NAR Molecular Medicine

BackgroundLMNA-related congenital muscular dystrophy (L-CMD) is a rare, life-threatening genetic disorder caused by point mutations in the LMNA gene, for which no effective treatment currently exists. It is characterized by early-onset muscle weakness, dropped-head syndrome, hypotonia, cardiac complications, and restrictive lung disease, frequently leading to premature death. The LMNA c.745C>T (p.R249W) mutation is the most prevalent amongst L-CMD patients. Given its monogenic nature, L-CMD represents a compelling candidate for gene therapy approaches. ResultsIn this study, we investigated the therapeutic potential of adenine base editing (ABE) to correct the pathogenic LMNA c.745C>T (p.R249W) mutation in human myoblasts. We evaluated multiple ABE variants and single-guide RNAs (sgRNAs), identifying optimal combinations that achieved efficient and specific correction of the mutant allele. However, we found that editing can also introduce an adjacent bystander mutation, c.743T>C (p.L248P). To determine the functional consequences of base editing, we established clonal cell lines reverted to wild type or harboring the p.L248P variant. Whereas wild-type edited cells showed a clear correction for all the studied parameters that were abnormal in R249W myoblasts, we found that L248P cells show nuclear abnormalities resembling those of R249W mutant cells, and their cellular function is partially compromised. These results demonstrate that ABE can effectively target the LMNA c.745C>T mutation but also reveal the significant impact of bystander edits on cellular physiology. ConclusionsOur findings provide proof-of-concept for the application of base editing as a therapeutic strategy for L-CMD, while underscoring the necessity of precise editing technologies to ensure both efficacy and safety in future clinical translation.

Duchenne muscular dystrophy is a lethal neuromuscular disorder caused by the absence of dystrophin, for which no curative treatment is available. RNA-based approaches have shown promising results; however, their evaluation is hindered by the lack of robust and rapid delivery methods for differentiated human muscle cells, which represent the most physiologically relevant in vitro models for assessing therapeutic strategies. Here, we establish a versatile lipid nanoparticle platform enabling efficient delivery of diverse RNA therapeutics across a range of human muscle models, including myotubes, induced pluripotent stem cell-derived myotubes, myoblasts, cardiomyocytes, and 3D engineered skeletal muscle tissues. Remarkably, a single commercially available lipid nanoparticle formulation supports delivery of cargos spanning more than 300-fold in size, from short antisense oligonucleotides ([~]20 nt) to complex CRISPR-based editors (up to [~]6.7 kb), including Cas9 nucleases, adenine base editors, and CRISPRa systems. This enables efficient gene correction and transcriptional modulation, resulting in dystrophin restoration or compensatory utrophin upregulation in relevant Duchenne muscular dystrophy models. Together, our results establish a single lipid nanoparticle formulation as a versatile platform for RNA delivery in human muscle systems and provide a practical framework for the rapid preclinical assessment of emerging therapies for Duchenne muscular dystrophy and other neuromuscular disorders. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/741396v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@a18d71org.highwire.dtl.DTLVardef@4d0faforg.highwire.dtl.DTLVardef@140b078org.highwire.dtl.DTLVardef@1a235_HPS_FORMAT_FIGEXP M_FIG C_FIG Created in BioRender. Leclerc, D. (2026) https://BioRender.com/90h0fme

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that disrupt the reading frame and abolish expression of functional dystrophin protein. Antisense oligonucleotides (ASO) can restore production of partially functional dystrophins by inducing exon skipping to restore the reading frame of dystrophin transcripts. While exon skipping is an FDA approved therapeutic strategy, there are currently no approved therapies for patients amenable to exon 44 skipping (8% of DMD patients). Here, we carried out a discovery campaign to identify phosphorothioate (PS) ASOs that efficiently induce exon 44 skipping and to define key sequence and chemistry features associated with activity. A tiling and micro-tiling approach with 18mer fully PS and 2-O-methoxyethyl (2MOE) modified ASOs in patient-derived myotubes identified five exonic target regions that promote skipping. ASO activity was strongly correlated across skeletal muscle and iPSC-derived cardiomyocytes, indicating similar exon 44 splicing regulation across cell types. Optimization studies showed that for 2MOE PS ASOs, 16-20mers were generally most active, while longer ASOs often had reduced activity due in part to impaired productive uptake into cells. Swapping out 2MOE modifications at both terminal positions for locked nucleic acids (LNAs) rarely improved activity and could also reduce it. Finally, promising candidates were tested in a humanized mouse model with an exon 44 skippable deletion, where one ASO outperformed others, inducing dose-dependent exon 44 skipping and dystrophin restoration in mouse models. These findings define practical design rules for exon 44-targeted ASOs and provide a foundation for therapeutic development.

