RESUMO
The circadian clock is encoded by a negative transcriptional feedback loop that coordinates physiology and behavior through molecular programs that remain incompletely understood. Here, we reveal rhythmic genome-wide alternative splicing (AS) of pre-mRNAs encoding regulators of peptidergic secretion within pancreatic ß cells that are perturbed in Clock-/- and Bmal1-/- ß-cell lines. We show that the RNA-binding protein THRAP3 (thyroid hormone receptor-associated protein 3) regulates circadian clock-dependent AS by binding to exons at coding sequences flanking exons that are more frequently skipped in clock mutant ß cells, including transcripts encoding Cask (calcium/calmodulin-dependent serine protein kinase) and Madd (MAP kinase-activating death domain). Depletion of THRAP3 restores expression of the long isoforms of Cask and Madd, and mimicking exon skipping in these transcripts through antisense oligonucleotide delivery in wild-type islets reduces glucose-stimulated insulin secretion. Finally, we identify shared networks of alternatively spliced exocytic genes from islets of rodent models of diet-induced obesity that significantly overlap with clock mutants. Our results establish a role for pre-mRNA alternative splicing in ß-cell function across the sleep/wake cycle.
Assuntos
Processamento Alternativo , Relógios Circadianos/genética , Exocitose , Glucose/metabolismo , Secreção de Insulina/genética , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/fisiologia , Animais , Proteínas CLOCK/genética , Proteínas CLOCK/fisiologia , Células Cultivadas , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanilato Quinases/genética , Guanilato Quinases/metabolismo , Homeostase , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Proteínas Nucleares/fisiologia , Obesidade/genética , Obesidade/metabolismo , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Fatores de Transcrição/fisiologiaAssuntos
Distrofina/genética , Morfolinos/uso terapêutico , Distrofia Muscular de Duchenne/terapia , Mutação , Reparo Gênico Alvo-Dirigido/métodos , Pesquisa Translacional Biomédica , Animais , Distrofina/metabolismo , Predisposição Genética para Doença , Humanos , Morfolinos/efeitos adversos , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/fisiopatologia , Fenótipo , Recuperação de Função Fisiológica , Reparo Gênico Alvo-Dirigido/efeitos adversos , Resultado do TratamentoRESUMO
Limb-girdle muscular dystrophy type 2C is caused by autosomal recessive mutations in the γ-sarcoglycan (SGCG) gene. The most common SGCG mutation is a single nucleotide deletion from a stretch of five thymine residues in SGCG exon 6 (521ΔT). This founder mutation disrupts the transcript reading frame, abolishing protein expression. An antisense oligonucleotide exon-skipping method to reframe the human 521ΔT transcript requires skipping four exons to generate a functional, internally truncated protein. In vivo evaluation of this multi-exon skipping, antisense-mediated therapy requires a genetically appropriate mouse model. The human and mouse γ-sarcoglycan genes are highly homologous in sequence and gene structure, including the exon 6 region harboring the founder mutation. Herein, we describe a new mouse model of this form of limb-girdle muscular dystrophy generated using CRISPR/Cas9-mediated gene editing to introduce a single thymine deletion in murine exon 6, recreating the 521ΔT point mutation in Sgcg These mice express the 521ΔT transcript, lack γ-sarcoglycan protein and exhibit a severe dystrophic phenotype. Phenotypic characterization demonstrated reduced muscle mass, increased sarcolemmal leak and fragility, and decreased muscle function, consistent with the human pathological findings. Furthermore, we showed that intramuscular administration of a murine-specific multiple exon-directed antisense oligonucleotide cocktail effectively corrected the 521ΔT reading frame. These data demonstrate a molecularly and pathologically suitable model for in vivo testing of a multi-exon skipping strategy to advance preclinical development of this genetic correction approach.
Assuntos
Éxons/genética , Edição de Genes , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/terapia , Animais , Sequência de Bases , Modelos Animais de Doenças , Fibrose , Camundongos Transgênicos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Mutação Puntual/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sarcoglicanas/genética , Sarcoglicanas/metabolismo , Sarcolema/metabolismoRESUMO
Myotonic dystrophy (DM) is the most common autosomal dominant muscular dystrophy and encompasses both skeletal muscle and cardiac complications. DM is nucleotide repeat expansion disorder in which type 1 (DM1) is due to a trinucleotide repeat expansion on chromosome 19 and type 2 (DM2) arises from a tetranucleotide repeat expansion on chromosome 3. Developing representative models of DM in animals has been challenging due to instability of nucleotide repeat expansions, especially for DM2, which is characterized by nucleotide repeat expansions often greater than 5,000 copies. To investigate mechanisms of human DM, we generated cellular models of DM1 and DM2. We used regulated MyoD expression to reprogram urine-derived cells into myotubes. In this myogenic cell model, we found impaired dystrophin expression, in the presence of muscleblind-like 1 (MBNL1) foci, and aberrant splicing in DM1 but not in DM2 cells. We generated induced pluripotent stem cells (iPSC) from healthy controls and DM1 and DM2 subjects, and we differentiated these into cardiomyocytes. DM1 and DM2 cells displayed an increase in RNA foci concomitant with cellular differentiation. iPSC-derived cardiomyocytes from DM1 but not DM2 had aberrant splicing of known target genes and MBNL sequestration. High-resolution imaging revealed tight association between MBNL clusters and RNA foci in DM1. Ca2+ transients differed between DM1- and DM2 iPSC-derived cardiomyocytes, and each differed from healthy control cells. RNA-sequencing from DM1- and DM2 iPSC-derived cardiomyocytes revealed distinct misregulation of gene expression, as well as differential aberrant splicing patterns. Together, these data support that DM1 and DM2, despite some shared clinical and molecular features, have distinct pathological signatures.
