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Differential splicing of the large sarcomeric protein nebulin during skeletal muscle development.

ABSTRACT: We studied differential splicing of nebulin, a giant filamentous F-actin binding protein (M(r) approximately 700-800kDa) that is found in skeletal muscle. Nebulin spans the thin filament length, its C-terminus is anchored in the Z-disc, and its N-terminal region is located toward the thin filament pointed end. Various lines of evidence indicate that nebulin plays important roles in thin filament and Z-disc structure in skeletal muscle. In the present work we studied nebulin in a range of muscle types during postnatal development and performed transcript studies with a mouse nebulin exon microarray, developed by us, whose results were confirmed by RT-PCR. We also performed protein studies with high-resolution SDS-agarose gels and Western blots, and structural studies with electron microscopy. We found during postnatal development of the soleus muscle major changes in splicing in both the super-repeat region and the Z-disc region of nebulin; interestingly, these changes were absent in other muscle types. Three novel Z-disc exons, previously described in the mouse gene, were upregulated during postnatal development of soleus muscle and this was correlated with a significant increase in Z-disc width. These findings support the view that nebulin plays an important role in Z-disc width regulation. In summary, we discovered changes in both the super-repeat region and the Z-disc region of nebulin, that these changes are muscle-type specific, and that they correlate with differences in sarcomere structure.

SUBMITTER: Buck D

PROVIDER: S-EPMC2856706 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Differential splicing of the large sarcomeric protein nebulin during skeletal muscle development.

Buck Danielle D Hudson Bryan D BD Ottenheijm Coen A C CA Labeit Siegfried S Granzier Henk H

Journal of structural biology 20100220 2

We studied differential splicing of nebulin, a giant filamentous F-actin binding protein (M(r) approximately 700-800kDa) that is found in skeletal muscle. Nebulin spans the thin filament length, its C-terminus is anchored in the Z-disc, and its N-terminal region is located toward the thin filament pointed end. Various lines of evidence indicate that nebulin plays important roles in thin filament and Z-disc structure in skeletal muscle. In the present work we studied nebulin in a range of muscle ...[more]

PMID: 20176113

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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.

Dataset Information

Differential splicing of the large sarcomeric protein nebulin during skeletal muscle development.

Publications

Differential splicing of the large sarcomeric protein nebulin during skeletal muscle development.

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