NAR Molecular Medicine

RNA oxidation has been implicated in neurodegeneration, but the underlying mechanism for such effects is unclear. Recently, we demonstrated extensive RNA oxidation within the neurons in multiple sclerosis (MS) brain. In this report we identified selectively oxidized mRNAs in neuronal cells that pertained to neuropathological pathways. N-acetyl aspartate transferase 8 like (NAT8L) mRNA is one such transcript, whose translated product enzymatically synthesizes N-acetyl aspartic acid (NAA), a neuronal metabolite important for myelin synthesis. We reasoned that impediment of translation of an oxidized NAT8L mRNA will result in reduction in its cognate protein, thus lowering NAA level. This assertion is directly supported by our studies on a model cellular system, an MS animal model and postmortem human MS brain. Reduced NAA level in the brain hampers myelin integrity making neuronal axons more susceptible to damage, which contributes in MS neurodegeneration. Overall, this work provides a framework for mechanistic understanding of the link between RNA oxidation and neurodegenerative diseases.

Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disorder caused by loss of dystrophin. Upregulation of utrophin (UTRN), a dystrophin paralogue, is a promising therapeutic avenue. Here, we present a CRISPR-Cas9-mediated strategy to increase utrophin expression by disrupting microRNA (miR) binding sites (BS). Using a Cas9/gRNA ribonucleoprotein (RNP) complex we disrupted several miR BS in DMD myoblasts and selected the Let-7c BS has crucial for UTRN repression. Interestingly, Cas9/gRNA indels were as efficient as the complete removal of Let-7c BS in upregulating UTRN expression, without any major off-targets. In three-dimensional human DMD cultures, Cas9/gRNA-mediated editing resulted in significant utrophin upregulation and functional improvements of calcium dysregulation and muscle contraction. Finally, Let-7c BS disruption in mdx animals by systemic rAAVs mediated delivery of Cas9 and gRNA resulted in utrophin upregulation and amelioration of the muscle histopathological phenotype. These findings provide the foundations for a universal (mutation-independent) gene editing therapeutic strategy for DMD. One Sentence SummaryCRISPR-Cas9 has the potential to upregulate utrophin to treat all DMD patients.

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.

MEG3, a long non-coding RNA (lncRNA), has been shown to play a critical role in regulating apoptosis. Its downregulation inhibits apoptosis in cancer cells, whereas its upregulation has been associated with cell death in both cardiovascular disease and, more recently, Alzheimers Disease. Here we show that MEG3 is upregulated in Myotonic Dystrophy 1 (DM1). Specifically, we show MEG3 upregulation by several-fold in DM1 human muscle cells and in two DM1 mouse models, HSA-LR and LC15. In human DM1 muscle cells we observe nuclear retention of MEG3 and an increase in its transcript diversity. Furthermore, we observe a general trend of nuclear retention in DM1 affecting lncRNAs and microRNAs (miRNAs), in contrast to mRNAs, when compared to healthy cells. This altered nuclear retention may contribute to the pathological effects of non-coding RNA dysregulation in DM1. Importantly, we demonstrate that treatment with antisense conjugates targeting the repeat expansion causing DM1, an approach currently being tested in Clinical Trials, corrects MEG3 levels in HSA-LR mice, without additional therapeutic interventions targeting MEG3.

Facioscapulohumeral muscular dystrophy (FSHD) is a potentially devastating muscle disease caused by de-repression of the toxic DUX4 gene in skeletal muscle. FSHD patients may benefit from DUX4 inhibition therapies, and although several experimental strategies to reduce DUX4 levels in skeletal muscle are being developed, no approved disease modifying therapies currently exist. We developed a CRISPR-Cas13b system that cleaves DUX4 mRNA and reduces DUX4 protein level, protects cells from DUX4-mediated death, and reduces FSHD-associated biomarkers in vitro. In vivo delivery of the CRISPR-Cas13b system with adeno-associated viral vectors reduced acute damage caused by high DUX4 levels in a mouse model of severe FSHD. However, protection was not sustained over time, with decreases in Cas13b and guide RNA levels between 8 weeks and 6 months after injection. In addition, wild-type mice injected with AAV6.Cas13b showed muscle inflammation with infiltrates containing Cas13b-responsive CD8+ cytotoxic T cells. Our RNA-seq data confirmed that several immune response pathways were significantly increased in human FSHD myoblasts transfected with Cas13b. Overall, our findings suggest that CRISPR-Cas13b is highly effective for DUX4 silencing but successful implementation of CRISPR/Cas13-based gene therapies may require strategies to mitigate immune responses.

