Study On The Mechanism Of METTL21C-Mediated IGF2BP1 Lysine Methylation On Regulating Chicken Myoblast Proliferation

Methyltransferase like 21C(METTL21C)is a lysine methyltransferase.It is specifically and highly expressed in mammalian skeletal muscle tissue and is involved in maintaining skeletal muscle homeostasis,but its research in poultry skeletal muscle is relatively rare.This study was based on METTL21 C interacting proteins screened by liquid chromatography mass spectrometry(LC-MS/MS).Insulin-like growth factor 2 mRNA-binding protein 1(IGF2BP1)was selected as the research object,which is closely related to cell proliferation among interacting proteins.The protein-protein interaction between METTL21 C and IGF2BP1 was verified,and then METTL21C-mediated IGF2BP1 methylation was analyzed.Chicken myoblasts was used as model to explore the role of METTL21C-mediated IGF2BP1 methylation on chicken myoblast proliferation.At the protein modification level,the molecular mechanism of METTL21C-mediated IGF2BP1 methylation on affecting chicken myoblast proliferation was revealed.This study will provide a reference for analyzing the function of METTL21 C in poultry skeletal muscle growth and development.The main results were as follows:1.Based on LC-MS/MS detection result,IGF2BP1 was selected as the research object.The pCDNA3.1-HA-Igf2bp1 fusion overexpression vector was successfully constructed.Co-IP result showed that the protein-protein interaction between METTL21 C and IGF2BP1 had existed.2.METTL21C-mediated lysine dimethylation and trimethylation were detected by Western Blot and LC-MS/MS technology.We speculated that IGF2BP1 protein may exist lysine methylation.Further IGF2BP1 protein was purificated,and the result showed that METTL21 C mediated and enhanced lysine trimethylation of IGF2BP1.3.The tissue expression pattern of Igf2bp1 showed that Igf2bp1 have a low expression level in three types of muscle tissues including breast muscle,leg muscle and cardiac muscle.The temporal expression pattern of Igf2bp1 gene in chicken myoblasts was established,and the result displayed that the expression of Igf2bp1 gradually decreased as the cell proliferation.We speculated that Igf2bp1 may play a negative regulatory role in skeletal muscle growth and development.4.Igf2bp1 overexpression vector was transfected into chicken myoblasts.The results showed that Igf2bp1 inhibited myoblast proliferation and the expression level of muscle development related genes was reduced.When Mettl21 c and Igf2bp1 were co-expressed,the combination of METTL21 C and IGF2BP1 attenuated the inhibitory effect of IGF2BP1 on myoblast proliferation.Conclusion: Methyltransferase METTL21 C regulates chicken myoblast proliferation by mediating and enhancing the lysine trimethylation modification of its interaction protein IGF2BP1.