NAR Molecular Medicine

BackgroundMetazoan adenosine-to-inosine (A-to-I) mRNA editing temporospatially diversifies the neuronal transcriptome and proteome. The limited read length from next-generation sequencing (NGS) constrains the quantification of the potentially differential editing levels across different splicing isoforms, restricting our understanding of the extent to which RNA editing contributes to molecular diversity and its interplay with splicing. MethodsWe employed reverse transcription nested PCR (RT-nPCR) and developed a novel interfering-Primer PCR (iPrimer PCR) technique to distinguish different transcripts of any gene. We selected multiple essential genes exhibiting RNA editing in coding sequences (CDSs) or untranslated regions (UTRs) for isoform-specific amplification and Sanger sequencing. ResultsNine different Adar isoforms together with pre-mRNA had distinct editing levels at the S>G auto-recoding site, which was predicted to have isoform-specific effects on catalytic activities. Although pre-mRNA editing might exert isoform-dependent promotion/suppression of splicing, closely located editing sites, such as those in neuronal genes qvr and stj, still exhibited high correlation in editing levels due to co-editing. iPrimer strategy further discovered differential recoding levels between the long/short 3UTR isoforms of gene jef. ConclusionsWe provide the first comprehensive solution for isoform-specific PCR amplification of any gene, enabling quantification of RNA editing level of different isoforms. Our results offer insights into how RNA editing interplays with splicing, and highlight its complicated role in expanding molecular diversity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/725286v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1ebc82org.highwire.dtl.DTLVardef@1ea365dorg.highwire.dtl.DTLVardef@1971aceorg.highwire.dtl.DTLVardef@160d053_HPS_FORMAT_FIGEXP M_FIG C_FIG We developed isoform-specific PCR followed by Sanger sequencing, and achieved the quantification of differential RNA editing levels in different transcripts of a gene.

Circulating long non-coding RNAs (lncRNAs) have emerged as compelling cancer biomarkers. However, the structural features that mediate their extracellular stability and protein interactions remain largely unexplored. Here, we present the first systematic investigation of G-quadruplex (G4) motifs within cancer-dysregulated circulating lncRNAs and exploit these structures as molecular handles to identify associated RNA-binding protein (RBP) networks. From 283 circulating lncRNAs curated from the Lnc2Cancer 3.0 database, putative G-quadruplex-forming sequences (PQSs) were identified computationally using QGRS Mapper and G4Hunter, yielding four prioritized candidates -- AGAP2-AS1, LINC00683, DLG1-AS1, and KRTAP5-AS1 -- spanning 2G to 4G architectures. In vitro transcribed PQSs were validated for parallel G4 formation by circular dichroism spectroscopy, native polyacrylamide gel electrophoresis with thioflavin T staining, and reverse transcriptase stop assays, conducted under both standard buffer and simulated body fluid conditions to approximate the circulatory milieu. Electrophoretic mobility shift assays and isothermal titration calorimetry demonstrated nanomolar-affinity interactions between the G4-containing RNA and human serum albumin (HSA), the most abundant circulating protein. Cross-referencing G4-interacting proteins from the G4IPDB database with lncRNA-protein associations from LncTarD and NPInter, combined with RPISeq interaction predictions, identified ten candidate RBPs. A STRING-based protein-protein interaction (PPI) network was constructed at a confidence threshold of [≥]0.7 and refined iteratively using experimental stability data to exclude proteins associated exclusively with the structurally weaker KRTAP5-AS1. The resulting network, centered on ELAVL1, IGF2BP1, hnRNPA2B1, and FUS, highlights a coordinated post-transcriptional regulatory module relevant to oncogenesis. This work establishes a novel, experimentally validated framework wherein G4 motifs serve as entry points for decoding the protein interactome of circulating lncRNAs, with implications for cancer diagnostics and RNA-targeted therapeutic strategies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/728349v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1bba7dborg.highwire.dtl.DTLVardef@1095a53org.highwire.dtl.DTLVardef@1092eddorg.highwire.dtl.DTLVardef@1e3b7e7_HPS_FORMAT_FIGEXP M_FIG C_FIG

