS as well as of combining NGS with functional mRNA studies to achieve a molecular diagnosis for patients with predicted splice-site mutations in JS-associated genes. It also emphasizes the need for functional reassessment of VUS when genomic data are expected to guide reproductive decision-making within affected families.

Nonsense-mediated mRNA decay (NMD) is a highly conserved RNA surveillance mechanism in eukaryotic cells. It plays a key role in safeguarding the accuracy of gene expression by eliminating mRNAs with premature termination codons (PTCs) in the cellular transcriptome. Furthermore, NMD fine-tunes gene expression by regulating the stability of numerous transcripts that harbor NMD-inducing features other than PTC. Emerging evidence has established NMD factors as key regulators in diverse biological processes, including embryonic development, tissue homeostasis, and tumor biology. This review focuses on the dual roles of NMD in tumorigenesis, cancer therapy, and their underlying mechanisms. NMD can promote tumorigenesis and progression by degrading PTC-containing mRNA variants arising from mutated tumor suppressor genes or by suppressing neoantigen expression. Conversely, NMD can exert tumor-suppressive effects by eliminating aberrant transcripts of certain oncogenes. The function of NMD in tumor dynamics is highly dependent on multiple factors, including the tumor's genetic background, the expression and mutation status of NMD factors, alternative splicing coupled to NMD (AS-NMD), and the tumor microenvironment. Moreover, we summarize cancer therapeutic strategies targeting NMD, especially NMD inhibitors (e.g., NMDI-1, NMDI-14, and 5-azacytidine). The synergistic potentials of combining NMD inhibition with nonsense mutation readthrough therapy, immune checkpoint blockade, and chemotherapy in cancer therapy are summarized. Finally, we provide perspectives on future research directions, emphasizing that a deeper understanding of the context-specific mechanisms of NMD in different tumors is crucial for developing precise anti-cancer therapies.

Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by social communication deficits, restricted interests, and repetitive behaviors. Emerging evidence links several autism susceptibility genes to the nonsense-mediated decay (NMD) pathway, which maintains the homeostasis of gene transcription and protein translation in the nervous system. However, the role of Suppressor with morphogenetic effect on genitalia 7 (Smg7), an essential NMD factor, in brain function and ASD remains largely unknown. Here, we generated an Emx1-Cre-mediated conditional Smg7 knockout (Smg7cko) mouse model to investigate its neurological consequences. We found that both male and female Smg7cko mice exhibited autism-like behaviors, including impaired social interaction and communication, repetitive behaviors, anxiety-like traits, and learning and memory deficits. These phenotypes were accompanied by neuronal hyperexcitability and increased dendritic spine density in layer II/III pyramidal neurons of the hippocampus and the medial prefrontal cortex (mPFC). Notably, Smg7 deletion led to pronounced upregulation of Protein Kinase D1 (PKD1) transcripts, an NMD target, in these brain regions. Strikingly, adeno-associated virus (AAV)-mediated PKD1 knockdown (AAVsh-PKD1) in the hippocampus and mPFC significantly rescued social deficits in Smg7-deficient mice. Together, these findings identify Smg7 as a key regulator of neuronal function and behavior, and reveal PKD1 upregulation as a pathogenic mechanism underlying ASD-like phenotypes, providing new insight into NMD deficiency in ASD pathophysiology and a potential therapeutic target.

