Abstract
Duck skeletal muscle serves as an important source of high-quality protein for humans, and a wide array of duck meat products are available. The investigation of RNA N6-methyladenosine (m(6)A) modifications in the growth and development of duck skeletal muscle is still in the early stages, with many regulatory functions and underlying mechanisms yet to be thoroughly examined. This study focused on duck embryonic skeletal muscle, highlighting the role of the RNA methyltransferase METTL14 in regulating muscle cell proliferation, differentiation, and muscle fiber hypertrophy. Using techniques such as glyoxal and nitrite-mediated deamination of unmethylated adenosines sequencing (GLORI-seq), RNA immunoprecipitation sequencing (RIP-seq), and luciferase reporter assays, we explored the interaction of METTL14 with the target mRNAs TET1 and TET2, as well as the mediation of their m(6)A modifications. These results indicate that METTL14 inhibits the proliferation and differentiation of duck primary myoblasts (DPMs) while facilitating the hypertrophy of muscle fibers. Through the integrated application of Dual-luciferase reporter assays and Single-base elongation- and ligation-based qPCR (SELECT), we established that METTL14 binds to the m(6)A motifs on TET1 and TET2 mRNAs, mediating their m(6)A modifications and thus influencing their expression levels. Specifically, TET1 promoted DPMs proliferation and inhibited differentiation, whereas TET2 suppressed DPMs proliferation and facilitated differentiation. The interactions between the METTL14/m(6)A/TET1 and METTL14/m(6)A/TET2 axes synergistically regulate the differentiation process of DPMs. These findings contribute to a more comprehensive understanding of the epigenetic regulatory framework governing duck skeletal muscle development, offering critical insights into the genetic mechanisms involved.