Skeletal muscle dynamically adapts to physiological stimuli such as exercise through coordinated molecular and structural remodeling processes. Circulating microRNAs (miRNAs) represent promising non-invasive biomarkers of exercise responsiveness and skeletal muscle physiological states; however, most analytical frameworks rely solely on annotated miRNAs and overlook novel candidates. Here, we present nf-sarcopipe, a modular Nextflow pipeline that integrates de novo and reference-guided miRNA discovery with transcriptomic analysis and regulatory network reconstruction. The pipeline is organized into three complementary modules: 1) Preprocessing, 2) miRNA Discovery, and 3) Target Prediction & mRNA Integration. Using publicly available datasets from active and sedentary young women, the pipeline identified reproducible miRNA signatures and prioritized a small set of structurally supported, high-confidence de novo candidates. Previously reported exercise-associated miRNAs compiled from the literature were additionally incorporated for comparative candidate evaluation. Although the available datasets were derived from different tissues, confounding-aware analyses enabled the identification of coherent transcriptional signatures associated with exercise responsiveness. Integrative miRNA-mRNA analysis uncovered consistent regulatory interactions linking circulating miRNAs--both novel and known--to pathways involved in immune response, extracellular matrix remodeling, autophagy, and skeletal muscle adaptation. Together, these results establish nf-sarcopipe as a robust and scalable framework for complementary miRNA discovery and for investigating regulatory mechanisms associated with exercise-induced skeletal muscle adaptation.

Background: Low-frequency heteroplasmic mitochondrial DNA (mtDNA) variants are associated with aging and neurological diseases, including Parkinson's disease (PD). Targeted deep mtDNA sequencing using PacBio HiFi long reads has the potential to resolve heteroplasmy across the full mitochondrial genome with high accuracy. Methods: To validate Vega PacBio sequencing for detecting mtDNA heteroplasmy, we analyzed four predefined mixtures of two mtDNA haplotypes. We generated a single long-range PCR amplicon covering the entire mitochondrial genome. These amplicons were mixed at predefined ratios (minor mixture haplotype component: 5%, 2%, 1%, and 0.1%). Variant calling was performed using Mutserve2, and accuracy was assessed by calculating the F1 score from comparisons between expected and detected variants. Full-length mtDNA PacBio sequencing was applied to investigate heteroplasmy across fibroblast passages derived from five LRRK2 p.Gly2019Ser variant carriers (n=3 affected with PD and n=2 unaffected carriers). Changes in mtDNA heteroplasmy level and variant load were assessed longitudinally using a linear mixed model. Results: The single-amplicon approach enabled full-length haplotype resolution without amplification bias associated with overlapping PCR strategies. The F1 score of the predefined mixtures was 1.0 for heteroplasmy levels between 5% and 1% and remained high (0.91) at 0.1%. We detected n=10/62 variants discordant with the Illumina reference at the 0.1% mixture, but sensitivity remained very high at 1.00 in that mixture. Detected minor variants closely matched expected heteroplasmy levels, with average variant levels of 0.057 (5%), 0.022 (2%), 0.011 (1%), and 0.001 (0.1%). Across twelve fibroblast passages, we observed fewer mtDNA heteroplasmic variants ({beta}=-3.2, p=0.026). Increased heteroplasmic variant load over time was also associated with older age ({beta}=1.50, p=0.001) and PD affection status ({beta}=5.0, p=1.0 x 10-4) in LRRK2 variant carriers. Notably, we observed distinct patterns of heteroplasmic variants that either increased or decreased in heteroplasmy level across passages. Conclusion: PacBio HiFi sequencing, combined with a single-amplicon strategy, enables accurate full-length mtDNA heteroplasmy detection and longitudinal analysis, providing a valuable tool for studying mitochondrial variation and dynamics in disease.