The majority of RNAs transcribed from the genome are non-coding RNAs (ncRNAs) that are involved in regulating the expression of protein-coding genes. However, a growing body of research highlights several novel microproteins encoded by unconventional ncRNAs such as long non-coding RNAs and circular RNAs (circRNAs) as important regulators of disease and development. Although several circRNAs have recently been reported to translate into functional peptides in diverse tissues, their roles in skeletal muscle remain largely unexplored. In this study, polyribosome-associated RNA sequencing and publicly available translatable circRNAs from the riboCIRC database were curated to discover potential protein-coding circRNAs in mouse C2C12 skeletal muscle cells. We validated a few circRNAs with high potential of translating into proteins in mouse C2C12 cells, including circular Smad1 (circSmad1) that encodes a 194 amino acid peptide called circSmad1-194aa. Interestingly, silencing of circSmad1 in C2C12 cells resulted in loss of myotube fusion and maturation. CircSmad1-194aa was found to contain the DNA-binding SMAD1-MH1 domain that localized into the nucleus during myogenesis. Moreover, CircSmad1-194aa associates with the BMP-responsive element (BRE) in the Id1 promoter that is known to inhibit Myod1-driven myoblast differentiation. We propose that circSmad1-194aa promotes myogenesis by masking Id1-BRE from SMAD complex interaction, leading to suppression of ID1 expression and upregulation of MYOD1. Together, our findings identify circSmad1-194aa as a novel regulator of skeletal muscle differentiation and highlight the potential for the discovery of other functional circRNA-derived peptides in muscle pathophysiology. HIGHLIGHTSO_LIRNA-seq discovers hundreds of polysome-associated circRNAs in mouse C2C12 myoblasts C_LIO_LIProtein-coding circRNAs exhibit myogenesis-specific expression C_LIO_LICircSmad1 is abundant, methylated, and translated into a 194aa polypeptide C_LIO_LICircSmad1-encoded polypeptide promotes myogenesis C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/708385v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@2074cforg.highwire.dtl.DTLVardef@d4fd98org.highwire.dtl.DTLVardef@1554735org.highwire.dtl.DTLVardef@1b6124a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Oligonucleotide therapeutics are an established class of drugs for the treatment of genetic disorders. Their clinical development is challenging, however, as they typically distribute poorly to extra-hepatic tissues after systemic injection. Here we tested the heteroduplex oligonucleotide (HDO) platform for systemic delivery of SMN2 splice-switching oligonucleotides of 2-O-methoxyethyl phosphorothioate or phosphorodiamidate morpholino oligomer chemistries. We first showed that splice-switching HDO cargoes correct SMN2 splicing in cells derived from spinal muscular atrophy (SMA) patients, and validated extra-hepatic activity in spinal cord and muscle in a mouse model of SMA following systemic delivery. Our study raises prospects for delivery of nusinersen, the 2-O-methoxylethyl phosphorothioate oligonucleotide therapy approved for SMA and currently delivered by intrathecal injection, by systemic injection exploiting the HDO chemistry platform. Our findings also suggest that oligonucleotide drugs lacking convincing in vivo efficacy in muscle tissue could be delivered effectively by the HDO technology.

RNase 2 is the most abundant human RNase A member in macrophages and its expression is activated upon exposure to viruses. Here, we explored the protein role by co-transcriptomics analysis of wild-type (WT) and RNase 2-knock-out (KO) macrophages in absence/presence of a virus-derived single-stranded RNA (ssRNA40). Results revealed that RNase 2 is key for maintaining cell homeostasis. Lacking RNase 2 induced the expression of stress-response markers under basal conditions and abolished the antiviral response of cells exposed to ssRNA40. In contrast, the up-regulated genes in WT macrophages participate in pro-inflammatory signaling response through TLR8-dependent pathways and antiviral immunity, with activation of MAPK and JAK-STAT pathways. Complementarily, we identified five top tRNA-derived small RNAs (tDRs) in response to ssRNA40 related to RNase 2, showing a preferential cleavage sites at CA and uridine rich regions of anticodon loops. Results highlight the essential roles of RNase 2 in antiviral response and inflammatory processes.

