Project description:Increasing evidence suggests that Long non-coding RNAs (LncRNAs) represent a new class of regulators of stem cells. However, the roles of LncRNAs in stem cell maintenance and myogenesis remain largely unexamined. For this study, hundreds of novel intergenic LncRNAs were identified that are expressed in myoblasts and regulated during differentiation. One of these LncRNAs, termed LncMyoD, is encoded next to the Myod gene and is directly activated by MyoD during myoblast differentiation. Knockdown of LncMyoD strongly inhibits terminal muscle differentiation largely due to a failure to exit the cell cycle. LncMyoD directly binds to IGF2-mRNA-binding-protein 2 (IMP2) and negatively regulates IMP2-mediated translation of proliferation genes such as N-Ras and c-Myc. While the RNA sequence of LncMyoD is not well-conserved between human and mouse, its locus, gene structure and function is preserved. The MyoD-LncMyoD-IMP2 pathway elucidates a mechanism as to how MyoD blocks proliferation to create a permissive state for differentiation. In order to perform an unbiased search for downstream signaling pathways perturbed by LncMyodD downregulation, microarrays were performed on myoblasts treated with control vs LncMyoD shRNAs. Total RNA was extracted using the TRIzol reagent (Invitrogen) and purified with Qiagen RNeasy separation columns (Qiagen) from myoblasts treated with control vs. LncMyoD shRNA. First-strand cDNA was synthesized and hybridized to GeneChip Mouse Genome 430 2.0 Array (Affymetrix).
Project description:Increasing evidence suggests that Long non-coding RNAs (LncRNAs) represent a new class of regulators of stem cells. However, the roles of LncRNAs in stem cell maintenance and myogenesis remain largely unexamined. For this study, hundreds of novel intergenic LncRNAs were identified that are expressed in myoblasts and regulated during differentiation. One of these LncRNAs, termed LncMyoD, is encoded next to the Myod gene and is directly activated by MyoD during myoblast differentiation. Knockdown of LncMyoD strongly inhibits terminal muscle differentiation largely due to a failure to exit the cell cycle. LncMyoD directly binds to IGF2-mRNA-binding-protein 2 (IMP2) and negatively regulates IMP2-mediated translation of proliferation genes such as N-Ras and c-Myc. While the RNA sequence of LncMyoD is not well-conserved between human and mouse, its locus, gene structure and function is preserved. The MyoD-LncMyoD-IMP2 pathway elucidates a mechanism as to how MyoD blocks proliferation to create a permissive state for differentiation. In order to perform an unbiased search for downstream signaling pathways perturbed by LncMyodD downregulation, microarrays were performed on myoblasts treated with control vs LncMyoD shRNAs.
Project description:The Igf2 mRNA binding protein2/Imp2 was selectively deleted from adult mouse muscle; two phenotypes were observed: modestly decreased accrual of skeletal muscle mass after weaning and reduced wheel running activity but normal forced treadmill performance. Reduced voluntary activity occurs when fed a high fat diet but is normalized when consuming standard chow. The reduced muscle mass is due to diminished autocrine Igf2 production, reduced Akt1 activation, disinhibition of Gsk3α and reduced protein synthesis, without altered mTOR complex1 activity. The diet-dependent reduction in spontaneous exercise is accompanied by suboptimal muscle fatty acid oxidation, caused by reduced PPARα mRNA and protein, the former an Imp2 client. Nevertheless, in contrast to global Imp2 deficiency, muscle specific Imp2 inactivation does not alter glucose tolerance or the hypoglycemic effect of insulin. Imp2 deficiency in skeletal muscle reduces autocrine production of Igf2 and fiber growth and disorders nutrient metabolism so as to reduce voluntary physical activity.
Project description:Soleus muscle has the most abundant H19 expression compared to other skeletal muscle tissues. In order to identify genes regulated by long noncoding RNA H19 in skeletal muscle, we performed RNA-Seq with dissected WT and H19KO soleus muscles from 21 week old mice. Among the differentially expressed genes, we found skeletal muscle - overexpressed gene DUSP27, which potentially plays an important role in regulating skeletal muscle glucose metabolisim by regulating the activitiy of AMPK, might be a target of H19 mediated regulation.