Exercise training improves skeletal muscle insulin sensitivity, yet its effects on white adipose tissue remain incompletely understood. We investigated how adiposity and exercise training influence insulin-stimulated glucose uptake in skeletal muscle and abdominal subcutaneous adipose tissue (ASAT), alongside adaptations in gene expression and DNA-methylation. Ten monozygotic twin pairs discordant for BMI underwent [18F]FDG-PET/CT imaging of skeletal muscle (vastus lateralis, VL) and ASAT during a euglycemic-hyperinsulinaemic clamp before and after six months of exercise training. VL and ASAT biopsies were analyzed using mRNA-sequencing and reduced representation bisulfite sequencing. Exercise training improved whole-body and VL insulin sensitivity in leaner and heavier co-twins (p<0.05), without altering ASAT insulin sensitivity or body weight. Whole body adiposity exerted a stronger impact on ASAT molecular profiles than on skeletal muscle. At baseline, heavier co-twins displayed widespread ASAT transcriptional alterations enriched for inflammatory, proliferative and extracellular matrix pathways compared with leaner co-twins. In heavier co twins, exercise training attenuated inflammatory and proliferative signatures in ASAT and induced transcriptomic convergence with the leaner co twins. These changes were accompanied by marked shifts in transcription factor activity and context specific DNA methylation changes. In contrast, VL exhibited more modest transcriptomic and epigenetic responses relative to ASAT, particularly in heavier co-twins. In conclusion, six months of exercise training improved whole-body and VL insulin sensitivity while in ASAT many of the obesity associated transcriptomic programmes were reversed. These findings highlight adipose tissue as a major site of obesity- and exercise-responsive molecular plasticity and reveal tissue-specific regulatory mechanisms that contribute to the metabolic benefits of exercise training.

The ribosome, long regarded as a passive, uniform machine, has only recently been recognised as a direct regulator of translation. Mass spectrometry and sequencing approaches have shown that heterogeneity in ribosome composition exists, which can actively regulate the translational process. One source of this heterogeneity is the modification of ribosomal RNA (rRNA), primarily pseudouridylation (pseU) and 2'-O-methylation (2OMe), mediated by specific H/ACA and C/D box small nucleolar RNAs (snoRNAs). Here, we investigate how the stoichiometry of rRNA modifications varies during embryonic stem cell differentiation. Using the modification basecalling capability of Nanopore direct RNA sequencing, we have identified distinct stoichiometric changes in modification patterns between pluripotent and differentiated cells, revealing highly dynamic, site-specific regulation. Further, profiling of snoRNA expression during trilineage differentiation revealed differential expression of H/ACA and C/D box snoRNAs responsible for a subset of these dynamic modifications. By integrating rRNA and snoRNA sequencing approaches, we have built a comprehensive profile of rRNA modification dynamics during early embryonic cell fate decisions, highlighting potential regulatory mechanisms for ribosome heterogeneity during development. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=191 HEIGHT=200 SRC="FIGDIR/small/743918v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@48ca91org.highwire.dtl.DTLVardef@eb0418org.highwire.dtl.DTLVardef@159fc8corg.highwire.dtl.DTLVardef@d34f19_HPS_FORMAT_FIGEXP M_FIG C_FIG

