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Alternative Splicing of Four Trafficking Genes Regulates Myofiber Structure and Skeletal Muscle Physiology.

ABSTRACT: During development, transcriptional and post-transcriptional networks are coordinately regulated to drive organ maturation. Alternative splicing contributes by producing temporal-specific protein isoforms. We previously found that genes undergoing splicing transitions during mouse postnatal heart development are enriched for vesicular trafficking and membrane dynamics functions. Here, we show that adult trafficking isoforms are also expressed in adult skeletal muscle and hypothesize that striated muscle utilizes alternative splicing to generate specific isoforms required for function of adult tissue. We deliver morpholinos into flexor digitorum brevis muscles in adult mice to redirect splicing of four trafficking genes to the fetal isoforms. The splicing switch results in multiple structural and functional defects, including transverse tubule (T-tubule) disruption and dihydropyridine receptor alpha (DHPR) and Ryr1 mislocalization, impairing excitation-contraction coupling, calcium handling, and force generation. The results demonstrate a previously unrecognized role for trafficking functions in adult muscle tissue homeostasis and a specific requirement for the adult splice variants.

SUBMITTER: Giudice J

PROVIDER: S-EPMC5140099 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Alternative Splicing of Four Trafficking Genes Regulates Myofiber Structure and Skeletal Muscle Physiology.

Giudice Jimena J Loehr James A JA Rodney George G GG Cooper Thomas A TA

Cell reports 20161101 8

During development, transcriptional and post-transcriptional networks are coordinately regulated to drive organ maturation. Alternative splicing contributes by producing temporal-specific protein isoforms. We previously found that genes undergoing splicing transitions during mouse postnatal heart development are enriched for vesicular trafficking and membrane dynamics functions. Here, we show that adult trafficking isoforms are also expressed in adult skeletal muscle and hypothesize that striate ...[more]

PMID: 27851958

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Project description:BackgroundAs the interest in manned spaceflight increases, so does the requirement to understand the transcriptomic mechanisms that underlay the detrimental physiological adaptations of skeletal muscle to microgravity. While microgravity-induced differential gene expression (DGE) has been extensively investigated, the contribution of differential alternative splicing (DAS) to the plasticity and functional status of the skeletal muscle transcriptome has not been studied in an animal model. Therefore, by evaluating both DGE and DAS across spaceflight, we set out to provide the first comprehensive characterization of the transcriptomic landscape of skeletal muscle during exposure to microgravity.MethodsRNA-sequencing, immunohistochemistry, and morphological analyses were conducted utilizing total RNA and tissue sections isolated from the gastrocnemius and quadriceps muscles of 30-week-old female BALB/c mice exposed to microgravity or ground control conditions for 9 weeks.ResultsIn response to microgravity, the skeletal muscle transcriptome was remodeled via both DGE and DAS. Importantly, while DGE showed variable gene network enrichment, DAS was enriched in structural and functional gene networks of skeletal muscle, resulting in the expression of alternatively spliced transcript isoforms that have been associated with the physiological changes to skeletal muscle in microgravity, including muscle atrophy and altered fiber type function. Finally, RNA-binding proteins, which are required for regulation of pre-mRNA splicing, were themselves differentially spliced but not differentially expressed, an upstream event that is speculated to account for the downstream splicing changes identified in target skeletal muscle genes.ConclusionsOur work serves as the first investigation of coordinate changes in DGE and DAS in large limb muscles across spaceflight. It opens up a new opportunity to understand (i) the molecular mechanisms by which splice variants of skeletal muscle genes regulate the physiological adaptations of skeletal muscle to microgravity and (ii) how small molecule splicing regulator therapies might thwart muscle atrophy and alterations to fiber type function during prolonged spaceflight.

Project description:Rationale: The adult skeletal muscle can self-repair efficiently following mechanical or pathological damage due to its remarkable regenerative capacity. However, regulatory mechanisms underlying muscle regeneration are complicated and have not been fully elucidated. Alternative splicing (AS) is a major mechanism responsible for post-transcriptional regulation. Many aberrant AS events have been identified in patients with muscular dystrophy which is accompanied by abnormal muscle regeneration. However, little is known about the correlation between AS and muscle regeneration. It has been reported that RNA binding motif protein 24 (Rbm24), a tissue-specific splicing factor, is involved in embryo myogenesis while the role of Rbm24 in adult myogenesis (also called muscle regeneration) is poorly understood. Methods: To investigate the role of Rbm24 in adult skeletal muscle, we generated Rbm24 conditional knockout mice and satellite cell-specific knockout mice. Furthermore, a cardiotoxin (CTX)-induced injury model was utilized to assess the effects of Rbm24 on skeletal muscle regeneration. Genome-wide RNA-Seq was performed to identify the changes in AS following loss of Rbm24. Results: Rbm24 knockout mice displayed abnormal regeneration 4 months after tamoxifen treatment. Using RNA-Seq, we found that Rbm24 regulated a complex network of AS events involved in multiple biological processes, including myogenesis, muscle regeneration and muscle hypertrophy. Moreover, using a CTX-induced injury model, we showed that loss of Rbm24 in skeletal muscle resulted in myogenic fusion and differentiation defects and significantly delayed muscle regeneration. Furthermore, satellite cell-specific Rbm24 knockout mice recapitulated the defects in regeneration seen in the global Rbm24 knockout mice. Importantly, we demonstrated that Rbm24 regulated AS of Mef2d, Naca, Rock2 and Lrrfip1 which are essential for myogenic differentiation and muscle regeneration. Conclusions: The present study demonstrated that Rbm24 regulates dynamic changes in AS and is essential for adult skeletal muscle regeneration.

Dataset Information

Alternative Splicing of Four Trafficking Genes Regulates Myofiber Structure and Skeletal Muscle Physiology.

Publications

Alternative Splicing of Four Trafficking Genes Regulates Myofiber Structure and Skeletal Muscle Physiology.

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