Biallelic disease-causing variants in IGHMBP2 cause spinal muscular atrophy with respiratory distress type I (SMARD1) and Charcot-Marie-Tooth type 2S (CMT2S). We present 12 unrelated patients with clinically suspected IGHMBP2-related-disease, each carrying a variant deep in intron 8 of IGHMBP2 (c.1235+1076G>A (n=6), c.1235+450G>A (n=5), and c.1235+894C>A (n=1)), along with a known deleterious variant in trans. To assess aberrant pathogenic splicing induced by these deep intronic variants in a relevant model, patient-derived induced pluripotent stem cells were differentiated into motor neurons (iMNs). Long-read RNA sequencing revealed introduction of different pseudoexons by each variant: c.1235+450G>A (626bp), c.1235+1076G>A (112bp and 77bp) and c.1235+894C>A (182bp). Although each variant utilizes a unique splice acceptor site, they all activate the same cryptic donor site, enabling a therapeutic approach to redirect aberrant splicing for all the variants using a single shared antisense oligonucleotide (ASO). Treatment of iMNs with this single ASO restored full-length IGHMBP2 protein in c.1235+894G>A and c.1235+1076G>A by decreasing the use of the novel acceptor site. In contrast, ASO treatment did not correct the splicing in c.1235+450G>A, suggesting that additional splice correction will be needed for this specific variant. A CRISPR interference screen of IGHMBP2 loss-of-function in iMNs identified ribonucleoprotein complex biogenesis (RNP), and rRNA and tRNA processing as top pathways implicated in motor neuron vulnerability. Proteomics and transcriptomics analysis of successfully treated patient iMNs revealed correction of RNP biogenesis and rRNA processing defects. This study highlights the importance of characterizing deep intronic variants in disease-relevant cells to assist the diagnostic process and inform therapeutics development. One Sentence SummaryIntron 8 of IGHMBP2 is a hotspot for splice activating pathogenic variants causing SMARD1 and CMT2S, which can be targeted with a single antisense oligonucleotide to correct the aberrant splicing, increase protein and restore cellular function in patient derived motor neurons.

Neurodegenerative diseases are complex disorders characterised by progressive neuronal loss and widespread transcriptomic dysregulation; however, the coordinated interactions among coding and non-coding RNAs that contribute to disease progression remain incompletely understood. In this study, RNA-seq datasets from disease-relevant neuronal populations and brain regions representing Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS) were analysed using an integrative network-based framework. Differential expression analysis coupled with weighted gene co-expression network analysis identified modules significantly correlated with disease and prioritised highly connected hub genes. Integration of these hub genes with curated RNA interaction database enabled the construction of candidate lncRNA-miRNA-mRNA regulatory networks. Functional enrichment analysis revealed Gene Ontology biological processes associated with synaptic signalling, mitochondrial function, RNA metabolism and neuroinflammatory responses across neurodegenerative conditions. The inferred regulatory networks suggested both disease-specific and shared post-transcriptional regulatory modules involving key hub genes and non-coding RNAs. Additionally, putative sequence variants were identified within untranslated regions of selected hub genes, suggesting potential alterations in miRNA-mediated regulations. Therefore, this study provides a systems-level view of transcriptomic dysregulation across major neurodegenerative diseases and identifies candidate regulatory interactions and molecular targets for future functional investigation

