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

Abstract

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.

Regulation of monocyte induced cell migration by the RNA binding protein, FXR1

“…6,16,18,22 CDKN1A mRNA was previously demonstrated to be downregulated by FXR1 22 ; accordingly, our microarray data also showed that depletion of FXR1 in serum-grown cells promotes CDKN1A mRNA levels (Fig. 2B, SC , Table S2A).…”

“…2B, SC), which is in agreement with previously published data demonstrating CDKN1A increase upon FXR1 depletion. 22 These data reveal that distinct mRNAs are regulated in the absence of FXR1 in THP1 cells.…”

“…5,6,16,[19][20][21][22]24 Our previous data demonstrated a role for an isoform of FXR1 in quiescent (extended > 24 h serum-starved) cells, in upregulating translation (independent of RNA level changes) of a critical cytokine, TNFa 17 that can promote monocyte functions, signaling and differentiation. 26,[28][29][30][31] In early (24 h) serumstarved THP1 acute monocytic leukemic cells, our data revealed altered gene expression mechanisms 33 that are distinct from those in quiescent (extended > 24 h serum-starved) cells.…”

“…6 FXR1 is similar to Fragile-X-Mental Retardation Protein 1 (FMR1), 4,[7][8][9][10][11][12][13][14] and is implicated at multiple levels of post-transcriptional control, including translation, mRNA stability and transport. 7-10, 12-13, 15-18 FXR1 is associated with negative regulation of specific growth factor and cytokine mRNAs in myocytes 5,[19][20][21][22] and macrophages, 16 which can control development, cell differentiation and cell state specific functions. However, our previous studies demonstrated that a spliced isoform of FXR1, FXR1a, promotes specific mRNA translation independent of RNA levels, in association with an altered microRNP (microRNA-protein complex), in distinct conditions, such as oocytes and quiescent (> 24 h, 30-48 h extended serum-starved) mammalian cells.…”

“…Cytokines like TNFa are transcribed and detected in these conditions; however, the role of FXR1 in regulating specific mRNA levels and expression in serum grown and in early 24 h serum-starved THP1 cells remains to be outlined. Given the effect of FXR1 on cell differentiation 5,[20][21][22] and cancer 6 and its regulation of TNFa cytokine mRNA expression in quiescent, extended serum-starved cells, we examined the role of FXR1 in regulating mRNA levels in serum grown cells and in early (24 h) serum-starved monocytic leukemic cells.…”