Dataset Information

Effects of age and hindlimb immobilization and remobilization on fast troponin T precursor mRNA alternative splicing in rat gastrocnemius muscle.

ABSTRACT: Fast skeletal muscle troponin T (TNNT3) is an important component of the skeletal muscle contractile machinery. The precursor mRNA (pre-mRNA) encoding TNNT3 is alternatively spliced, and changes in the pattern of TNNT3 splice form expression are associated with alterations in thin-filament calcium sensitivity and force production during muscle contraction and thereby regulate muscle function. Interestingly, during aging, the muscle force/cross-sectional area is reduced, suggesting that loss of mass does not completely account for the impaired muscle function that develops during the aging process. Therefore, in this study, we tested the hypothesis that age and changes in muscle loading are associated with alterations in Tnnt3 alternative splicing in the rat gastrocnemius muscle. We found that the relative abundance of several Tnnt3 splice forms varied significantly with age among 2-, 9-, and 18-month-old rats and that the pattern correlated with changes in body mass rather than muscle mass. Hindlimb immobilization for 7 days resulted in dramatic alterations in splice form relative abundance such that the pattern was similar to that observed in lighter animals. Remobilization for 7 days restored the splicing pattern toward that observed in the nonimmobilized limb, even though muscle mass had not yet begun to recover. In conclusion, the results suggest that Tnnt3 pre-mRNA alternative splicing is modulated rapidly (i.e., within days) in response to changes in the load placed on the muscle. Moreover, the results show that restoration of Tnnt3 alternative splicing to control patterns is initiated prior to an increase in muscle mass.

SUBMITTER: Ravi S

PROVIDER: S-EPMC4876695 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Effects of age and hindlimb immobilization and remobilization on fast troponin T precursor mRNA alternative splicing in rat gastrocnemius muscle.

Ravi Suhana S Schilder Rudolf J RJ Berg Arthur S AS Kimball Scot R SR

Applied physiology, nutrition, and metabolism = Physiologie appliquee, nutrition et metabolisme 20151016 2

Fast skeletal muscle troponin T (TNNT3) is an important component of the skeletal muscle contractile machinery. The precursor mRNA (pre-mRNA) encoding TNNT3 is alternatively spliced, and changes in the pattern of TNNT3 splice form expression are associated with alterations in thin-filament calcium sensitivity and force production during muscle contraction and thereby regulate muscle function. Interestingly, during aging, the muscle force/cross-sectional area is reduced, suggesting that loss of m ...[more]

PMID: 26799695

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Project description:BackgroundLong-term exposure to microgravity will cause skeletal muscle atrophy, which can cause serious harm to astronauts in space travel. Therefore, it is important to explore skeletal muscle atrophy's molecular mechanism for its prevention and treatment. However, as an important regulatory approach of skeletal muscle physiology, the role of alternative splicing in skeletal muscle atrophy, especially skeletal muscle atrophy caused by disuse, is unclear.MethodsWe established a rat hindlimb unloading model and performed RNA sequencing on soleus muscle, which was seriously atrophied during unloading. Several bioinformatics methods were used to identify alternative splicing events and determine their gene functions.ResultsMany alternative splicing events were found to occur at different time points (12 h, 24 h, 36 h, 3 days, and 7 days) after hindlimb unloading. These differential alternative splicing events mainly occurred in the gene's coding domain sequence region, and 59% of the alternative splicing events caused open reading frameshift. Bioinformatics analysis results showed that genes with different alternative splicing events were enriched in multiple pathways related to muscle atrophy, including the insulin signaling pathway, endocytosis, mitophagy, and ubiquitin-proteasome pathway. Moreover, alternative splicing of several deubiquitinase genes persisted during skeletal muscle atrophy induced by unloading. Additionally, we identified 10 differentially expressed RNA binding proteins during skeletal muscle atrophy induced by unloading, mainly containing Xpo4, Eif4e2, P4ha1, Lrrfip1, Zc3h14, Emg1, Hnrnp h1, Mbnl2, RBfox1, and Mbnl1. Hnrnp h1 and Mbnl2 were significantly downregulated, and RBfox1 and Mbnl1 were significantly upregulated during skeletal muscle atrophy caused by unloading.ConclusionsTo the best of our knowledge, the present study is the first to propose alternative splicing alterations related to disuse-induced muscle atrophy, emphasizing that alternative splicing is a new focus of attention in the occurrence of muscle atrophy.

Project description:BackgroundMuscleblind-like proteins (MBNL) have been involved in a developmental switch in the use of defined cassette exons. Such transition fails in the CTG repeat expansion disease myotonic dystrophy due, in part, to sequestration of MBNL proteins by CUG repeat RNA. Four protein isoforms (MblA-D) are coded by the unique Drosophila muscleblind gene.Methodology/principal findingsWe used evolutionary, genetic and cell culture approaches to study muscleblind (mbl) function in flies. The evolutionary study showed that the MblC protein isoform was readily conserved from nematods to Drosophila, which suggests that it performs the most ancestral muscleblind functions. Overexpression of MblC in the fly eye precursors led to an externally rough eye morphology. This phenotype was used in a genetic screen to identify five dominant suppressors and 13 dominant enhancers including Drosophila CUG-BP1 homolog aret, exon junction complex components tsunagi and Aly, and pro-apoptotic genes Traf1 and reaper. We further investigated Muscleblind implication in apoptosis and splicing regulation. We found missplicing of troponin T in muscleblind mutant pupae and confirmed Muscleblind ability to regulate mouse fast skeletal muscle Troponin T (TnnT3) minigene splicing in human HEK cells. MblC overexpression in the wing imaginal disc activated apoptosis in a spatially restricted manner. Bioinformatics analysis identified a conserved FKRP motif, weakly resembling a sumoylation target site, in the MblC-specific sequence. Site-directed mutagenesis of the motif revealed no change in activity of mutant MblC on TnnT3 minigene splicing or aberrant binding to CUG repeat RNA, but altered the ability of the protein to form perinuclear aggregates and enhanced cell death-inducing activity of MblC overexpression.Conclusions/significanceTaken together our genetic approach identify cellular processes influenced by Muscleblind function, whereas in vivo and cell culture experiments define Drosophila troponin T as a new Muscleblind target, reveal a potential involvement of MblC in programmed cell death and recognize the FKRP motif as a putative regulator of MblC function and/or subcellular location in the cell.

Dataset Information

Effects of age and hindlimb immobilization and remobilization on fast troponin T precursor mRNA alternative splicing in rat gastrocnemius muscle.

Publications

Effects of age and hindlimb immobilization and remobilization on fast troponin T precursor mRNA alternative splicing in rat gastrocnemius muscle.

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