Abstract/SummaryRetroelements, including retrotransposons, endogenous retroviruses, and their fragments, as well as rare co-opted or domesticated retroelements, can contribute to neurodegenerative disorders and aging through modulation of gene expression and induction of neuroinflammation. Paternally Expressed Gene 10 (PEG10) is a retroelement-derived human gene that has recently been identified as a putative driver of Amyotrophic Lateral Sclerosis (ALS) and Angelmans Syndrome. PEG10 has been reported to bind nucleic acid and undergoes a complex self-processing pathway that results in gene expression changes when the protein accumulates in cells. Here, we report that PEG10 has selectivity for binding U/G-rich RNAs and influences widespread gene expression changes. PEG10 overexpression mimics the loss of TDP-43 in broad changes to gene expression, including dysregulation of mRNA splicing pathways. Specific changes to mRNA splicing were largely unique between TDP-43 knockdown and PEG10 overexpression, as classic TDP-43 targets including STMN2 were not altered by PEG10. Instead, we identified a unique role for PEG10 in regulating splicing of neuregulin 3 (NRG3), a ligand for the neuronal receptor ERBB4. In SH-SY5Y cells and in human neurons overexpressing PEG10, NRG3 protein levels were decreased along cellular processes, suggesting that these cells are less competent at signaling through the NRG3/ERBB4 axis. Using human patient data, we observed similar changes to NRG3 splicing in UBQLN2-mediated ALS, where PEG10 is accumulated, as well as in some cases of sporadic ALS. In conclusion, the retroelement-derived gene PEG10 plays an unexpected role in regulating splicing of neuronal transcripts, which mimics some of the transcript changes observed in human ALS patient samples. Ultimately, this work has implications for the study of PEG10, and mRNA splicing in neurological diseases associated with elevated PEG10 abundance. HighlightsO_LIPEG10 NC expression influences abundance of transcripts implicated in ALS C_LIO_LIPEG10 NC expression leads to an exon skipping event in neuregulin 3 (NRG3) C_LIO_LINRG3 expression is decreased along dendrites of PEG10 NC expressing human neurons C_LIO_LIExpression of PEG10 NC mimics changes observed in human ALS C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/727000v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@1a957d2org.highwire.dtl.DTLVardef@c4b15corg.highwire.dtl.DTLVardef@15825faorg.highwire.dtl.DTLVardef@25533d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Structured RNAs cause human diseases but remain challenging to target selectively with small molecules. Here, we report a chemoinformatics-guided discovery framework that integrates fingerprint-based molecular design, experimental validation, and mechanistic profiling to identify small molecules that bind highly structured, disease-associated RNAs. Using an RNA-binder fingerprint derived from known ligands, a Tversky similarity screen of >8 million compounds yielded a 150-member library enriched in chemical space for RNA-active scaffolds. Target engagement and cell-based assays identified multiple selective ligands for the pathogenic expanded triplet repeat, r(CUG)exp, that causes myotonic dystrophy type 1 (DM1) by binding and sequestering the RNA-binding protein muscleblind-like 1 (MBNL1). Biophysical and single-molecule analyses revealed that the small molecules bind the 1x1 nucleotide U/U internal loops formed when r(CUG)exp folds, partially block MBNL1 binding, and modulate RNA folding equilibria. Two optimized scaffolds rescued MBNL1-dependent splicing in patient-derived myotubes with micromolar potency and minimal cytotoxicity. This study establishes a generalizable, data-driven platform for discovering drug-like RNA-binding lead small molecules and demonstrates its application to the toxic repeat expansion RNA underlying DM1. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/723748v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1a87b41org.highwire.dtl.DTLVardef@340a14org.highwire.dtl.DTLVardef@81b583org.highwire.dtl.DTLVardef@1b3ba14_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

