A defined N6-methyladenosine (m6A) profile conferred by METTL3 regulates muscle stem cell/myoblast state transitions

Abstract

Muscle-specific adult stem cells (MuSCs) are required for skeletal muscle regeneration. To ensure efficient skeletal muscle regeneration after injury, MuSCs must undergo state transitions as they are activated from quiescence, give rise to a population of proliferating myoblasts, and continue either to terminal differentiation, to repair or replace damaged myofibers, or self-renewal to repopulate the quiescent population. Changes in MuSC/myoblast state are accompanied by dramatic shifts in their transcriptional profile. Previous reports in other adult stem cell systems have identified alterations in the most abundant internal mRNA modification, N6-methyladenosine (m6A), conferred by its active writer, METTL3, to regulate cell state transitions through alterations in the transcriptional profile of these cells. Our objective was to determine if m6A-modification deposition via METTL3 is a regulator of MuSC/myoblast state transitions in vitro and in vivo. Using liquid chromatography/mass spectrometry we identified that global m6A levels increase during the early stages of skeletal muscle regeneration, in vivo, and decline when C2C12 myoblasts transition from proliferation to differentiation, in vitro. Using m6A-specific RNA-sequencing (MeRIP-seq), a distinct profile of m6A-modification was identified, distinguishing proliferating from differentiating C2C12 myoblasts. RNAi studies show that reducing levels of METTL3, the active m6A methyltransferase, reduced global m6A levels and forced C2C12 myoblasts to prematurely differentiate. Reducing levels of METTL3 in primary mouse MuSCs prior to transplantation enhanced their engraftment capacity upon primary transplantation, however their capacity for serial transplantation was lost. In conclusion, METTL3 regulates m6A levels in MuSCs/myoblasts and controls the transition of MuSCs/myoblasts to different cell states. Furthermore, the first transcriptome wide map of m6A-modifications in proliferating and differentiating C2C12 myoblasts is provided and reveals a number of genes that may regulate MuSC/myoblast state transitions which had not been previously identified.

Regulation of RNA N6-methyladenosine modification and its emerging roles in skeletal muscle development

“…Using mouse C2C12 myoblasts and muscle-specific adult stem cells (MuSCs) as models, recent studies have suggested that RNA m 6 A methylation plays an important role in myoblast proliferation and differentiation in vitro (Figure 3). The m 6 A modifications are closely linked to genes associated with transcriptional regulation during proliferation, and positively regulate myogenic differentiation in C12C12 skeletal myoblasts [103,104].…”

“…METTL3 regulates m 6 A levels in MuSCs and myoblasts, controlling their transition to different cell states. Knockdown of METTL3 can slow the proliferation of primary MuSCs in mouse and enhance their engraftment [103]. Meanwhile, m 6 A modifications have been found to be enriched within the 5'-UTR of MyoD mRNA.…”

“…To date, a few studies have evaluated the genome-wide m 6 A modification on skeletal muscle tissue in vivo, the dynamic change of m 6 A methylation in skeletal muscle development and the correlation between m 6 A and skeletal muscle are explored [14,103,110]. Many muscle-specific m 6 A sites have been identified in mammals [41,110,111].…”

METTL3-Mediated m6A Methylation Regulates Muscle Stem Cells and Muscle Regeneration by Notch Signaling Pathway

“…Interestingly, in myoblasts, METTL3 can promote MyoD mRNA maintenance in proliferative myoblasts for skeletal muscle differentiation [12]. Another study reported that METTL3 regulates the myoblast transition from proliferation to differentiation [13]. And depletion of FTO in myoblasts led to impaired skeletal muscle development [14].…”

Dynamic m6A mRNA Methylation Reveals the Role of METTL3/14-m6A-MNK2-ERK Signaling Axis in Skeletal Muscle Differentiation and Regeneration

“…For example, Kudou et al (2017) reported that the myogenic potential is maintained partly by the Mettl3mediated stabilization of processed MyoD mRNA through m 6 A modification of the 5 UTR during proliferative phases. Gheller et al (2020) showed that global m 6 A levels were increased after skeletal muscle injury at a time point corresponding to rapid muscle stem cell (MuSC) proliferation, and global m 6 A levels in primary mouse myoblasts or C2C12 myoblasts were shown to increase during proliferation and to decline during differentiation in vitro. METTL3 knockdown affected MuSC engraftment after primary transplantation (Gheller et al, 2020).…”