Alternative splicing (AS) coupled with nonsense-mediated decay (NMD) is an important post-transcriptional mechanism that regulates the expression of many genes, including serine/arginine-rich (SR) proteins across eukaryotes. In plants, SR proteins participate in diverse developmental processes and stress responses, particularly in abscisic acid (ABA) signaling. However, the functional differences among individual splice isoforms of SR proteins remain poorly understood. Here, we investigated SCL30a, a plant-specific SR protein in Arabidopsis thaliana. By integrating third-generation long-read transcriptome sequencing, NMD stability assays, and subcellular localization analyses, we identified five alternatively spliced SCL30a transcripts. Among them, SCL30a.2 and SCL30a.3 contain premature termination codons (PTCs), display nucleocytoplasmic localization, and are rapidly degraded through the NMD pathway. In contrast, the other three isoforms, SCL30a.1, SCL30a.4, and SCL30a.5, retain an intact RS domain and localize exclusively to the nucleus. Functional analyses showed that SCL30a acts as a positive regulator of ABA signaling. Loss-of-function mutants of SCL30a displayed reduced ABA sensitivity in both root growth and seed germination assays, whereas complementation or overexpression of three stable isoforms of SCL30a (SCL30a.1, SCL30a.4, and SCL30a.5) enhanced ABA responsiveness. Transcriptome analysis further showed that the expression of a subset of ABA-related genes, particularly SnRK2.6, was significantly altered in ABA-treated scl30a mutants and SCL30a.1-OE lines compared with WT plants. In addition, genetic evidence showed that overexpression of SnRK2.6 rescued the ABA-insensitive phenotype of the scl30a mutant. Together, these findings suggest that SnRK2.6 may function as a candidate downstream component associated with SCL30a-mediated ABA responses.

The selective degradation of aberrant mRNAs plays a vital role in ensuring cellular survival under stress conditions. Here, we investigated the role of OsFKBP20-1b, a splicing factor, in dehydration stress response in rice (Oryza sativa). We show that OsFKBP20-1b associates with the core nonsense-mediated mRNA decay (NMD) components, UP-FRAMESHIFT1 (OsUPF1) and OsUPF2, enhances their stability, thereby supporting the efficient degradation of aberrant transcripts during dehydration stress. These associations were demonstrated using bimolecular fluorescence complementation (BiFC), co-immunoprecipitation (Co-IP), and in vitro binding assays. Integrative analyses combining ribosome profiling and transcriptome sequencing further revealed that OsFKBP20-1b influences both alternative splicing (AS) patterns and translational dynamics of stress-responsive transcripts. Notably, loss of OsFKBP20-1b compromises OsUPF1- and OsUPF2-mediated decay of aberrant mRNAs under dehydration conditions. Consistent with these molecular defects, osfkbp20-1b mutant plants exhibited heightened sensitivity to dehydration stress. Together, our findings identify OsFKBP20-1b as a key regulator linking pre-mRNA splicing with cytoplasmic RNA surveillance during dehydration stress, thereby providing mechanistic insight into post-transcriptional control of stress adaptation in rice. These results advance our understanding of RNA quality control pathways in plants and suggest potential molecular targets for improving drought-resilience in crops.

Translating transcriptomics analysis into diagnostic workflows: clinical variant identification and interpretation in hypothesis-driven and hypothesis-free approaches

SUMMARYThe alternative splicing landscape of cancer transcriptomes remains poorly characterized, since short read sequencing cannot resolve complete transcript structures. Using the Oxford Nanopore cDNA platform, we generated nearly 2 billion long reads (median 25.8 million per sample) from 71 human samples, including 48 acute myeloid leukemia or myelodysplastic syndrome samples, 25 of which had splicing-factor gene mutations (in SRSF2, U2AF1, or SF3B1). An additional 23 samples were from sorted hematopoietic cell populations from healthy individuals. We identified 174,162 novel isoforms absent from the reference transcriptome, and proteomic validation confirmed that many are translated. We also identified isoforms enriched in spliceosome-mutant samples, and found proteomic evidence of frequent nonsense-mediated decay regulation of novel transcripts. This dataset is a valuable community resource, enabling detection of new transcripts in short read data sets. An interactive portal to explore splicing patterns in these data is available at https://leylab.org/isoforms/.

