Long Non-coding RNA Linc-RAM Promotes Skeletal Muscle Cell Differentiation By Regulating Glycogen Phosphorylase Activity

RNA deep sequencing and functional genomics analysis demonstrate significant number of noncoding RNAs encoded in the genome of human and other model organism.Although increasing lines of evidence show that lncRNA have profound functions in regulating various aspect of cellular biology,development and disease,only a few number of lncRNAs are functionally investigated in detail.In particular,we have known little about the functions of lncRNAs in regulating cell metabolism.We have previously identified a MyoD-regulated skeletal muscle specifically expressed Linc-RAM which significantly enhance myogenic lineage differentiation by interacting with MyoD in nucleus.We later found that Linc-RAM mainly located in cytoplasm and higher express in glycolytic muscle compared to oxidative muscle,suggesting that Linc-RAM implicates in muscle cell metabolism regulation during myogenic cell differentiation.To investigate the molecular mechanism underlining Linc-RAM-mediated myogenic cell differentiation,we firstly identified Linc-RAM binding proteins in cytoplasm by using RNA pull-down followed MS.We found glycogen phosphorylase(PYGM)is a binding protein of Linc-RAM,which break down glycogen into glucose-1-phosphate.RNA immunoprecipitation assay(RIP)confirmd that Linc-RAM binds with PYGM.Indeed,Linc-RAM and PYGM directly interacted with each other by EMSA(Electrophoresis mobility shift assay)and BlAcore(Biomolecular Interaction Analysis)assay.The up-regulation of PYGM expression during myogenic cell differentiation indicated its key role in the process.In fact,perturbation PYGM expression or its enzymatic activity impacted myogenic cell differentiation.Mechanistically,we found Linc-RAM promotes myogenic cell differentiation by regulating PYGM activity.Key enzymes expression change after perturbation Linc-RAM and PYGM suggested F-1,6-P(FDP)play an important role in myogenic cell differentiation.Our findings illustrate the magnitude and diversity of cytoplasmic lcRNAs and highlight the important roles of lncRNAs in cell metabolism.MicroRNAs(miRNAs)have recently been implicated in muscle stem cell function.miR-127 is known to be predominantly expressed in skeletal muscle,but its roles in myogenic differentiation and muscle regeneration are unknown.Here,we show that miR-127 is upregulated during C2C12 and satellite cell(SC)differentiation and,stably expressing miR-127,demonstrate that overexpression of miR-127 in C2C12 cells enhances myogenic cell differentiation.To investigate the function of miR-127 during muscle development and regeneration in vivo,we generated miR-127 transgenic mice.These mice exhibited remarkably accelerated muscle regeneration compared with wild-type mice by promoting SC differentiation.Mechanistically,we demonstrated that the gene encoding sphingosine-1-phosphate receptor 3(SIPR3),a G protein-coupled receptor for sphingosine-1-phosphate,is a target of miR-127 required for its function in promoting myogenic cell differentiation.Importantly,overexpression of miR-127 in muscular dystrophy model mdx mice considerably ameliorated the disease phenotype.Thus,our findings suggest that miR-127 may serve as a potential therapeutic target for treatment of skeletal muscle disease in humans.