Biology subjects

Furling, D.

Publications and source records attributed to Furling, D..

9 recordsLinked to original sources

bioRxiv2026

Single-nucleotide resolution profiling reveals dynamic and site-specific m⁶A regulation during human myogenesis

BackgroundMyogenic differentiation of muscle stem cells required for skeletal muscle growth and regeneration relies on the precise spatiotemporal coordination of multiple gene-expression programs. Over the past decades, considerable progress has been made in defining the transcriptional networks that govern myogenesis. In particular, myogenic transcription factors integrate extrinsic and intrinsic cues to drive skeletal muscle development and regeneration. In contrast, post-transcriptional layers of regulation have remained comparatively underexplored. Among these, the epitranscriptome - comprising more than 170 chemical RNA modifications - has recently emerged as a major regulatory axis in cellular differentiation. N-methyladenosine (mA), the most abundant internal modification of mRNA, is now recognized as a key regulator of stem-cell fate decisions across multiple biological systems. By dynamically modulating RNA stability, translation, and other processing steps, mA enables precise fine-tuning of gene-expression programs in response to physiological cues. These effects are mediated through mA-binding RNA-binding proteins (RBPs), also referred to as "readers", which selectively recognize methylated transcripts and translate the modification into functional cellular outcomes. Despite its central role in other tissue contexts, the contribution of mA to skeletal muscle physiology - and particularly to human muscle stem cell biology - remains poorly characterized. This gap represents a critical and largely unexplored frontier in muscle biology. MethodsTo define the epitranscriptome landscape during the myogenic differentiation of human skeletal muscle cells, we performed GLORI on proliferating and differentiated human primary myoblasts. This recently developed approach enables detection of mA at single-nucleotide resolution together with quantitative measurement of methylation stoichiometry at individual sites. mA-modified positions were mapped across the transcriptome in both cellular states and integrated with matched RNA-seq datasets data to contextualise mA features relative to transcript abundance. To place these findings in a broader context, we compared our human mA maps with previously published mouse muscle cell datasets and overlaid them with available CLIP-seq datasets of mA RNA-binding proteins. ResultsWe identified tens of thousands of high-confidence mA sites in proliferating and differentiated human myogenic cells. While many sites were shared between states, myogenic differentiation was accompanied by epitranscriptomic remodeling, affecting both the distribution and stoichiometry of mA marks. Transcripts exhibiting differential mA regulation were enriched for pathways related to myogenic development, cytoskeletal remodeling, signalling, nucleic-acid-associated processes and energy metabolism. Joint analysis of mA site number, methylation stoichiometry, and expression levels indicated the presence of distinct mA regulatory frameworks at the transcript level. Genes associated with myogenesis and regulatory pathways were distributed across these categories, consistent with the coexistence of multiple regulatory programs during myogenic differentiation. In addition, cross-species comparisons revealed the presence of human-specific methylation patterns. Integration with published mA RNA-binding protein datasets uncovered distinct subsets of mA sites with varying binding profiles, suggesting state-dependent interpretation of mA marks. Together, these data constitute, to our knowledge, the first single-nucleotide-resolution atlas of mA methylation in human skeletal muscle, revealing a previously uncharacterized epitranscriptomic landscape and providing a foundational resource for studies of human myogenesis.

molecular biology↗

bioRxiv2025

Dysregulated MEG3 in Myotonic Dystrophy 1: nuclear retention, pathological role, and therapeutic correction by antisense conjugates.

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.

molecular biology↗

bioRxiv2024

Alternative splicing of SORBS1 affects neuromuscular junction formation and stability in myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is a multisystemic neuromuscular disease characterized by a CTG repeat expansion in the 3" untranslated region of the gene coding for the dystrophia myotonica protein kinase (DMPK). Presence of expanded CTG repeats in DMPK-mRNAs leads to the sequestration of RNA binding factors such as the Muscleblind like (MBNL) proteins resulting in widespread splicing defects contributing to progressive muscle weakness and myotonia. Here, we show that abnormal splicing of SORBS1 exon 25 found in skeletal muscle of myotonic dystrophy type 1 patients is a critical contributor to neuromuscular junction (NMJ) formation and maintenance. Forced exclusion of SORBS1 exon 25 in mice results in NMJ degeneration with marked denervation and postsynaptic destabilization. In zebrafish, misregulation of sorbs1 exon 25 results in reduced motor function and abnormal AChR cluster morphology. Finally, we observed that forcing SORBS1 exon 25 exclusion in hiPSC-derived skeletal muscle cells reduces the formation of large AChR clusters upon agrin stimulation. Thus, our study identifies MBNL regulated SORBS1 alternative splicing during skeletal muscle development as a critical event for NMJ formation and maintenance. The aberrant splicing of SORBS1 exon 25 in DM1 expands our understanding of how splicing dysregulation compromises neuromuscular system communication, shedding light on the broader impact of mRNA splicing regulation on NMJ integrity.