MYH9-related disease (MYH9-RD) is an autosomal dominant disorder characterized by thrombocytopenia, giant platelets, and variable systemic manifestations including nephropathy. While most pathogenic MYH9 variants are missense substitutions causing dominant-negative effects, the pathogenic potential of non-missense variants, particularly those affecting splicing, remains unclear. MYH9 (NM_002473.6) variants registered as "DM" (disease-causing variant) or "DM?" (possible disease-causing variant) in HGMD® were curated. After excluding missense and non-analyzable variants, ten intronic or single-base deletions were selected. Splicing effects were assessed by minigene assays in HEK293T cells and compared with SpliceAI predictions. Clinical information was reviewed. Aberrant splicing was confirmed in three variants: c.3838-2A>G, c.5765+2T>A, and c.5765+2T>G. The c.3838-2A>G variant caused in-frame skipping of exon 29, non-truncating variant, whereas the latter two induced inclusion of a cryptic 50-bp exon with premature termination codons in the final exon that can escape nonsense-mediated decay. These spliceogenic variants were associated with MYH9-RD phenotypes producing abnormal proteins that likely exert dominant-negative effects. The remaining seven variants showed no splicing abnormalities and were reported in non-MYH9-RD contexts, suggesting limited evidence for pathogenicity. SpliceAI predictions were concordant with experimental results. This study provides systematic evidence that some aberrant splicing MYH9 variants can lead to MYH9-RD with dominant-negative pathogenesis. Conversely, several variants previously annotated as "DM?" showed no functional or clinical relevance, arguing against a pathogenic role and supporting their consideration as likely benign or uncertain significance. Integrating in silico prediction with experimental validation improves variant interpretation and has implications for the clinical management of MYH9-RD.

Alternative splicing is a fundamental mechanism of gene regulation that generates transcriptomic and proteomic diversity, and its dysregulation is widely implicated in human diseases. The voltage-gated sodium channel (VGSC) NaV1.7, encoded by SCN9A, plays an essential role in nociceptive signaling, and alterations in its activity are closely associated with inherited pain disorders. In particular, alternative splicing of mutually exclusive exons, 5N and 5A, occurs in human, represents a key regulator of pain sensation in both growth and pathological processes. In this study, we examined the splicing regulatory landscape of exon 5N/5A in the mouse Scn9a gene and identified two potent splicing silencers, including ESS18, which is predominately regulated by the RNA-binding protein HuR. We further discovered a previously unreported Scn9a-Δ5 isoform whose exon 5 skipping induces nonsense-mediated decay, thereby reducing NaV1.7 expression. Based on these findings, we designed a series of MOE/PS-modified antisense oligonucleotides (ASOs) targeting exon 5N/5A and adjacent regions. Among these, ASO 5N(24-43) robustly promoted exon 5 skipping in vitro. Intracerebroventricular administration of this ASO in adult mice significantly enhanced tolerance to thermal and mechanical stimuli, correlating with extensive exon 5 skipping across multiple central nervous system regions. Our results reveal key cis-regulatory elements controlling Scn9a exon 5 splicing and demonstrate the therapeutic potential of exon-skipping ASOs for modulating NaV1.7 expression in pain management.

SummaryGammaherpesvirus reactivation drives a collapse of host mRNA splicing fidelity that extends across the transcriptome, with exon skipping affecting up to ∼57% of expressed genes, exceeding the effects of depletion of any of 186 splicing factors. Combining five Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV) reactivation systems across B cell and epithelial models with deep poly(A)+ RNA sequencing of purified lytic cells, we find that most induced isoforms are predicted to undergo nonsense-mediated decay or to lose conserved protein domains, broadly compromising cell cycle, innate immune and RNA-processing pathways. The phenotype arises independently of viral DNA replication, indicating early host remodeling. A screen of EBV early genes identifies BRRF1 as a key driver: through a CIY(Y/E) motif conserved in KSHV ORF49, BRRF1 engages the nuclear CCR4-NOT complex through its CNOT9 and CNOT1 subunits, hijacking this canonically cytoplasmic deadenylation hub for nuclear disruption of host splicing.