METTL16 inhibits differentiation and promotes proliferation and slow myofibers formation in chicken myoblasts.

N6-methyladenosine (m6A) plays a crucial regulatory role in muscle growth and development. In our previous studies, we identified a m6A methyltransferase, Methyltransferase like 16 (METTL16), which is associated with chicken muscle development and muscle fiber type conversion. To further understand the regulatory role of METTL16 in chicken muscle function, we analyzed its expression in muscle tissues with different myofiber type compositions and in chicken primary myoblasts (CPMs) at various stages. We also manipulated METTL16 expression in CPMs to examine its effects on cell proliferation, differentiation, muscle fiber type formation, and global m6A RNA methylation status. Our results showed that METTL16 expression increased during myoblast proliferation and gradually decreased in the late differentiation stage. Furthermore, METTL16 exhibited specific expression in slow-twitch muscles. Cell Counting Kit-8 assays, 5-Ethynyl-2'-deoxyuridine staining, RT-qPCR, Western blot, and immunofluorescence analyses showed that METTL16 promotes myoblast proliferation while inhibiting myoblast differentiation. We also observed that METTL16 induces the upregulation of slow-twitch myosin heavy chain (MyHC) and slow-twitch-specific genes in myotubes, while downregulating fast-twitch MyHC and fast-twitch-specific genes. Furthermore, both interference and overexpression of METTL16 led to changes in overall cellular m6A modification levels and Methyltransferase like 3 (METTL3) expression levels. These findings confirm that METTL16 plays a key role in myoblast proliferation, differentiation, and muscle fiber type formation in chickens. Considering the role of myoblasts in chicken muscle growth and meat quality regulation, METTL16 may serve as a key target for molecular selection in chicken meat traits.