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1. Methyltransferase-like 21C (METTL21C) and insulin-like growth factor 2  mRNA -binding protein 1 (IGF2BP1) play important roles in the proliferation of chicken myoblasts. However, it remains unclear whether there is protein-protein interaction between METTL21C and IGF2BP1 to regulate proliferation of chicken myoblasts. 2. In this study, the  Igf2bp1  gene was amplified from  cDNA  of liver tissue of Lueyang black-bone chicken to construct the overexpression vector HA- Igf2bp1 . The HA- Igf2bp1  and Flag- Mettl21c  vectors were individually transfected and co-transfected into HEK293T, respectively. Co-immunoprecipitation (Co-IP) assay indicated a protein-protein interaction between METTL21C and IGF2BP1. 3. Using the Western blotting and LC-MS/MS, it was found that METTL21C could mediate the lysine methylation modification of IGF2BP1. Furthermore, the His-tagged overexpression vector HA- Igf2bp1 -His was constructed, transfected and co-transfected with Flag- Mettl21c  into HEK293T. His-tagged IGF2BP1 was purified by nickel ion affinity chromatography. Western blotting revealed that IGF2BP1 was successfully purified, and the trimethylation modification level of co-transfection group was significantly elevated compared with the single-transfection  Igf2bp1  group. 4.  Mettl21c  and  Igf2bp1  overexpression vectors were transfected and co-transfected into primary chicken myoblasts, respectively. The results of 5-ethynyl-2’-deoxyuridine assay and the expression level of Pax7 and MyoD indicated that overexpression of  Igf2bp1  alone inhibited the chicken myoblast proliferation, whereas co-expression of  Mettl21c  and  Igf2bp1  eliminated the inhibitory effects of  Igf2bp1 , thereby favouring cell proliferation and differentiation. 5. The results, for the first time, revealed that METTL21C mediated the lysine trimethylation modification of IGF2BP1 to regulate the proliferation of chicken myoblasts, which provided a new insight into in-depth analysis of the molecular mechanism of METTL21C methylation involved in regulating the growth and development of skeletal muscle in Lueyang black-bone chicken.

METTL21C mediates lysine trimethylation of IGF2BP1 to regulate chicken myoblast proliferation