Facioscapulohumeral muscular dystrophy (FSHD) is caused by incomplete epigenetic silencing of a D4Z4 macrosatellite array, leading to pathogenic misexpression of DUX4 in skeletal muscle. Therapeutic development of small molecule drugs for FSHD has been hampered by screens that yield key myogenic regulators as candidates and a lack of mechanistic knowledge regarding their effects on DUX4. To uncover more specific targets, we performed a candidate-based screen which identified several epigenetic facilitators of DUX4 expression in primary FSHD myocytes, including the chromatin remodeling factor BAZ1A. Here, we used a compound that we recently identified as a BAZ1A inhibitor and potent DUX4 suppressor to interrogate the role of BAZ1A at the FSHD locus. Our data suggest a model in which BAZ1A binds to D4Z4 in FSHD muscle, changing the chromatin landscape of the array. BAZ1A binding leads to reduced occupancy of the HP1 repressor, increased occupancy of the p300 coactivator, and increased H3K27 acetylation, promoting transcription of both DUX4 and the long non-coding RNA DBE-T from the disease locus. DBE-T, in turn, recruits the histone methyltransferase ASH1L, which establishes H3K36 methylation in cis, further promoting DUX4 transcription. BAZ1A inhibition disrupts this powerful feed-forward loop, supporting the development of more metabolically stable inhibitors. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/732407v1_ufig1.gif" ALT="Figure 1000"> View larger version (28K): org.highwire.dtl.DTLVardef@13bba8dorg.highwire.dtl.DTLVardef@5485c4org.highwire.dtl.DTLVardef@11a2e02org.highwire.dtl.DTLVardef@1c2a6b2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Pathogenic RYR1 variants are associated with a set of rare neuromuscular disorders termed RYR1-related disorders (RYR1-RD). Clinical manifestations of RYR1-RD include proximal/axial muscle weakness, delayed motor milestones, impaired mobility, muscle pain, and fatigue. Muscle-specific microRNAs (miRNAs) are mostly expressed in muscle tissue and can be detected peripherally in plasma. Using a digital detection system, here we identified and quantified differential amounts of miRNAs in six adult (four monoallelic and two biallelic) RYR1-RD patient plasma samples compared to controls. Overall, 51 differentially expressed miRNAs were identified and hsa-miR-4454+hsa-miR-7975, in particular, was significantly overexpressed relative to controls (+ 39-fold, P=0.00285). Exploration of these differentially expressed miRNAs warrant further investigation as potential biomarkers of RYR1-RD.

Background: Biallelic variants in GFM2, encoding mitochondrial elongation factor G2 (mtEFG2), a GTPase involved in the termination stage of mitochondrial translation, cause autosomal recessive combined oxidative phosphorylation deficiency. Noncoding structural variants may be missed by exome sequencing but can disrupt splicing and provide opportunities for variant-specific therapeutic rescue. We investigated the molecular mechanism underlying suspected Leigh syndrome in an infant with mitochondrial disease and evaluated whether splice-switching oligonucleotide (SSO) treatment could correct the pathogenic splicing defect. Methods: The proband underwent exome sequencing followed by short-read and long-read whole genome sequencing. RNA sequencing, reverse-transcription PCR, quantitative PCR, and cycloheximide treatment were used to characterize the effect of the identified intronic duplication on GFM2 splicing and transcript stability. Patient-derived fibroblasts were treated with SSOs targeting the aberrant splice junction. Rescue was assessed by RNA studies, western blotting, and spectrophotometric measurement of cytochrome c oxidase (COX). Results: Whole genome sequencing identified a paternally-inherited GFM2 missense variant, NM_032380.5:c.2195C>T p.(Pro732Leu), in trans to a maternally-inherited 221-nucleotide intronic duplication, NM_032380.5:c.2029-741_2029-521dup. RNA studies revealed a 87-nucleotide pseudoexon, generated by activation of a cryptic acceptor splice site within the duplicated sequence. The resulting transcript harbored a premature termination codon (PTC) and underwent nonsense-mediated decay, as confirmed by cycloheximide rescue. Together with reduced mtEFG2 protein levels on western blot, the findings supported a loss-of-function mechanism. Enzymatic analysis of affected fibroblasts showed reduced activity of the mtDNA-dependent complex IV subunit COX, with preservation of the nuclear-encoded complex II enzyme succinate dehydrogenase and the control enzyme citrate synthase, consistent with impaired mitochondrial translation. A SSO targeting the aberrant intron-pseudoexon junction nearly abolished pseudoexon inclusion, restored correctly spliced GFM2 transcript from the duplication-containing allele, increased mtEFG2 protein levels, and significantly improved COX activity. Conclusions: This study identifies a pathogenic intronic GFM2 duplication that causes mitochondrial disease through pseudoexon activation and nonsense-mediated decay. The findings demonstrate the value of integrated genome and transcriptome analysis for exome-negative mitochondrial disease and provide in-vitro proof of concept that SSOs can restore transcript processing, protein expression, and mitochondrial respiratory-chain function in patient-derived cells.