Assuntos
Predisposição Genética para Doença/genética , Proteína MyoD/metabolismo , Distrofia Miotônica/genética , Distrofia Miotônica/patologia , Cálcio/metabolismo , Linhagem Celular , Distrofina/metabolismo , Expressão Gênica , Variação Genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Distrofia Miotônica/classificação , Distrofia Miotônica/urina , Splicing de RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Exon skipping uses chemically modified antisense oligonucleotides to modulate RNA splicing. Therapeutically, exon skipping can bypass mutations and restore reading frame disruption by generating internally truncated, functional proteins to rescue the loss of native gene expression. Limb-girdle muscular dystrophy type 2C is caused by autosomal recessive mutations in the SGCG gene, which encodes the dystrophin-associated protein γ-sarcoglycan. The most common SGCG mutations disrupt the transcript reading frame abrogating γ-sarcoglycan protein expression. In order to treat most SGCG gene mutations, it is necessary to skip 4 exons in order to restore the SGCG transcript reading frame, creating an internally truncated protein referred to as Mini-Gamma. Using direct reprogramming of human cells with MyoD, myogenic cells were tested with 2 antisense oligonucleotide chemistries, 2'-O-methyl phosphorothioate oligonucleotides and vivo-phosphorodiamidate morpholino oligomers, to induce exon skipping. Treatment with vivo-phosphorodiamidate morpholino oligomers demonstrated efficient skipping of the targeted exons and corrected the mutant reading frame, resulting in the expression of a functional Mini-Gamma protein. Antisense-induced exon skipping of SGCG occurred in normal cells and those with multiple distinct SGCG mutations, including the most common 521ΔT mutation. These findings demonstrate a multiexon-skipping strategy applicable to the majority of limb-girdle muscular dystrophy 2C patients.
Assuntos
Morfolinos/genética , Sarcoglicanopatias/genética , Sarcoglicanopatias/terapia , Sarcoglicanas/genética , Células Cultivadas , Reprogramação Celular , Éxons , Fibroblastos/metabolismo , Terapia Genética , Humanos , Microscopia de Fluorescência , Mutação , Cultura Primária de Células , Splicing de RNA , Fases de Leitura , Sarcoglicanopatias/metabolismo , Sarcoglicanas/metabolismo , Transdução Genética , Urina/citologiaRESUMO
Exon skipping uses antisense oligonucleotides (ASOs) to alter transcript splicing for the purpose of rescuing or modulating protein expression. In this issue of the JCI, Lee and colleagues developed and evaluated an ASO-dependent method for treating certain molecularly defined diseases associated with alterations in lamin A/C (LMNA) splicing. Exon skipping by ASOs is gaining traction as a therapeutic strategy, and the use of ASOs is now being applied to bypass mutations and generate modified but functional proteins for an array of genetic disorders.
Assuntos
Processamento Alternativo/efeitos dos fármacos , Lamina Tipo A/biossíntese , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Progéria/tratamento farmacológico , Progéria/metabolismo , RNA Mensageiro/metabolismo , Animais , HumanosRESUMO
BACKGROUND: Cellular models of muscle disease are taking on increasing importance with the large number of genes and mutations implicated in causing myopathies and the concomitant need to test personalized therapies. Developing cell models relies on having an easily obtained source of cells, and if the cells are not derived from muscle itself, a robust reprogramming process is needed. Fibroblasts are a human cell source that works well for the generation of induced pluripotent stem cells, which can then be differentiated into cardiomyocyte lineages, and with less efficiency, skeletal muscle-like lineages. Alternatively, direct reprogramming with the transcription factor MyoD has been used to generate myotubes from cultured human fibroblasts. Although useful, fibroblasts require a skin biopsy to obtain and this can limit their access, especially from pediatric populations. RESULTS: We now demonstrate that direct reprogramming of urine-derived cells is a highly efficient and reproducible process that can be used to establish human myogenic cells. We show that this method can be applied to urine cells derived from normal individuals as well as those with muscle diseases. Furthermore, we show that urine-derived cells can be edited using CRISPR/Cas9 technology. CONCLUSIONS: With progress in understanding the molecular etiology of human muscle diseases, having a readily available, noninvasive source of cells from which to generate muscle-like cells is highly useful.
Assuntos
Reprogramação Celular , Desenvolvimento Muscular , Doenças Musculares/fisiopatologia , Proteína MyoD/metabolismo , Urina/citologia , Técnicas de Cultura de Células , Diferenciação Celular , Células Cultivadas , Células Clonais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Humanos , Doenças Musculares/metabolismo , Distrofias Musculares/metabolismo , Distrofias Musculares/fisiopatologiaRESUMO
The New Directions in the Biology and Disease of Skeletal Muscle is a scientific meeting, held every other year, with the stated purpose of bringing together scientists, clinicians, industry representatives and patient advocacy groups to disseminate new discovery useful for treatment inherited forms of neuromuscular disease, primarily the muscular dystrophies. This meeting originated as a response the Muscular Dystrophy Care Act in order to provide a venue for the free exchange of information, with the emphasis on unpublished or newly published data. Highlights of this years' meeting included results from early phase clinical trials for Duchenne Muscular Dystrophy, progress in understanding the epigenetic defects in Fascioscapulohumeral Muscular Dystrophy and new mechanisms of muscle membrane repair. The following is a brief report of the highlights from the conference.