Actinomycin D (Act D) is a global transcriptional inhibitor widely used in research and clinical practice; however, its effects on RNA-binding protein (RBP) dynamics remain poorly understood. Analysis of an RNA-seq dataset from Act D-treated HeLa cells revealed a compensatory stress response enriched in RNA metabolism, processing, and translation. Here, we investigated the effects of Act D on the subcellular localization of RBPs using HuR as a model mRNA stabilizing RBP. Short-term Act D treatment markedly increased cytoplasmic HuR localization in HCT116 and HeLa cells where the protein is known to be active. Analysis of known pathways regulating HuR nucleocytoplasmic translocation did not fully explain this redistribution, suggesting alternative mechanisms. To identify proteins proximal to HuR following Act D treatment, we performed TurboID labeling followed by LC-MS/MS in HCT116 cells. Several proteins involved in RNA regulation were identified. Probabilistic modeling highlighted FUS, an RBP with established roles in phase-separated granule dynamics, as a candidate proximal protein. The Act D-dependent interaction between HuR and FUS was interrogated using molecular dynamics simulations and validated with proximity ligation assays. Furthermore, increased cytoplasmic localization of RBPs following Act D treatment was accompanied by formation of granular structures that were relatively fluid and could be disrupted by hypotonic shock. Collectively, our findings demonstrate that Act D induces cytoplasmic redistribution of multiple RBPs and their sequestration into dynamic granular structures, revealing a previously unrecognized cellular response to transcriptional inhibition. Graphical AbstractAct D induced cytoplasmic re-localization of HuR along with FUS and other RBPs in dynamic, hypotonic shock-sensitive granular structures. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/745449v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@515322org.highwire.dtl.DTLVardef@128f0d5org.highwire.dtl.DTLVardef@db4302org.highwire.dtl.DTLVardef@10c4a2e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Context. Teprotumumab (TEP) is an emerging treatment for thyroid eye disease (TED), but real-world evidence outside the United States remains limited, and detailed changes in orbital components have not been fully clarified. Objective. To evaluate the effectiveness of TEP based on clinical manifestations and magnetic resonance imaging (MRI) findings, and compare it with that of intravenous glucocorticoid (IVGC) therapy. Methods: The TEP cohort included all 18 patients who started TEP therapy at Kyoto University Hospital by July 31, 2025. A historical IVGC cohort included 20 patients matched to the TEP cohort. Results: During 24 weeks of TEP therapy, proptosis measured using a Hertel exophthalmometer improved from 22 (20-22) to 19 (16-21) mm (p = 0.025), and clinical activity score decreased from 4 (3-5) to 1 (0-1) point (p < 0.001). Among 15 patients with diplopia, a reduction of at least 1 point in Gorman score was observed in 9 patients (60.0%). Thyroid-stimulating antibody titers decreased from 1,180% (349-4,710) to 282% (132-504) (p = 0.013). MRI-based comparisons with the IVGC cohort showed that TEP reduced both extraocular muscle and orbital fat areas, whereas IVGC reduced extraocular muscle area but conversely increased orbital fat area. Inflamed extraocular muscles identified on MRI were enlarged at baseline and showed marked shrinkage after both therapies. Conclusion: TEP showed robust effectiveness in Japanese real-world patients with TED. MRI-based analyses revealed distinct effects of TEP and IVGC on orbital fat and identified inflamed extraocular muscles as treatment-responsive components.

Duchenne muscular dystrophy (DMD) is an X-linked muscle wasting disorder marked by lack of dystrophin expression. Symptoms include loss of ambulation, respiratory problems, and cardiac complications with heart failure being the leading cause of death. Dystrophin transduces force from the actin cytoskeleton to the extracellular matrix to protect cells during muscle contraction. Restoration of dystrophin expression by gene transfer holds promise in addressing the root cause of disease. We compared the changes to transcriptional profiles after gene transfer by adeno-associated virus or lentivirus to examine whether viral treatment alone impacts cell homeostasis. We delivered GFP to cardiomyocytes differentiated from induced pluripotent stem cells (iPSCs) with DMD mutations. Global transcriptional profiling revealed a downregulation of metabolic genes after lentiviral transduction compared to untreated controls. In both AAV and lentivirus-treated DMD iPSC-cardiomyocytes, we observed an activation of the p53 DNA damage response in addition to a downregulation of cell cycle genes, suggesting stress-induced G2/M checkpoint arrest following viral delivery. These findings demonstrate that gene therapy mediated by viral vectors activates cell stress pathways. Interventions to mitigate these stress responses may be necessary for safe and effective gene transfer in diseased cells.

