Depletion of HuR in murine skeletal muscle enhances exercise endurance and prevents cancer-induced muscle atrophy
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE134241 | GEO | 2019/08/21
REPOSITORIES: GEO
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Project description:HuR promotes myogenesis by stabilizing MyoD, Myogenin and p21 mRNAs during the fusion step of muscle cells to form myotubes. Here we show that HuR, via a novel mRNA destabilizing activity, promotes the early steps of myogenesis by reducing the expression of the cell cycle promoter nucleophosmin (NPM). Depletion of HuR stabilizes the NPM mRNA, increases NPM protein levels and inhibits myogenesis, while its overexpression elicits the opposite effects. NPM mRNA destabilization involves the association of HuR with the decay factor KSRP as well as the ribonuclease PARN and the exosome complex. The C-terminus of HuR mediates the formation of the HuR-KSRP complex and is sufficient for maintaining a low level of the NPM mRNA as well as promoting the commitment of muscle cells to myogenesis. We therefore propose a model whereby the downregulation of the NPM mRNA, mediated by the HuR/KSRP complex and its associated ribonucleases, is required for proper myogenesis. RNA was extracted from C2C12 treated with siRNA against HuR and compared to control siRNA
2014-04-26 | E-GEOD-57113 | biostudies-arrayexpress
Project description:HuR promotes myogenesis by stabilizing MyoD, Myogenin and p21 mRNAs during the fusion step of muscle cells to form myotubes. Here we show that HuR, via a novel mRNA destabilizing activity, promotes the early steps of myogenesis by reducing the expression of the cell cycle promoter nucleophosmin (NPM). Depletion of HuR stabilizes the NPM mRNA, increases NPM protein levels and inhibits myogenesis, while its overexpression elicits the opposite effects. NPM mRNA destabilization involves the association of HuR with the decay factor KSRP as well as the ribonuclease PARN and the exosome complex. The C-terminus of HuR mediates the formation of the HuR-KSRP complex and is sufficient for maintaining a low level of the NPM mRNA as well as promoting the commitment of muscle cells to myogenesis. We therefore propose a model whereby the downregulation of the NPM mRNA, mediated by the HuR/KSRP complex and its associated ribonucleases, is required for proper myogenesis.
2014-04-26 | GSE57113 | GEO
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Project description:Skeletal muscles are composed of a heterogeneous collection of fiber types with different physiological adaption in response to a stimulus and disease-related conditions. Each fiber has a specific molecular expression of myosin heavy chain molecules (MyHC). So far MyHCs are currently the best marker proteins for characterization of individual fiber types and several proteome profiling studies have helped to dissect the molecular signature of whole muscles and individual fibers. Herein, we describe a mass spectrometric workflow to measure skeletal muscle fiber type-specific proteomes. To bypass the limited quantities of protein in single fibers, we developed a Proteomics high-throughput Fiber Typing (ProFiT) approach enabling profiling of MyHC in single fibers. Aliquots of protein extracts from separated muscle fibers were subjected to capillary LC-MS gradients to profile MyHC isoforms in a 96-well format. Muscle fibers with the same MyHC protein expression were pooled and subjected to proteomic, pulsed-SILAC and phosphoproteomic analysis. Our fiber type-specific quantitative proteome analysis confirmed the distribution of fiber types in the soleus muscle, substantiates metabolic adaptions in oxidative and glycolytic fibers, and highlighted significant differences between the proteomes of type IIb fibers from different muscle groups, including a differential expression of desmin and actinin-3. A detailed map of the Lys-6 incorporation rates in muscle fibers showed an increased turnover of slow fibers compared to fast fibers. In addition, labeling of mitochondrial respiratory chain complexes revealed a broad range of Lys-6 incorporation rates, depending on the localization of the subunits within distinct complexes.
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Project description:Purpose: Reduced Representation Bisulfite Sequencing (RRBS) DNA input requirements become a challenge when working with small pools of tissue-specific cell types. We describe an application of the RRBS method to assess DNA methylation on low-DNA input from human slow-twitch (MHC I) and fast-twitch (MHC IIa) skeletal muscle fibers. Methods : Fiber-type specific (MHC I and MHC IIa muscle fibers) total DNA was extracted from vastus lateralis muscle biopsies of 8 young physically active men (~25 yrs). A total of 16 DNA samples were generated : 8 DNA samples from pure MHC I and 8 DNA samples from pure MHC IIa muscle fibers. An equal quantity of DNA (4 ng) from each sample was combined to generate a "pooled" DNA sample representing all 8 subjects for each fiber type. Two fiber-type specific "pooled" samples of 32 ng of DNA were generated for library construction and sequencing, creating a Type 1 (MHC I muscle fibers) and Type 2a (MHC IIa muscle fibers) sample. Sequencing was performed using the HiSeq 2500 (Illumina) with 50 bp paired-end read parameters. Minimum sequencing read coverage of 5 (5x) was used as the cutoff for CpG-sites inclusion in the DNA methylation analysis. Fisher’s exact test was performed on CpG-sites that overlapped (i.e. identified in both samples) Type 1 and Type 2a samples to obtain p-values that indicate the likelihood of the site being a differentially methylated CpG-site (DMS). DMS with p<0.05 were classified as hypermethylated or hypomethylated if they were more or less methylated than the Type 1 sample, which was used as the reference sample. Results: The 32 ng of DNA from fiber-type specific muscle samples (Type 1 and 2a) used in this study ensured similar sequencing quality as compared to other studies using greater DNA input (>50 ng). Mapping ratios of ~47% and bisulfite conversion rates of ~97-98% were obtained.The unique and best alignment was successfully assessed for each of 17,376,728 CpG-sites in the Type 1 sample and 17,006,993 in the Type 2a sample, which represents ~30% of the total CpG number in the human genome. We identified 143,160 differentially methylated CpG-sites (DMS) across 14,046 genes among MHC I and MHC IIa muscle fibers. The analysis revealed that some genes predominantly expressed in MHC I were hypermethylated in MHC IIa muscle fibers. Conclusion: This study validates a low-DNA input RRBS method for human skeletal muscle samples to investigate the methylation patterns at a fiber-type specific level. These are the first fiber-type specific methylation data reported from human skeletal muscle. Considering the metabolic and structural differences between MHC I and MHC IIa muscle fibers, this technique could provide novel insights into the skeletal muscle methylation profile in relation to health, performance, disease or disuse.
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