The interferon (IFN) response is crucial for antiviral activity, but its overstimulation can lead to a wide range of autoimmune disorders. The cytoplasmic pattern recognition receptor RIG-I detects viral double-stranded RNAs (dsRNAs) and endogenous polymerase III transcripts carrying a 5'-triphosphate (5'-ppp) or 5'-diphosphate (5'-pp) moiety, triggering phosphorylation of IRF3 and IFN immune response. While many viral RNAs initiate with 5'-ppp-adenosine (5'-pppA) and most endogenous Pol III transcripts in higher eukaryotes start with 5'-ppp-guanosine (5'-pppG), no apparent reason for this bias has been identified so far. Here we demonstrate that dsRNAs initiating with 5'-pppA trigger stronger RIG-I/IFN response than those starting with 5'-pppG. We show that several GTP-binding proteins interact preferentially with 5'-pppG RNAs. Finally, supplementation with guanosine, but not adenosine, which rapidly increases intracellular concentrations of GTP and ATP, respectively, eliminates the difference in immunogenicity between 5'-pppG and 5'-pppA RNAs. Our findings suggest that 5'-pppG RNAs may enable certain RNA viruses and Pol III transcripts to limit detection by innate immune receptors. These results offer new insights into the sequence-dependent activation of the RIG-I/IFN pathway and have important implications for both antiviral immunity and the role of Pol III-derived RNAs in autoimmune diseases.

Myoblast proliferation and differentiation are tightly controlled by epigenetic mechanisms, yet how clinically used epigenetic modulators influence myogenic cell fate remains incompletely understood. Here, we demonstrate that the histone deacetylase inhibitor and chemical chaperone 4-phenylbutyric acid (4-PBA) selectively promotes myoblast proliferation without inducing differentiation in C2C12 cells. Mechanistically, 4-PBA increases histone H3 acetylation at lysines 18 and 27 via downregulation of HDAC5, resulting in activation of NF-{kappa}B p65. Chromatin immunoprecipitation identifies early growth response 1 (Egr-1) as a direct transcriptional target of NF-{kappa}B p65. Transcriptomic analyses reveal that Egr-1 regulates extracellular matrix- and myogenesis-associated gene programs, including multiple collagen genes. Consistently, 4-PBA induces a transcriptional signature that significantly overlaps with Egr-1-dependent gene expression. Functional studies further establish that the NF-{kappa}B p65 - Egr-1 axis is required for 4-PBA-mediated transcriptional remodeling in proliferating myoblasts. Together, these findings uncover an epigenetic mechanism by which 4-PBA modulates myoblast proliferation through HDAC5-dependent histone acetylation and NF-{kappa}B p65 - Egr-1 driven transcriptional programs, providing insight into how epigenetic therapeutics influence skeletal muscle cell behavior. HighlightO_LI4-PBA enhances murine myoblast proliferation independently of differentiation induction. C_LIO_LI4-PBA enhances H3K18 and H3K27 acetylation through downregulation of HDAC5. C_LIO_LINF-{kappa}B p65 activates Egr-1 by directly binding to the Egr-1 promoter region. C_LIO_LI4-PBA stimulates the HDAC5 - NF-{kappa}B p65 - Egr-1 axis drives extracellular matrix-related gene profiles. C_LI

Circular RNAs (circRNAs) originate from backsplicing of numerous genes in animals, but the functions of most circRNAs remain elusive. We previously demonstrated that circZNF827 forms a complex with hnRNPL/K and its host gene-encoded protein ZNF827 that acts in the nucleus to transcriptionally repress the nerve growth factor receptor (NGFR/p75NTR) gene during neuronal differentiation (Hollensen, 2020) [1]. To explore the mechanism of action, and to assess a potential role of the circZNF827-hnRNP complex on additional loci, we scrutinized the genome-wide consequences of circZNF827 and/or hnRNPL knockdown at the transcriptomic and epigenetic level. RNA-sequencing and CUT&RUN confirmed that NGFR and additional loci are transcriptionally repressed by the circZNF827-protein complex, and that these are primarily enriched for H3K27me3 signatures. Only a fraction of the massive transcriptomic changes could be ascribed to a direct circZNF827 transcription-regulated phenotype, suggesting that initial key regulatory events elicited by the circZNF827-hnRNP complex likely lead to a secondary response, which further augments neuronal differentiation.