Regeneration of skeletal muscle preserves muscle mass and function, which decline with age. Here, we sought to identify long noncoding (lnc)RNAs involved in skeletal muscle myogenesis and potentially relevant to muscle aging. Cross-sectional analysis of skeletal muscle transcriptomes from healthy 22-through 89-year-old individuals revealed lncRNA LANCL1-AS1 among the top declining transcripts. Conversely, LANCL1-AS1 increased robustly during skeletal myogenesis and promoted myogenic differentiation in culture. Affinity pulldown by ChIRP followed by mass spectrometry revealed that LANCL1-AS1 associated with the mitochondrial protein LRPPRC, enhancing the formation of the chaperone complex LRPPRC-SLIRP, which maintains longer poly(A) tails of mitochondrial (mt-)mRNAs and stabilizes mt-mRNAs. Importantly, while myoblasts from old rhesus monkey muscle expressed lower levels of LANCL1-AS1 and mt-mRNAs, and displayed lower mitochondrial activity than young monkey myoblasts, overexpressing LANCL1-AS1 in old myoblasts restored mitochondrial activity and myogenesis. We propose that the age-associated reduction in LANCL1-AS1 contributes to impaired mitochondrial function and reduced myogenic capacity in aging skeletal muscle.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two related neurodegenerative disorders that display overlapping features. The hexanucleotide repeat expansion GGGGCC (G4C2) in the C9ORF72 gene is the most common cause of ALS and FTD, which results in the accumulation of dipeptide-repeat protein aggregates. Regulation of protein synthesis at the level of the initiation factor eIF2 has been suggested as a transversal event contributing to neurodegeneration in ALS and FTD. eIF2 phosphorylation blocks protein synthesis to alleviate protein misfolding overload, but conversely it can reduce the expression of synaptic proteins resulting in neuronal dysfunction. Dibenzoylmethane (DBM) is a small molecule that reverses the translational attenuation mediated by eIF2 phosphorylation which has been shown to alleviate neurodegeneration in prion-infected mice and Tau transgenic animals. Here we investigated the efficacy of the oral administration of DBM in protecting a mouse model of C9ORF72 pathogenesis. Treatment of mice with 0.5% of DBM mixture in powdered food ad libitum was sufficient to prevent cognitive impairment in C9ORF72 mice. Unexpectedly, DBM treatment did not modify the content of poly(GA) and poly(GR) protein inclusion in the hippocampus and brain cortex. Proteomic profiling of brain tissue indicated that DBM administration corrected nearly 70% of the changes in gene expression triggered by expanded G4C2, where the main pathways modified by DBM were related to cytoskeleton organization, ALS, and metabolic processes. Most proteins corrected by DBM in our C9ORF72 model were also altered in the brain of human FTD/ALS patients. Overall, our results reinforce the idea that targeting protein synthesis with small molecules in patients carrying C9ORF72 mutations may result in improved cognitive capacity.

Background: Small nuclear RNAs (snRNAs) are RNA components of the major and minor spliceosomes that play a core role in splice-site recognition and control of the splicing process. Variants in genes that produce snRNAs are increasingly recognised as major contributors to rare disorders, including neurodevelopmental disorders (NDD) and retinal dystrophies (collectively termed RNUopathies, a subset of spliceosomopathies). Clinical interpretation of variants in snRNAs is, however, challenging and existing guidance to support clinical variant classification does not adequately capture the unique features of snRNAs that necessitate a bespoke approach. Methods: We quantified the elevated background mutation rate in snRNA genes using de novo variants from 12,007 trios and assessed mutation density in 76,215 genome sequenced individuals in gnomAD. We convened a panel of clinical, research, and industry scientists with wide-ranging expertise in clinical variant interpretation and classification and expert knowledge in snRNA genes to draft and refine a guidance document. Results: We detail important considerations for variant classification in snRNA genes. These include: the difficulties of variant identification which requires genome or targeted sequencing approaches, the large number of gene paralogs with high sequence identity that complicate read mapping and variant calling, and historical inaccuracies in snRNA gene annotation. Further we show a ~50-fold increase in de novo mutation rate in snRNA genes compared to intergenic sequence and discuss the implications of this for variant classification. We provide a set of specific recommendations for classifying variants in snRNA genes. Finally, we introduce RNUdb, an interactive web-based tool to support snRNA variant annotation and classification. Conclusions: We provide the first guidance for clinical variant classification in snRNA genes and anticipate that this will support routine screening and analysis of snRNA genes in clinical genetic testing.