Duchenne muscular dystrophy (DMD) is the most common, lethal X-linked neuromuscular disorder of childhood and is caused by mutations in the Dmd gene that disrupt dystrophin expression. Although adeno-associated virus-mediated gene therapies hold tremendous promise for DMD treatment, their clinical applications have been limited by dose-dependent vector and genome-level toxicities. Here, we developed and tested a single-vector adenine base editing strategy as a potentially safer genome editing approach to recode the pathogenic nonsense mutation into a benign missense mutation in mdx4cvDMD mouse model. Delivered using a muscle-tropic adeno-associated virus (MyoAAV) at a clinically-feasible dose (4E13 VG/kg), this strategy enabled detectable molecular recoding of the mdx4cv mutation in mice ranging in age from 3 days to 6 months. Yet, the overall efficiency and therapeutic impact of in vivo base editing with this system was highest in mice treated at the juvenile stage, with animals administered MyoAAV vectors at 3 weeks of age showing robust recovery of dystrophin expression and significant improvement in muscle contractile properties only one month later. Notably, introduction of adenine base editors either earlier in development, in neonatal mice, or later, in adulthood, yielded substantially lower editing efficiencies, particularly in muscle satellite cells whose editing is essential to ensure durable rescue of dystrophin expression in growing and regenerating muscle. Taken together, these results demonstrate the therapeutic potential of single-vector adenine base editing for DMD and underscore the importance of recipient age and disease stage in achieving optimal treatment outcomes for this and other genetic muscle disorders.

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.

Influenza A virus (IAV) causes significant morbidity and mortality worldwide. Understanding how viral RNAs may regulate host genes through microRNA-like mechanisms can clarify pathogenesis and reveal therapeutic targets. In this study, we screened all eight IAV H3N2 RNA segments (PB2, PB1, PA, HA, NP, NA, M, and NS) using an ab initio computational pipeline; five segments (PB2, PB1, PA, HA, and M) met the VMir scoring threshold for further analysis, while NP, NA, and NS were excluded due to low pre-miRNA scores. Mature miRNAs were identified using MatureBayes, and target genes in the human genome were predicted with the miRDB server. From these targets, we selected two genes per qualifying segment (10 genes total) based on their functional relevance to influenza infection and supporting literature; all selected genes are unique to their respective segment. We identified 10 segment-specific target genes (IFNL1, DDX60, SAMHD1, MAVS, IRF4, BIRC2, AGO1, MAP3K1, NOD1, and TNFAIP1) and one common target across all five analyzed segments (CADM2). Gene Ontology and pathway analyses showed enrichment in interferon signaling, RIG-I-like receptor pathways, antiviral restriction, RNA interference, and inflammatory responses. Literature supports roles for these genes in pulmonary and antiviral innate immunity. Our findings provide a basis for experimental validation and may help the research community better understand influenza virus pathogenesis and identify novel therapeutic candidates. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/725090v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@2b14adorg.highwire.dtl.DTLVardef@5a9b2eorg.highwire.dtl.DTLVardef@81ffc1org.highwire.dtl.DTLVardef@be119b_HPS_FORMAT_FIGEXP M_FIG C_FIG