Alternative polyadenylation (APA) at the 3UTR of transcripts contributes to the cell transcriptome. APA is suppressed by the nuclear RNA binding protein, PABPN1. Aging-associated reduced PABPN1 levels in skeletal muscles lead to muscle wasting. Muscle weakness in oculopharyngeal muscular dystrophy (OPMD) is caused by short alanine expansion in PABPN1 exon1. The expanded PABPN1 forms nuclear aggregates, an OPMD hallmark. Whether the expanded PABPN1 affect APA and how contributes to muscle pathology is unresolved. To investigate these questions, we developed a procedure including RNA library preparation and a simple pipeline calculating APA-shift ratio as a readout for PABPN1 function. Using the mouse OPMD model we demonstrate similar results between previously published PAS utilization and APA-shift results. Studying APA-shift in two OPMD models and in OPMD patients we show that the expression of the expanded PABPN1 does not correlate with APA-shift. Instead, APA-shift is correlated with reduced expression levels of PABPN1 isoforms, amongst the isoform lacking exon1. Further we show that with our protocol APA-shift is enriched in muscle transcripts, moreover in OPMD patients. We suggest that muscle weakness in OPMD is caused by PABPN1 loss-of-function leading to APA-shift that primarily affects in muscle transcripts.

MotivationTraditional methods for detecting large-scale mitochondrial DNA (mtDNA) deletions (LSMDs) in cells present challenges, i.e. a priori information, high DNA inputs, poor sensitivity and are not always quantitative. Mitigation can be achieved through high throughput DNA sequencing using e.g. Illumina and Oxford Nanopore Technologies (ONT), in combination with LSMD breakpoint identification and quantification using bioinformatic tools. Splice-aware RNA alignment tools increase the sensitivity for detecting LSMD breakpoints compared with DNA aligners. Long-read sequencing (LRS) also offers potential advantages over short read sequencing, e.g. greater read lengths and capturing variants on single reads. No existing pipelines capture the benefits of both a splice-aware alignment tool and LRS. ResultsWe developed "NanoDel", a LRS pipeline, to sensitively and accurately detect cellular LSMDs. Using artificial datasets, "NanoDel" was more sensitive and accurate than other pipelines. In samples diagnosed with mitochondrial disease, it identified both known and previously uncharacterised (including mixtures) of LSMDs, without a priori information. LSMD breakpoints were found in mt-co1, mt-cyb, mt-nd6 and mt-nd5 genes. Analysis of selected LSMDs revealed proximity to repeat and putative G-quadruplex motifs, and occurrence in a range of healthy and pathological tissues, indicating potential for a shared vulnerability landscape in mtDNA, shaped by sequence motifs and structural constraints. "NanoDel" combined with one-amplicon, not two-amplicon, LR-PCR offers a robust strategy with clinical application for detecting LSMDs across a variety of cell/tissue samples, and its application across a broader range of samples, will yield new mechanistic insights into LSMD formation, and further our understanding of mtDNA instability.

BackgroundHuntingtons disease (HD) is a fatal neurodegenerative disorder caused by a mutation in the huntingtin gene (HTT), characterized by an expanded CAG trinucleotide repeat. At the time of writing no cure or disease-modifying treatments exist. Currently, the most explored investigational therapeutic strategy targets HTT gene expression, either lowering both alleles (total lowering) or selectively lowering the mutant allele. These approaches require reliable pharmacodynamic biomarkers to measure an allele-selective knockdown. However, allele specific quantification of wild-type and mutant HTT RNA or protein remains a challenge. ResultsHere we optimized a droplet digital PCR (ddPCR) assay to distinguish between mutant and wild-type HTT (mHTT and wtHTT respectively) mRNA expression based on differential amplification of HTT mRNA molecules with different CAG repeat lengths under limited dNTP conditions. This assay, combined with our novel automated analysis pipeline reliably detects allele-specific expression in HD patient cell lines. We simulated various mHTT to wtHTT mRNA ratios by mixing RNA from respective homozygous cell lines to demonstrate the assays accuracy under varying allele ratios. We also validated the assays utility in 13 cell lines from HD patients and their family members. Additionally, we optimized a one-step RT-ddPCR method, offering a streamlined alternative to a two-step ddPCR method. We further confirmed the assays clinical relevance by demonstrating allele-selective siRNA mediated HTT lowering in HD patient fibroblast cell lines. ConclusionsOur optimized ddPCR assay, with its pipeline for automated data analysis, enables the precise quantification of allele-selective HTT mRNA knockdown. The method does not require prior knowledge of patients SNP genotypes, previously a prerequisite for assays aiming to determine the mHTT transcript expression in patient samples. Our HTT ddPCR assay is universally applicable regardless of patient genotype. The ability to accurately monitor allele-specific HTT mRNA expression levels holds great promise for developing effective treatments for Huntingtons disease.