Huntington disease (HD) is caused by a polyglutamine expanded huntingtin protein that exerts progressive cellular toxicity. However, the temporal sequence of pathogenic, particularly early and reversible versus late and irreversible events remain incompletely defined, despite their distinct therapeutic implications. To delineate this trajectory, we profiled the proteome of a huntingtin expressing cell model at early (72 h) and late (144 h) stages. Rather than a linear progression, pathogenicity unfolded in two discrete phases. At the early stage, cells exhibited a broad activation of RNA processing, splicing, and protein synthesis machinery, consistent with an adaptive response aimed at preserving gene expression fidelity under stress. By the late stage, this compensatory program had collapsed, giving rise to a dominant failure in mitochondrial energy metabolism. Notably, 85% of proteins altered at both time points reversed direction of change between stages, indicating that mutant huntingtin reprograms cellular function wholesale rather than amplifying a fixed set of perturbations. Detailed analysis of mitochondrial respiratory complexes revealed that terminal ATP generating components (cytochrome c oxidase and ATP synthase) were severely affected, whereas upstream electron transport elements were retained or upregulated. Leveraging this proteomic map, we applied an AI assisted, direction aware drug repurposing strategy. Of 1,712 differentially expressed proteins, 498 were druggable, and 89 mapped to approved agents with mechanisms concordant with the required correction. These included Complex I targeted agents (metformin, ME 344) and mitochondria directed therapeutics (SS 31, MitoQ), several of which have previously been evaluated in HD. Collectively, these findings define a biphasic course of huntingtin toxicity and highlight an early therapeutic window in which intervention is most likely to be applied, prior to irreversible deterioration of mitochondrial respiratory function.

Periodontal disease and COVID-19 are linked by convergent immunoinflammatory pathways, yet the molecular basis of their interaction remains poorly defined. Here, we present a comprehensive salivary microRNA profile from individuals with prior SARS-CoV-2 infection, sampled approximately 3-6 months after diagnosis and meeting criteria for long COVID, providing new insight into the post-viral oral microenvironment. Salivary miRNA sequencing revealed widespread repression in patients with PD, consistent with persistent immune dysregulation. Relative to COVID-19-negative/PD-negative controls, thirty-two miRNAs were differentially expressed in COVID-19-positive/PD-positive individuals, all significantly downregulated. A similar signature was observed in a post-vaccination cohort for the selected dysregulated miRNAs. Integrative pathway analyses identified these miRNAs as regulators of core inflammatory circuits, including Ras, MAPK, and NF{kappa}B signaling, converging on IL-1{beta}- and TNF-centered networks relevant to both PD and COVID-19. Mechanistically, restoration of three downregulated miRNAs, miR- miR-30e-3p 106-3p-3p, and miR-652-3p attenuated NF{kappa}B activation and cytokine release in TLR-stimulated human oral keratinocytes, while their functional suppression using inhibitors potentiates inflammation. These miRNAs were also predicted to target SARS-CoV-2 spike and nucleocapsid transcripts, an interaction validated by dual-luciferase reporter assays. Their overexpression further reduced spike and nucleocapsid expression in Beta- and Omicron-infected epithelial cells, as measured by flow cytometry and RT-qPCR confirming host miRNAs as potent endogenous SARS-CoV-2 restriction factor. Together, these findings identify salivary host miRNAs as mechanistic regulators of oral inflammatory tone and viral persistence, establishing a molecular link between periodontal inflammation and post-COVID oral pathology.

