METTL3-dependent m(6)A modification of GHR mRNA regulates mitochondrial function through mitochondrial biogenesis during myoblast differentiation.

N6-methyl-adenosine (m(6)A) methylation has recently been shown to play a critical role in muscle development. We recently revealed that local GHR knockdown impairs mitochondrial function by inhibiting mitochondrial biogenesis, thereby repressing myoblast differentiation. And we identified m(6)A modification peaks in the GHR mRNA of chicken muscle tissue. However, whether m(6)A modification may regulate GHR mRNA expression to impinge on mitochondrial function through mitochondrial biogenesis during myoblast differentiation is lagging. We first predicted three potential m(6)A modification sites (GHR-139, GHR-203, GHR-385) on GHR mRNA through SRAMP online prediction website. We then confirmed that GHR-139 is the METTL3-dependent m(6)A modification site. Further, METTL3-dependent m(6)A modification down-regulated the GHR mRNA and protein expression, and blunted the GHR mediated GH-GHR-IGFs axis signal transduction during myoblast differentiation. We next revealed that METTL3-dependent m(6)A modification down-regulated GHR mRNA to inhibit mitochondrial biogenesis and impair mitochondrial function during myoblast differentiation. On the other hand, overexpression of METTL3 alone also proved to inhibit the expression of GHR gene, while suppressing mitochondrial biogenesis and mitochondrial function. In terms of the m(6)A reader protein, we uncovered that m(6)A modification might regulate the GHR mRNA expression through three m(6)A reader proteins hnRNPR, hnRNPA3 and hnRNPM. In conclusion, our data corroborate that METTL3-dependent m(6)A modification down-regulates GHR mRNA expression to impair mitochondrial function by inhibiting mitochondrial biogenesis during myoblast differentiation.