PARK7, also known as DJ-1, plays a critical role in protecting cells by functioning as a sensitive oxidation sensor and modulator of antioxidants. DJ-1 acts to maintain mitochondrial function and regulate transcription in response to different stressors. In this study, we show that cell lines vary by their antioxidation potential at basal condition. The transcriptome of HEK293 cells was tested following knockdown (KD) of DJ-1 using siRNAs which reduced the DJ-1 transcripts to only 12% of the original level. We compared the expression levels of 14k protein coding transcripts, and 4.2k non-coding RNAs relative to control cells treated with non-specific siRNAs. Among the coding genes, [~]200 upregulated differentially expressed genes (DEGs) signified a coordinated antiviral innate immune response. Most genes were associated with regulation of type 1 interferons (IFN) and induction of inflammatory cytokines. About a quarter of these genes were also induced in cells treated by non-specific siRNAs that were used as a negative control. Beyond the antiviral response, 114 genes were specific to KD of DJ-1 with enrichment in RNA metabolism and mitochondrial functions. A smaller set of downregulated genes (58 genes) were associated with dysregulation in membrane structure, cell viability, and mitophagy. We propose that KD of DJ-1 diminish its protective potency against oxidative stress, rendering the cells labile and responsive to dsRNA signal by activation of a large number of genes, many of which drive apoptosis, cell death, and inflammatory signatures. The KD of DJ-1 highlights its crucial role in regulating genes associated with antiviral responses, RNA metabolism, and mitochondrial functions, apparently through alteration in STAT activity and downstream signaling. Given that DJ-1 is highly expressed in metastatic cancers, targeting DJ-1 could be a promising therapeutic strategy where manipulation of DJ-1 level may reduce cancer cell viability and enhance the efficacy of cancer treatments.

IntroductionDiabetic foot ulcers (DFU) affect 15% of diabetic patients globally. While microRNAs (miRNAs) are known regulators of wound healing, comprehensive bioinformatics analysis of their structural determinants and network interactions in DFU pathophysiology remains limited. ObjectiveTo perform integrative bioinformatics analysis of miRNA networks in DFU healing, characterizing structure-function relationships and identifying potential therapeutic targets through computational approaches. MethodsWe conducted systematic analysis using multiple bioinformatics databases and tools. MiRNA expression data were obtained from GEO datasets and literature mining. Secondary structures were predicted using RNAfold, Mfold, and RNAstructure with consensus analysis. Target prediction employed TargetScan, miRanda, and DIANA-microT. Protein-protein interaction networks were constructed using STRING. Pathway enrichment was performed with DAVID and Reactome. Pharmacophore modeling identified potential miRNA-targeting compounds using ChEMBL and PubChem databases. ResultsAnalysis identified 8 consistently dysregulated miRNAs across 15 DFU datasets (n=1,247 samples). Meta-analysis revealed miR-146a (fold-change: -3.2{+/-}0.8), miR-155 (+4.1{+/-}1.2), and miR-21 (-1.9{+/-}1.2) as key regulators. Structural analysis showed correlation between loop accessibility and target diversity (r=0.73, p<0.01). Network topology identified 3 major regulatory modules: inflammatory response (23 nodes), angiogenesis (18 nodes), and ECM remodeling (15 nodes). Drug-miRNA interaction analysis revealed 12 FDA-approved compounds with predicted miRNA-modulating activity, including metformin and curcumin analogs. ConclusionsThis comprehensive bioinformatics analysis reveals miRNA network architecture in DFU healing and identifies structure-based therapeutic targets. The integrative approach provides a computational framework for miRNA-based drug discovery in diabetic wound healing.