Purpose: Pathogenic variants in NFIX cause Marshall-Smith syndrome and Malan syndrome (MALNS). We identified a severe subtype of MALNS characterized by adolescent-onset musculoskeletal deterioration and investigated functional consequences of underlying variants. Methods: Clinical data were collected from seven individuals with pathogenic NFIX variants. Wild-type and mutated recombinant NFIX DNA-binding domains (DBDs) were evaluated using biochemical, structural, and DNA-binding assays. Results: Six individuals carrying R116W, R116P, K125E, or G147E NFIX substitutions developed progressive muscle wasting, markedly reduced body mass index, and rapidly progressive scoliosis after the typical childhood features of MALNS; two died from disease-related complications. A seventh individual with R116G did not develop this severe phenotype. Functional studies on recombinant NFIX DBDs showed complete or near-complete loss of DNA-binding activity for R116W, R116P, K125E, and G147E despite preserved protein folding, consistent with disrupted DNA recognition and a potential dominant-negative mechanism. In contrast, R116G exhibited a 7.7{degrees}C decrease in thermal stability, which may support haploinsufficiency mediated by protein degradation. Conclusion: Specific NFIX missense variants define a severe subtype of MALNS associated with progressive musculoskeletal deterioration. In vitro functional studies support variant-specific disruption of DNA binding, providing a mechanistic basis of genotype-phenotype correlations and informing prognosis, clinical surveillance, and therapy development.

Interleukin 27 (IL-27) is an anti-HIV cytokine that induces 14 novel microRNAs (miRNAs) in T cells. We previously reported that transfection of two of these miRNA mimics, miRTC10 and miRTC14, differentially induced interferons (IFN)A2, A8, A13, and L1 expression in human primary macrophages. However, the mechanism underlying this activation remains unclear. Here, we show that miRTC14 does not directly target IFN-regulatory genes but instead engages cytosolic RNA-sensing proteins. Using miRNA pull-down coupled with mass spectrometry and immunoblotting, we identified a metabolic enzyme, pyruvate carboxylase (PC) and laboratory of genetics and physiology 2 (LGP2/DHX58) as direct binding partners of miRTC14. Functional analyses revealed that miRTC14 induces IFN expression by more than100-fold (p < 0.001), whereas PC and LGP2 depletion markedly attenuated this response (50-100 fold reduction, p < 0.01). Reconstitution of PC and LGP2 in deficient HEK293 cells restored miRTC14-driven IFN induction. We found that miRTC14-induced IFN activation depends on sequence features at the duplex termini and is unlikely to arise from canonical miRNA-mediated gene silencing. These findings establish PC as a novel miRNA-binding protein and define a previously unrecognized RNA-sensing mechanism by which miRTC14 drives IFN production, linking metabolic enzymes to RNA sequence-dependent innate immunity.

Targeting GC-rich gene loci is a major challenge owing to their high duplex stability, repetitive sequence composition, and propensity to adopt alternative DNA structures. Triplex-forming oligonucleotides (TFOs) provide a programmable strategy towards the recognition of GC-rich DNA, but their application is restricted by the limited recognition capabilities of natural nucleobases in a cellular setting. Here, we overcome this barrier using parallel-binding TFOs containing the synthetic nucleobase 6-amino-5-nitropyridin-2-one (Z), which enables pH-independent recognition of G-C base pairs. Using two structurally distinct regulatory elements within the MYC promoter, we show that Z-modified TFOs form stable, sequence-selective triplexes that repress promoter activity by 50-80% in both episomal reporter assays and at endogenous gene loci. Notably, the greatest repression was observed at a GC-rich quadruplex-forming element that functions as a structural hub for transcription factor recruitment. To our knowledge, this represents the first demonstration that a simple nucleobase modification alone is sufficient to enable parallel-binding TFOs to repress expression of an endogenous gene, establishing a general strategy for targeting GC-rich regulatory elements through programmable DNA recognition. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/741700v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1d23b1corg.highwire.dtl.DTLVardef@126de76org.highwire.dtl.DTLVardef@d75631org.highwire.dtl.DTLVardef@15bd46d_HPS_FORMAT_FIGEXP M_FIG C_FIG