The Ryanodine Receptor 1 (RyR1) is the major Ca2+ release channel in skeletal muscle and plays a crucial role in excitation-contraction coupling. Pathogenic variants in RYR1 are the most common cause of congenital myopathy, for which there are no approved treatments. Patient-centric disease models may help to facilitate the design and screening of novel therapeutics in a human genomic context. In this report, we describe the differentiation of five dominant RYR1-related myopathy patient-derived induced pluripotent stem cell (iPSC) lines into muscle progenitor cells (MPCs), and subsequently into multinucleated myotubes in 2- and 3- Dimensional (D) culture models. In 2D, we show significantly reduced Ca2+ release in a patient line compared to a healthy control following stimulation with caffeine. In 3D engineered muscle tissues (EMTs), patient-relevant phenotypes including reduced twitch amplitude, delayed relaxation and altered force-frequency relationships were observed in a patient line compared to two healthy controls. We also show that the 2D cultures are a suitable platform for screening the efficacy and cellular toxicity of antisense oligonucleotide therapeutics. Together, these results suggest that iPSC-derived skeletal muscle cultures are useful models for understanding the pathobiology of RYR1-related myopathies and as a testbed for emerging treatments.

Oligonucleotide therapeutics such as antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) enable sequence-specific gene silencing but rely on endogenous cellular machinery and often require extensive chemical modification for stability and efficacy. DNAzymes offer a mechanistically distinct alternative through intrinsic catalytic RNA cleavage; however, their therapeutic translation has been limited by nuclease susceptibility, structural constraints, and synthetic challenges. Here, we report the development of Circular Active Nano DNAzyme (CANDe), an enzymatically synthesized circular DNAzyme platform designed to enhance stability without backbone modification. The therapeutic potential of CANDe constructs was investigated against Japanese Encephalitis Virus (JEV) infection associated host long-noncoding RNA JINR1 (LINC01518). CANDe constructs were generated via splint-assisted ligation and incorporate modular elements, including catalytic cores (8-17 or 10-23), target-binding arms, and structural stems. Circularization conferred marked resistance to exonuclease-mediated degradation compared to linear DNA, maintaining structural integrity under nuclease-rich conditions.,CANDe targeting the lncRNA JINR1 achieved effective JINR1 knockdown in SHSY-5Y with and without JEV infection. This was accompanied by reduced expression JEV RNA and titers. In line with this, CANDe constructs attenuated of virus-induced cytotoxicity and apoptosis. Among the constructs, 10-23-based CANDe targeting the JINR1-1 site exhibited the strongest overall activity. These findings establish CANDe as a modular, modification-free DNAzyme platform that combines catalytic efficiency with enhanced stability, enabling effective host-directed antiviral intervention. This approach highlights topological engineering as a viable alternative to chemical modification for advancing DNAzyme-based therapeutics.