molecular biology↗

bioRxiv2024

CCG interruptions are unstable and hypermethylated in DM1 patients

Myotonic dystrophy type 1 (DM1) exhibits highly heterogeneous clinical manifestations caused by an unstable CTG repeat expansion reaching up to 4,000 CTG. The dynamics of CTG repeats and clinical variability depends on the CTG repeat number, CNG repeat interruptions, DNA methylation, somatic mosaicism, but also gene modifiers. Around 10% of the DM1 population carries triplet repeat interruptions (CCG, CGG, CTC, CAG), which differ in number and nature between DM1 families. CCG interruptions have been associated with stabilization of the CTG repeat expansions and a milder phenotype in the DM1 population. However, the dynamics and precise role of interruptions in CTG repeat instability remain relatively underexplored due to the complexities of analyzing them. In this study, we showed that the number of CCG interruptions within the expansion varies within tissues and between generations using single-molecule real-time long-read sequencing. Although the interrupted expanded alleles showed global stabilization, the CCG interruptions themselves displayed variability across generations and within somatic tissues. Importantly, our findings reveal, for the first time, CCG hypermethylation within the CTG expansion, which is linked to downstream hypermethylation of the repeat. These results support the hypothesis that methylation of CCG interruptions within the expanded DMPK alleles may contribute to the stabilization of trinucleotide repeats.

molecular biology↗

bioRxiv2023

TALEN-induced contraction of CTG trinucleotide repeats in myotonic dystrophy type 1 cells

Trinucleotide repeat expansions are the cause of two dozen neurodegenerative and developmental disorders. One of these, myotonic dystrophy type 1 (Steinert disease, or DM1) is due to the expansion of a CTG triplet in the 3 UTR of the DMPK gene. We used highly specific DNA endonucleases to induce a double-strand break in the repeat tract to contract it below pathological length. Expression of a TALE Nuclease (TALEN) in human DM1 cells induced moderate CTG repeat contractions in 27% of the clones analyzed. These clones exhibited large internal deletions within the TALEN, occurring by homologous recombination between internal TALE repeats, inactivating the nuclease, and explaining its reduced efficacy. Taking advantage of the degeneracy of the genetic code, we recoded the TALEN sequence, to decrease internal redundancy and optimize codon usage. The new recoded TALEN showed increased efficacy in DM1 cells, with 68% of clones exhibiting a moderate to large contraction of the CTG repeat tract. In contrast, Staphylococcus aureus Cas9 (SaCas9) was unable to contract the CTG repeat tract. In parallel, we completely sequenced to very high coverage the DM1 genome using the PacBio technology. Several clones in which the TALEN was induced were also totally sequenced. In some of them, length changes of other long CTG repeats were detected, possibly corresponding to off-target effects, all of them in introns or intergenic regions. Repeat contractions were never associated with recombination of flanking markers, suggesting that contractions most probably occur by an intra-allelic mechanism such as single-strand annealing. TALENs should now be considered as a promising gene therapy approach, not only for DM1 but also for many other microsatellite expansion disorders.

genomics↗

bioRxiv2022

Identification of a CCG-enriched expanded allele in DM1 patients using Amplification-free long-read sequencing

Myotonic dystrophy type 1 (DM1) exhibits highly heterogeneous clinical manifestations caused by an unstable CTG repeat expansion reaching up to 4,000 CTG. The clinical variability depends on CTG repeat number, CNG repeat interruptions and somatic mosaicism. Currently, none of these factors are simultaneously and accurately determined due to the limitations of gold standard methods used in clinical and research laboratories. An amplicon method for targeting DM1 locus using Single-Molecule Real-Time sequencing was recently developed to accurately analyze expanded alleles. However, amplicon-based sequencing still depends on PCR and the inherent bias towards preferential amplification of smaller repeats can be problematic in DM1. Thus, an amplification-free long-read sequencing method was developed using the CRISPR/Cas9 technology in DM1. This method was used to sequence the DM1 locus in patients with CTG repeat expansion ranging from 130 to > 1000 CTG. We showed that elimination of PCR amplification improves the accuracy of measurement of inherited repeat number and somatic repeat variations, two important key factors in the DM1 severity and age at onset. For the first time, an expansion composed of over 85% CCG repeats was identified using this innovative method in a DM1 family with an atypical clinical profile. No-Amplification targeted sequencing represents a promising method that can overcome research and diagnosis shortcomings, with translational implications for clinical and genetic counseling in DM1.

