Abnormal splicing switch of DMD's penultimate exon compromises muscle fibre maintenance in myotonic dystrophy

International audienceMyotonic Dystrophy type 1 (DM1) is a dominant neuromuscular disease caused by nuclear-retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors resulting in alternative splicing misregulation and muscular dysfunction. Here we show that the abnormal splicing of DMD exon 78 found in dystrophic muscles of DM1 patients is due to the functional loss of MBNL1 and leads to the re-expression of an embryonic dystrophin in place of the adult isoform. Forced expression of embryonic dystrophin in zebrafish using an exon-skipping approach severely impairs the mobility and muscle architecture. Moreover, reproducing Dmd exon 78 missplicing switch in mice induces muscle fibre remodelling and ultrastructural abnormalities including ringed fibres, sarcoplasmic masses or Z-band disorganization, which are characteristic features of dystrophic DM1 skeletal muscles. Thus, we propose that splicing misregulation of DMD exon 78 compromises muscle fibre maintenance and contributes to the progressive dystrophic process in DM

Étude de la dérégulation de miR-1 dans le coeur de patients atteints de dystrophies myotoniques

Les dystrophies myotoniques sont les formes les plus courantes de dystrophies musculaires observées chez l adulte. Les patients souffrent de défauts de la conduction et du rythme cardiaques. Les DM sont dues à l expression d ARN mutés contenant des expansions de répétitions des nucléotides CUG ou CCUG. Il a été montré que ces expansions de répétitions interfèrent avec la fonction de MBNL1 et CUGBP1. Les microARN (miARN) jouent un rôle important dans le fonctionnement du cœur. Nous avons identifié une diminution significative de l expression de miR-1 dans des échantillons de cœur de patients DM. En accord avec cette diminution, les cibles de miR-1 sont surexprimées au niveau protéique dans le cœur de patients DM. De plus, nous avons observé une diminution de l expression de la forme mature de miR-1 alors que la quantité de son précurseur, pré-miR-1, est inchangée, suggérant un défaut de la maturation de pré-miR-1 dans le cœur de patients DM. La surexpression de longues répétitions CUG ou la déplétion de MBNL1 par shARN inhibent la maturation de pré-miR-1 suggèrant une régulation de la maturation de miR-1 par MBNL1. D autre part, nous avons montré que la voie Lin28-TUT4 inhibe la maturation de pré-miR-1 par Dicer. Finalement, MBNL1 et Lin28 entrent en compétition pour la liaison de pré-miR-1 suggérant un modèle selon lequel MBNL1 protégerait l uridylation de pré-miR-1 par TUT4 en empêchant Lin28 de se lier. En conclusion, nos résultats démontrent une dérégulation de la maturation d un miARN chez les patients DM. Enfin, nous proposons que la diminution de miR-1 dans le cœur de patients DM puisse participer aux défauts cardiaques observés chez les patients DM.Myotonic Dystrophy (DM) is the most common form of muscular dystrophy in adult and is characterized by several symptoms including cardiac conduction abnormalities and arrhythmia resulting in sudden death. DM is caused by expansions of CUG or CCUG repeats which interfere with pre-mRNAs processing through dysfunction of the MBNL1 and CUGBP1 RNA-binding proteins. Micro-RNAs (miRNA) play an important role in heart functions. Using microarray and qRT-PCR, we identified a downregulation of miR-1, in heart samples of Myotonic Dystrophic patients. In agreement with decreased level of miR-1 we found that targets of miR-1 are up-regulated at the protein level in DM1. Importantly, while we found a down-regulation of the mature miR-1, we observed no changes in the quantity of pre-miR-1 both in DM1 cell models and patient heart samples. We demonstrated that expanded CUG repeats and shRNA-mediated depletion of MBNL1 inhibit exogenous and endogenous pre-miR-1 processing. Furthermore, we found that MBNL1 binds UGC motifs located within the pre-miR-1 loop. Next, we observed that Lin28 binds pre-miR-1 loop and promotes uridylation of pre-miR-1 by TUT4. Finally, we demonstrated that MBNL1 and Lin28 compete for pre-miR-1 binding suggesting a model in which MBNL1 protects pre-miR-1 from uridylation by Lin28-TUT4. In conclusion, our results demonstrate for the first time that miRNA maturation is altered in Myotonic Dystrophic patients, and suggest a novel function to MBNL1 as a regulator of miRNA processing and a new target of Lin28-TUT4: miR-1. We propose that the down-regulation of miR-1 may be involved in the heart defects observed in DM patients.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Cells of Matter—In Vitro Models for Myotonic Dystrophy