BackgroundMYORG (myogenesis-regulating glycosidase) and STRADB (STE20-related kinase adapter protein beta) were previously identified as activity-mediated skeletal muscle genes with potential roles in frailty and sarcopenia. We hypothesized that, if these genes are sustained by neuromuscular contractile activity, their expression should be consistently downregulated in muscular dystrophies, conditions defined by progressive muscle degeneration and secondary functional disuse. MethodsWe performed a systematic cross-dataset transcriptomic analysis of five publicly available GEO microarray datasets of human skeletal muscle. Discovery analysis was conducted in GSE3307 (Affymetrix HG-U133A/B; samples spanning DMD, LGMD2A/B/I, BMD, FSHD, JDM, ALS, AQM versus healthy controls). Independent external validation was performed in GSE38417 (HG-U133 Plus 2.0, DMD; n=16/6), GSE11681 (HG-U133A/B, LGMD2A; n=8-10/9-10), GSE465 (HG-U95Av2/B/C, multi-disease), and GSE1007 (HG-U95B/C/E, DMD; n=10-11/11). Raw CEL files underwent array-level quality assessment using NUSE and RLE diagnostics prior to normalization. Seven poor-quality arrays were excluded (none from Control, DMD, or LGMD groups). Remaining arrays were processed by robust multi-array average (RMA) normalization, and differential expression was assessed by limma with Benjamini-Hochberg FDR correction. ResultsMYORG was significantly downregulated in DMD (log2 fold-change [logFC] = -0.93, adj.P<0.001), LGMD2A (logFC = -0.82, adj.P<0.01), LGMD2B (logFC = -1.01, adj.P<0.01), and LGMD2I (logFC = -1.03, adj.P<0.01) in GSE3307. STRADB was significantly reduced in DMD (logFC = -0.33, adj.P<0.05) and showed a near-significant trend in LGMD2I (logFC = - 0.42, adj.P = 0.061). MYORG downregulation in DMD was independently replicated in GSE38417 (logFC = -1.40, adj.P<0.001) and GSE1007 (logFC = -0.80, adj.P<0.001). STRADB was also significantly downregulated in GSE38417 DMD (logFC = -0.45, adj.P<0.001). Deoxygalactonojirimycin, an iminosugar and an FDA/EMA-approved pharmacological chaperone (migalastat/Galafold) for Fabry disease, has been reported to be a specific molecular interactor that stabilizes MYORG protein in skeletal muscle. ConclusionsThis multi-dataset study further supports the role of MYORG and STRADB as activity-sensitive muscle genes that are robustly downregulated in DMD and LGMD. The pharmacological interaction between migalastat and MYORG provides a mechanistically grounded rationale for investigating this approved agent as an adjunct therapy in muscular dystrophies, in combination with the existing standard of care. This also supports active investigation of iminosugar analogs to target MYORG as potential therapeutics for improving skeletal muscle function in dystrophies, frailty, and sarcopenia.

Myotonic Dystrophy type 1 (DM1) is an autosomal multisystem disorder manifested due to unstable CTG nucleotide repeat expansion within the 3'-untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Although progress towards understanding of molecular pathogenesis in muscle and heart has been made, the pathways that affect the brain in DM1 is fundamentally unknown. In addition, the congenital DM1 manifest even more complicated brain abnormalities. Despite the wealth of existing cellular and animal models, iPSCs based studies are being fostered as they replicate the human model more closely to the disease. In view of this context, we set out to characterize the differentiation potential of congenital DM1 patient derived iPSC lines towards neuronal cells. Using neurogenin2 (NGN2) induced direct reprogramming of iPSCs into neurons and chemically defined media-induced neural induction protocol, we find that congenital DM1 mutant iPSC derived neurons exhibited precocious differentiation, as evidenced by their expression of pan-neuronal markers TUJ1 and Map2, along with increased processes extension and neurite length. Moreover, unbiased RNA sequencing analyses and qPCR validation revealed precocious and enhanced expression of several neurogenic transcription factors including, Ascl1, NeuroG2, and NeuroD1. Furthermore, immunofluorescence imaging of MBNL1 and MBNL2, RNA-splicing factors, displayed enhanced nuclear aggregations, a hallmark of the DM1 disease, in the mutant lines. Moreover, investigation of RNA splicing events identified mis-splicing in many important genes/transcripts including RMST, ANK3 and MBD1 during the neural conversion of congenital DM1 lines. These studies reveal novel paradigms that may contribute to neurological pathogenesis in CDM1 patients. These studies also provide a strong foundation for future mechanistic investigation aimed at understanding CDM1 pathology and may open new avenues for the development of gene therapy approaches for individuals with DM1.