RNA dysregulation is a recognized contributor to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), the most common motor neuron (MN) disease. However, the molecular mechanisms linking defects in RNA metabolism to selective neuronal vulnerability remain poorly understood. Alterations in the cellular levels of R-loops -structures forming by reannealing of the nascent RNA with the template DNA during transcription- have been observed in neurodegeneration, but it is unclear how perturbations in R-loop homeostasis contribute to neuronal dysfunction. Here we investigate the molecular basis of a juvenile form of ALS dubbed ALS4 that is caused by mutations in the helicase SETX, which plays important roles in the resolution of R-loops and transcription termination. Using isogenic human induced pluripotent stem cell-derived MNs, we show that ALS4-associated SETX mutations induce progressive axonal defects and widespread transcriptomic alterations, including reduced expression or altered splicing of transcripts critical for neuronal function. ALS4 MNs exhibit a transcriptional signature marked by cellular stress, aberrant cell cycle re-entry, and compromised neuronal identity that is partially shared by other forms of ALS. Mechanistically, these defects are partly driven by downstream aberrant activation of the TGF-{beta} signaling pathway, whose pharmacological inhibition ameliorates axonal defects. Finally, our analyses support a link between mutant SETX ectopic activity at R-loops and the observed alterations in RNA expression and splicing, providing new insights into how RNA dysregulation can drive neuronal dysfunction Altogether, our work reveals how perturbations at the interface of transcription and R-loop metabolism can reshape neuronal identity and drive disease. TeaserDeregulation of TGF-{beta} signaling drives axonal defects and compromised motor neuron identity in senataxin-mediated ALS

Symmetry elements in nucleic acids are most strongly correlated with sites of biological function; however, their relevance to non-canonical structures remains underexplored. In this study, we demonstrate the presence and significance of trinucleotide symmetry elements within G-quadruplex (G4) motifs. Our central hypothesis is that the intra-strand mirror symmetry of trinucleotides has been evolutionarily selected to facilitate G4 formation builds on the established sequence-structure association of G-quadruplexes and the natural symmetry law governing nucleotide insertion during genome evolution. Using a conserved G4 motif in the first exon of the MTOR gene as a model, we showed remarkable trinucleotide symmetry preservation across primates and broader mammals, with functional G4 regions displaying locally elevated symmetry relative to the codon-biased exonic background. Analysis of experimentally validated oncogenic G4s, including c-MYC, BCL2, VEGF, and KRAS, revealed that mirror and reverse complement symmetries converge around biologically important G4s. To quantify this feature, we formulated two complementary descriptors: the mirror symmetry index (MSI) and its non-palindromic variant (nMSI). Across 14 oncogene-promoter wild-type G4s, the majority scored MSI [&ge;] 0.80 (mean 0.884), with only the loop-rich ATG7, BCR, and MDM2 motifs falling below this value, and the KRAS promoter G4 reached individual significance against its mononucleotide-preserving null distribution (p = 0.042). Most decisively, each wild-type G4 scored higher on MSI than its experimentally confirmed G4-abolished mutant in 12 of 14 paired comparisons (sign test, p = 0.0065; mean {Delta}MSI = +0.089, mean {Delta}nMSI = +0.192); the two reversals (BCL2 and HIF-1) are attributable to scrambled mutant controls that introduce more balanced trinucleotide compositions rather than to failure of the index. The directional trend was reproduced across three independently published datasets, with nMSI [&ge;] 0.50 separating G4-forming from non-G4 sequences at 77.8% sensitivity and 100% specificity, although the collective per-sequence signal from mononucleotide-preserving shuffles remained a non-significant trend (Stouffer combined Z = 1.197, p = 0.116). This first report of trinucleotide symmetry in G4 motifs posits that coordinated nucleotide insertion and quadruplet maintenance act as an evolutionary forcing mechanism that pre-organizes single strands for G4 folding.