Phosphorylation Of Ago2 At Serine-387 By MTORC1 Regulates MicroRNA-mediated Silencing

Background and Objectives: The mechanism of m RNA translation regulation has always been an important research hotspot in the field of cell biology.The research of m RNA translation helps us to grasp the mechanism of cell biological behavior and function,which involves almost all aspects of cell biology.It also helps us understand the molecular mechanisms underlying the development of many diseases,providing new theoretical and drug screening targets for exploring disease prevention and treatment.Eukaryotic messenger RNAs(m RNAs)are transcribed in the nucleus,capped,spliced,polyadenylated,and exported to the cytoplasm where they shape the cellular proteome finally.mechanistic target of rapamycin(m TOR)is an evolutionarily conserved serine/threonine kinase and forms two distinct complexes called m TOR complex 1(m TORC1)and m TOR complex 2(m TORC2).m TOR mediates various physiological and pathological processes,especially cell proliferation,protein synthesis,autophagy,and cancer development.m TORC1 phosphorylates 4E-BP to trigger its dissociation from e IF4 E and promotes m RNA translation.The mi RNA-mediated silencing complex(RISC)formed by the target m RNA,Ago2 protein and GW182 protein is an important mechanism for negative regulation of m RNA translation.So,how do cells coordinate these two opposite m RNA translational regulation mechanisms? Whether and how m TORC1 might regulate the function of RISC-mi RNAs largely remain unexplored.Previous studies mainly focused on the regulation of a single mi RNA by m TORC1.There were few studies regarding how m TORC1 might globally regulate RISC-mi RNAs functions.Our study made a preliminary study on the regulation of mi RNA-mediated gene silencing by m TORC1.Methods: 1.Co-IP and GST Pull-down assay were applied to investigate whether there is a physical interaction between Ago2 and Raptor or other components of m TORC1.Ago2 truncation plasmids with different structural domains were constructed to delineate Ago2 domain mediating its interaction with Raptor.2.The phosphorylation of Ago2 at serine-387 by m TORC1 was evaluated both in vivo and in vitro by endogenous Ago2 IP followed by site-specific phosphorylation antibody immunoblotting and in vitro kinase assay respectively.3.RIP,tethering assays and Co-IP were carried out to evaluate the ability of phosphorylated Ago2 in binding RNA and GW182.Results: 1.Both in vitro and in vivo experiments revealed that m TORC1 interacts to Ago2 directly,we also revealed that Raptor,a main component of in m TORC1,directly binds to PAZ domain of Ago2.2.The level of phosphorylation of Ago2 phosphorylation at serine-387 is associated with m TORC1 activity but not Akt in vivo.m TORC1 in vitro kinase assay reveals that,Ago2 serine-387 is probably the only site phosphorylated by m TORC1.3.Phosphorylated of Ago2 at serine-387 by m TORC1 blunted its ability to bind RNA and GW182.Conclusion: By directly phosphorylating at Ago2 serine-387,m TORC1 regulates its ability to bind RNA and GW182 protein,affecting the formation of mi RNA-mediated RISC and gene silencing.When m TORC1 activated,the phosphorylation level of Ago2 serine-387 was enhanced and the ability of Ago2 binding with RNA and GW182 protein was weakened,inhibiting gene silencing mediated by mi RNA.