A Novel Role for the RNA–Binding Protein FXR1P in Myoblasts Cell-Cycle Progression by Modulating p21/Cdkn1a/Cip1/Waf1 mRNA Stability

摘要

The Fragile X-Related 1 gene ( FXR1 ) is a paralog of the Fragile X Mental Retardation 1 gene ( FMR1 ), whose absence causes the Fragile X syndrome, the most common form of inherited intellectual disability. FXR1P plays an important role in normal muscle development, and its absence causes muscular abnormalities in mice, frog, and zebrafish. Seven alternatively spliced FXR1 transcripts have been identified and two of them are skeletal muscle-specific. A reduction of these isoforms is found in myoblasts from Facio-Scapulo Humeral Dystrophy (FSHD) patients. FXR1P is an RNA–binding protein involved in translational control; however, so far, no mRNA target of FXR1P has been linked to the drastic muscular phenotypes caused by its absence. In this study, gene expression profiling of C2C12 myoblasts reveals that transcripts involved in cell cycle and muscular development pathways are modulated by Fxr1 -depletion. We observed an increase of p21—a regulator of cell-cycle progression—in Fxr1 -knocked-down mouse C2C12 and FSHD human myoblasts. Rescue of this molecular phenotype is possible by re-expressing human FXR1P in Fxr1 -depleted C2C12 cells. FXR1P muscle-specific isoforms bind p21 mRNA via direct interaction with a conserved G-quadruplex located in its 3′ untranslated region. The FXR1P/G-quadruplex complex reduces the half-life of p21 mRNA. In the absence of FXR1P, the upregulation of p21 mRNA determines the elevated level of its protein product that affects cell-cycle progression inducing a premature cell-cycle exit and generating a pool of cells blocked at G0. Our study describes a novel role of FXR1P that has crucial implications for the understanding of its role during myogenesis and muscle development, since we show here that in its absence a reduced number of myoblasts will be available for muscle formation/regeneration, shedding new light into the pathophysiology of FSHD. Author Summary Muscle development is a complex process controlled by the timely expression of genes encoding crucial regulators of the muscle cell precursors called myoblasts. We know from previous studies that inactivation of the Fragile X related 1 ( FXR1 ) gene in various animal models (mouse, frog, and zebrafish) causes muscular and cardiac abnormalities. Also, FXR1P is reduced in a human myopathy called Fascio-Scapulo Humeral Dystrophy (FSHD), suggesting its critical role in muscle that findings presented in this study contribute to elucidating. Cell-cycle arrest is a prerequisite to differentiation of myoblasts into mature myotubes, which will form the muscle. One key regulator is the p21/Cdkn1a/Cip1/Waf1 protein, which commands myoblasts to stop proliferating, and this action is particularly important during muscle regeneration. In this study, we have identified FXR1P as a novel regulator of p21 expression. We show that FXR1P absence in mouse myoblasts and FSHD-derived myopathic myoblasts increases abnormally the levels of p21, causing a premature cell cycle exit of myoblasts. Our study predicts that FXR1P absence leads to a reduced number of myoblasts available for muscle formation and regeneration. This explains the drastic effects of FXR1 inactivation on muscle and brings a better understanding of the molecular/cellular bases of FSHD.