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

The steady state levels of mRNA are outcomes of a finely tuned interplay between RNA transcription and decay. Therefore, the modulation of RNA stability is generally assumed to influence RNA abundance in a positive direction. However, the correlation between mRNA transcription, translation and stability remains elusive. Here, we employed a newly developed simplified mRNA stability profiling technique to explore the role of mRNA stability in SH-SY5Y neuronal differentiation model. Transcriptome-wide mRNA stability analysis revealed neural-specific RNA stability kinetics, including stabilization of transcripts encoding regulators of neuronal morphogenesis and function and destabilization of mitochondrial electron transport and redox homeostasis. When we further examined the relationship between transcription, translation and mRNA stability, a bidirectional regulation of RNA stability was revealed, wherein mRNA stability could either exert the buffering effect on gene products or change in a same direction as transcription. Motif analysis unveiled SAMD4A as a major regulator of the dynamic changes in mRNA stability observed during differentiation. Knockdown of SAMD4A impaired neuronal differentiation and influenced the response to oxidative stress. Mechanistically, SAMD4A was found to alter the stability of several mRNAs to which it binds. Meanwhile, a dimorphic pattern of the correlation between gene expression and SAMD4A-regulated mRNA stability was observed, suggesting dynamic regulation mRNA stability during the neuronal differentiation guided by SAMD4A. The novel insights into the interplay between mRNA stability and cellular behaviors provide a foundation for understanding neurodevelopmental processes and neurodegenerative disorders and highlights dynamic mRNA stability as an important layer of gene expression regulation.

Spinal muscular atrophy (SMA) is caused by loss of SMN protein and is increasingly recognized as a multisystem disorder involving molecular pathology beyond motor neurons. Recently, we identified NRF2-KEAP1 signaling as dysregulated in SMA mice. Because NRF2 coordinates transcriptional programs that maintain cellular redox homeostasis and adaptive stress responses, we investigated whether NRF2 signaling is similarly altered in SMA type I patient-derived fibroblasts and whether it can be pharmacologically engaged. Compared with control fibroblasts, SMA fibroblasts displayed reduced basal expression of NRF2 target proteins, including NQO1 and xCT (SLC7A11), along with decreased levels of PGC1. Omaveloxolone (OMAV), a pharmacological NRF2 activator approved for the treatment of Friedreichs ataxia, increased cell viability and upregulated NRF2 target proteins in both control and SMA fibroblasts. Notably, OMAV produced a modest increase in SMN protein abundance and PGC1 levels selectively in SMA cells. Together, these findings support diminished NRF2 pathway output as a feature of SMA fibroblasts and demonstrate that OMAV induces NRF2 target proteins in this human SMA cellular model, consistent with enhanced cytoprotective signaling. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/712434v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1904bfeorg.highwire.dtl.DTLVardef@6d20e2org.highwire.dtl.DTLVardef@89f365org.highwire.dtl.DTLVardef@ca9638_HPS_FORMAT_FIGEXP M_FIG C_FIG

Neuronal differentiation is a complex process that entails extensive morphological, transcriptional, metabolic, and functional changes that dictate neuronal lineage commitment. Much less understood is the role that epigenetic and epi-transcriptional reprogramming plays in the process of neuronal differentiation and maturation. To depict the whole landscape of transcriptomics and epigenetic changes during neuronal differentiation and maturation, we differentiated SH-SY5Y cells and performed RNA sequencing on differentiated and undifferentiated cells. 728 differentially expressed genes (DEGs) enriched in synaptic signaling and cell morphogenesis pathways were observed. Moreover, transcriptome-wide mRNA stability profiling revealed that genes with altered stability were exceptionally enriched for redox homeostasis pathways. Mature neurons are known to be highly sensitive to oxidative stress, which is crucial in the pathophysiology of neurodegenerative disease. Our results suggest that this heightened sensitivity is regulated at the mRNA stability level (i.e., epigenetic) rather than at the transcriptional level. Alternative splicing analysis revealed the exon skipping and alternative mRNA isoforms enriched for morphogenesis related pathway. Alternatively, alternative 5 and 3 prime splicing site, intron retention and mutually exclusive exon events exclusively clustered in the translation and translation initiation pathways, suggesting the potential effect of alternative splicing on translation following neuronal maturation. Splice motif analysis revealed enriched motifs for RBPs that regulate various splice types and can be further correlated to distinct phenotypical changes during neuronal differentiation and maturation. Here we present an extensive exploration of the transcriptional and epigenetic changes and their potential association with the process of neuronal differentiation, providing a new insight into understanding the molecular mechanism of neuronal function and behavior.