Myotonic dystrophy type 1 (DM1 also known as Steinert disease) is a multisystemic disorder mainly characterized by myotonia, progressive muscle weakness and wasting, cognitive impairments, and cardiac defects. This autosomal dominant disease is caused by the expression of nuclear retained RNAs containing pathologic expanded CUG repeats that alter the function of RNA-binding proteins in a tissue-specific manner, leading ultimately to neuromuscular dysfunction and clinical symptoms. Although considerable knowledge has been gathered on myotonic dystrophy since its first description, the development of novel relevant disease models remains of high importance to investigate pathophysiologic mechanisms and to assess new therapeutic approaches. In addition to animal models, in vitro cell cultures provide a unique resource for both fundamental and translational research. This review discusses how cellular models broke ground to decipher molecular basis of DM1 and describes currently available cell models, ranging from exogenous expression of the CTG tracts to variable patients’ derived cells

TDP-43 condensation properties specify its RNA-binding and regulatory repertoire

Mutations causing amyotrophic lateral sclerosis (ALS) often affect the condensation properties of RNA-binding proteins (RBPs). However, the role of RBP condensation in the specificity and function of protein-RNA complexes remains unclear. We created a series of TDP-43 C-terminal domain (CTD) variants that exhibited a gradient of low to high condensation propensity, as observed in vitro and by nuclear mobility and foci formation. Notably, a capacity for condensation was required for efficient TDP-43 assembly on subsets of RNA-binding regions, which contain unusually long clusters of motifs of characteristic types and density. These “binding-region condensates” are promoted by homomeric CTD-driven interactions and required for efficient regulation of a subset of bound transcripts, including autoregulation of TDP-43 mRNA. We establish that RBP condensation can occur in a binding-region-specific manner to selectively modulate transcriptome-wide RNA regulation, which has implications for remodeling RNA networks in the context of signaling, disease, and evolution

Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds

International audienceMyotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant neuromuscular diseases caused by microsatellite expansions and belong to the family of RNA-dominant disorders. Availability of cellular models in which the DM mutation is expressed within its natural context is essential to facilitate efforts to identify new therapeutic compounds. Here, we generated immortalized DM1 and DM2 human muscle cell lines that display nuclear RNA aggregates of expanded repeats, a hallmark of myotonic dystrophy. Selected clones of DM1 and DM2 immortalized myoblasts behave as parental primary myoblasts with a reduced fusion capacity of immortalized DM1 myoblasts when compared with control and DM2 cells. Alternative splicing defects were observed in differentiated DM1 muscle cell lines, but not in DM2 lines. Splicing alterations did not result from differentiation delay because similar changes were found in immortalized DM1 transdifferentiated fibroblasts in which myogenic differentiation has been forced by overexpression of MYOD1. As a proof-of-concept, we show that antisense approaches alleviate disease-associated defects, and an RNA-seq analysis confirmed that the vast majority of mis-spliced events in immortalized DM1 muscle cells were affected by antisense treatment, with half of them significantly rescued in treated DM1 cells. Immortalized DM1 muscle cell lines displaying characteristic disease-associated molecular features such as nuclear RNA aggregates and splicing defects can be used as robust readouts for the screening of therapeutic compounds. Therefore, immortalized DM1 and DM2 muscle cell lines represent new models and tools to investigate molecular pathophysiological mechanisms and evaluate the in vitro effects of compounds on RNA toxicity associated with myotonic dystrophy mutations

Reversal of RNA toxicity in myotonic dystrophy via a decoy RNA-binding protein with high affinity for expanded CUG repeats

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