I.Understanding the mechanisms governing neuronal differentiation is essential for elucidating neurodevelopmental processes and identifying therapeutic targets for neurological disorders. In this study, we optimized serum-dependent induction conditions and benchmarked multiple RNA-seq pipelines to establish a robust in-vitro model of neurogenesis using P19 embryonal carcinoma cells. Retinoic acid (RA, 0.5 {micro}M) was used to induce neuronal differentiation under varying concentrations (1%, 2%, and 5%) of fetal bovine serum (FBS) obtained from three suppliers. Morphological observation and marker gene analysis (MAP2, OCT4) revealed that serum concentration strongly influenced aggregation, survival, and neuronal commitment, with 2-5% FBS yielding optimal neurogenic differentiation. Total RNA extracted on day 10 of differentiation was subjected to RNA sequencing, and the resulting datasets were analyzed using four independent bioinformatics workflows: a Linux-based R pipeline (HISAT2 + featureCounts + DESeq2), nf-core, Galaxy, and BGIs Dr. Tom platform. Differential gene expression analysis identified 9,943 differentially expressed genes (DEGs) (FDR < 0.05, |log2FC| > 1), enriched in synaptic assembly and axon development among upregulated genes, and in ribosome biogenesis and RNA processing among downregulated genes. Comparison across all pipelines revealed 62 consistently upregulated and 63 downregulated genes, representing a robust core signature of P19 neurogenesis. Together, these findings establish an optimized and reproducible framework for in-vitro neuronal differentiation and transcriptomic analysis, providing a foundation for mechanistic and disease-modeling studies in neurodevelopmental biology.

The early onset of labour increases mortality and developmental risks for a human newborn. Key genes in human labour have been investigated using multiple modalities, but their regulation by non-coding RNA (e.g. lncRNA and miRNA) remains incomplete. This study explores the three-way relationship between labour-associated transcription factors (TFs), miRNA and lncRNA suggested by the competing endogenous RNA (ceRNA) hypothesis, to understand the underlying regulatory framework. Experimentally validated miRNA-lncRNA interactions are modelled using five distinct machine learning (ML) architectures to predict 20469 labour-linked miRNA-lncRNA interactions. Known mRNA-ncRNA interactions from databases were included to construct a tripartite network, and a subset of 9989 labour-linked network motifs containing TFs were isolated and analysed. Gene enrichment of nodes in TF-lncRNA-miRNA network, as well as validation from public myometrial datasets indicate high significance in contractile pathways including immune signalling. Experimentally unconfirmed tripartite network motifs have been found, and we elaborate on their potential regulation in labour using 8 TF-lncRNA-miRNA network motifs. A unified ncRNA-TF regulatory atlas in labour has been synthesized, and a complete summary of the tripartite network motifs can be accessed and visualised using the user-friendly, public database.

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.

Human endogenous retroviruses (HERVs) are mobile genetic sequences derived from ancient retroviral infections. While typically silenced, their reactivation has been implicated in gene dysregulation, aging, and immune-related transcriptional pathogenesis of some neurodegenerative diseases. Parkinson's disease (PD) is the second most common neurodegenerative disorder, yet its etiology and HERV reactivation remain poorly understood. This study investigates locus-specific HERV expression in early-stage PD, including genetic and non-genetic cases (all PD), idiopathic PD without a known genetic cause (iPD), and PD driven by leucine-rich repeat kinase 2 mutations (LRRK2 PD). We analyzed RNA-seq whole-blood samples from 492 individuals (358 all PD, 256 were iPD, 63 LRRK2 PD, and 134 healthy controls (HC)). We identified 20 significantly dysregulated HERV loci in all PD versus HC. Five HERV loci were shared with iPD analysis, and one HERV locus was shared with LRRK2 PD. Notably, these shared loci included HERV-H and ERVLE elements, indicating robust disease-associated retroviral signals independent of disease subtype. We found that genes proximal to these HERVs revealed pathways implicated in PD pathogenesis. Immune cell deconvolution showed increased neutrophil abundance and decreased resting CD4+ memory T cells proportions across the PD cohorts when compared to HC, consistent with neutrophil-lymphocyte ratio observed in previous peripheral immunity studies. Transcriptomic HERV alterations are present in whole blood across PD populations and are associated with dysregulation of fundamental cellular pathways and peripheral immune remodeling. Our findings motivate experimental validation of locus-specific HERV expression as a candidate blood-based signature with potential to inform PD neuroinflammatory and neurodegenerative processes.