molecular biology↗

bioRxiv2021

The beneficial effect of chronic muscular exercise on muscle fragility is increased by Prox1 gene transfer in dystrophic mdx muscle

PurposeGreater muscle fragility is thought to cause the exhaustion of the muscle stem cells during successive degeneration/repair cycles, leading to muscle wasting and weakness in Duchenne muscular dystrophy. Chronic voluntary exercise can partially reduce the susceptibility to contraction induced-muscle injury, i.e., muscle fragility, as shown by a reduced immediate maximal force drop following lengthening contractions, in the dystrophic mdx mice. Here, we studied the effect of Prospero-related homeobox factor 1 gene (Prox1) transfer (overexpression) on fragility in chronically exercised mdx mice, because Prox1 promotes slower type fibres in healthy mice and slower fibres are less fragile in mdx muscle. Methodsmdx mice received or not Prox1 transfer into the tibialis anterior muscle and performed voluntary running into a wheel during 1 month. We also performed Prox1 transfer in sedentary mdx mice. In situ maximal force production of the muscle in response to nerve stimulation was assessed before, during and after 10 lengthening contractions. Molecular and cellular parameters were also evaluated. ResultsInterestingly, Prox1 transfer reduced the force drop following lengthening contractions in exercised mdx mice (p < 0.05 to 0.01), but not in sedentary mdx mice. It also increased the muscle expression of Myh7 (p < 0.001), MHC-2x (p < 0.01) and Trpc1 (p < 0.01), whereas it reduced that one of Myh4 (p < 0.001) and MHC-2b (p < 0.01) in exercised mdx mice. Moreover, Prox1 transfer decreased the maximal force (p < 0.01) before lengthening contraction in exercised mdx mice (p < 0.01), and reduced muscle weight (p < 0.0001) despite increased Mstn expression (p < 0.001). ConclusionOur results indicate that the beneficial effect of Prox1 transfer on muscle fragility is only observed in chronically exercised mdx mice. Thus, Prox1 transfer combined to chronic exercise have the potential to substantially slow the progression of the dystrophic disease in the long term.

physiology↗

bioRxiv2019

Desmin is a modifier of dystrophic muscle features in Mdx mice

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease, caused by dystrophin deficiency. Desmin is like dystrophin associated to costameric structures bridging sarcomeres to extracellular matrix that are involved in force transmission and skeletal muscle integrity. In the present study, we wanted to gain further insight into the roles of desmin which expression is increased in the muscle from the mouse Mdx DMD model. We show that a deletion of the desmin gene (Des) in Mdx mice (DKO, Mdx:desmin-/-) induces a marked worsening of the weakness (reduced maximal force production) as compared to Mdx mice. Fragility (higher susceptibility to contraction-induced injury) was also aggravated and fatigue resistance was reduced in DKO mice. Moreover, in contrast to Mdx mice, the DKO mice did not undergo a muscle hypertrophy because of smaller and less numerous fibers, with reduced percentage of centronucleated fibres. Interestingly, Desmin cDNA transfer with adeno-associated virus in 1-month-old DKO mice and newborn Mdx mice improved muscle weakness. Overall, desmin plays important and beneficial roles on muscle performance, fragility and remodelling in dystrophic Mdx mice.

pathology↗

bioRxiv2018

Dysregulation of circular RNAs in myotonic dystrophy type 1

Circular RNAs (circRNAs) constitute a recently re-discovered class of non-coding RNAs functioning as sponge for miRNAs and proteins, affecting RNA splicing and regulating transcription. CircRNAs are generated by \"back-splicing\", linking covalently 3- and 5-ends of exons. Thus, circRNA levels might be deregulated in conditions associated to altered RNA-splicing. Indeed, increasing evidence indicates their role in human diseases. Specifically, myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by expanded CTG-repeats in the DMPK gene, resulting in abnormal mRNA-splicing. In this investigation, circRNAs expressed in DM1 skeletal muscles were identified by analyzing RNA-sequencing data-sets followed by qPCR validation. In muscle biopsies, out of 9 tested, 4 transcripts showed an increased circular fraction: CDYL, HIPK3, RTN4_03 and ZNF609. The circular fraction values correlated positively with skeletal muscle strength and Receiver-Operating-Characteristics curves showed that these four circRNAs allow to distinguish DM1 patients from controls. The identified circRNAs were also detectable in peripheral-blood-mononuclear-cells (PBMCs) and plasma of DM1 patients, but they were not regulated significantly, indicating a tissue-selectivity of the identified modulations. Finally, increased circular fractions of RTN4_03 and ZNF609 were also observed in differentiated myogenic cell lines derived from DM1 patients.\n\nIn conclusion, this proof-of-principle study identified circRNA dysregulation in DM1 patients.

molecular biology↗