Project description:Skeletal muscle atrophy is a serious and highly prevalent condition that remains poorly understood at the molecular level. Previous work found that skeletal muscle atrophy involves an increase in skeletal muscle Gadd45a expression, which is necessary and sufficient for skeletal muscle fiber atrophy. However, the direct mechanism by which Gadd45a promotes skeletal muscle atrophy was unknown. To address this question, we biochemically isolated skeletal muscle fiber proteins that associate with Gadd45a as it induces skeletal muscle atrophy in living mice. We found that Gadd45a interacts with multiple proteins in skeletal muscle fibers, including, most prominently, the MAP kinase kinase kinase MEKK4. Furthermore, by forming a complex with MEKK4 in skeletal muscle fibers, Gadd45a increases MEKK4 protein kinase activity, which is sufficient to induce skeletal muscle fiber atrophy and required for Gadd45a-mediated skeletal muscle fiber atrophy. Together, these results identify a direct biochemical mechanism by which Gadd45a induces skeletal muscle atrophy and provide new insight into way that skeletal muscle atrophy occurs at the molecular level.
Project description:Aged skeletal muscle is markedly affected by fatty muscle infiltration and strategies to reduce the occurrence of adipocytes within skeletal muscle, the intramuscular adipose tissue (IMAT), are urgently needed. Fibroblast growth factor-2 (FGF-2) is a critical growth factor for muscle tissue. Here, we show that FGF-2 not only stimulates muscle growth, but also promotes intramuscular adipogenesis. Using multiple screening assays for upstream and downstream signaling of microRNA (miR)-29a we located the secreted protein and adipogenic inhibitor SPARC to an FGF-2 signaling pathway that is conserved between skeletal muscle cells from mice and humans and that is activated in skeletal muscle from aged mice. FGF-2 induces the miR-29a/SPARC axis through transcriptional activation of FRA-1 which binds and activates an evolutionary conserved AP-1 site element proximal in the miR-29a promoter. Genetic deletions in muscle cells and AAV-mediated overexpression of FGF-2 or SPARC in mouse skeletal muscle revealed that this axis regulates differentiation of fibro/adipogenic progenitors in vitro and intramuscular fat formation in vivo. Thus, our data highlight an ambivalent role of FGF-2 for adult skeletal muscle and reveal a novel pathway to combat fat accumulation in aged skeletal muscle.
Project description:Growing evidences are suggesting that extra-long genes in mammals are vulnerable for full-gene length transcription and dysregulation of long genes is a mechanism underlying human genetic disorders. Skeletal muscle expresses Dystrophin which is 2.26 Mbp in length; however, how long-distance transcription is achieved is totally unknown. We had discovered RNA-binding protein SFPQ preferentially binds to long pre-mRNAs and specifically regulates the cluster of neuronal genes > 100 kbp. Here we investigated the roles of SFPQ for long gene expression, target specificities, and also physiological functions in skeletal muscle. Loss of Sfpq selectively downregulated genes >100 kbp including Dystrophin and caused progressive muscle mass reduction and metabolic myopathy characterized by glycogen accumulation and decreased abundance of mitochondrial oxidative phosphorylation complexes. Functional clustering analysis identified metabolic pathway related genes as the targets of SFPQ. These findings indicate target gene specificities and tissue-specific physiological functions of SFPQ in skeletal muscle.
Project description:Growing evidences are suggesting that extra-long genes in mammals are vulnerable for full-gene length transcription and dysregulation of long genes is a mechanism underlying human genetic disorders. Skeletal muscle expresses Dystrophin which is 2.26 Mbp in length; however, how long-distance transcription is achieved is totally unknown. We had discovered RNA-binding protein SFPQ preferentially binds to long pre-mRNAs and specifically regulates the cluster of neuronal genes > 100 kbp. Here we investigated the roles of SFPQ for long gene expression, target specificities, and also physiological functions in skeletal muscle. Loss of Sfpq selectively downregulated genes >100 kbp including Dystrophin and caused progressive muscle mass reduction and metabolic myopathy characterized by glycogen accumulation and decreased abundance of mitochondrial oxidative phosphorylation complexes. Functional clustering analysis identified metabolic pathway related genes as the targets of SFPQ. These findings indicate target gene specificities and tissue-specific physiological functions of SFPQ in skeletal muscle.