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

Background and Objectives:The mammalian target of rapamycin(mTOR)is a member of the serine–threonine kinase family of protein kinases,belongs to the phosphatidylinositol 3-kinase-related kinase family of protein kinases.In cells,mTOR serves as a core component of two distinct protein complexes,namely mTOR complex 1 and mTOR complex 2.mTOR has emerged as a key regulator in the cellular response to signals of growth factors,nutrients,and energy states regulating many biological processes such as cell growth,cell survival,cell viability,cell proliferation,autophagy,anabolism of biological macromolecules(including proteins,nucleic acids,lipids,etc.)mTORC1 regulates protein synthesis mainly through the phosphorylation of 4E-BP1/2 and P70 S6 K,which promote mRNA translation.In addition to the positive regulation of mTORC1 by mRNA translation,MicroRNA(miRNA)also negatively regulates mRNA translation.MiRNAs are short non-coding RNAs,are a family of 21–25 nucleotide single-stranded small RNAs which encoded by endogenous genes.The mature miRNA is often derived from one arm of the precursor hairpin,and is released from the primary transcript through stepwise processing by two ribonuclease-III(RNase III)enzymes.Small RNAs need to be loaded onto an Argonaute protein(AGO protein)to form the effector complex referred to as RNA-induced silencing complex(RISC),RISC can bind to the target mRNA and inhibit its translation.Argonaute(Ago)protein plays an central role in RISC functioning.In mammalian AGO family,AGO family,including AGO1,AGO2,AGO3,and AGO4,AGO2 has been revealed as the only member with endonuclease activity,become an important molecule for direct cleavage of mRNA by RISC.AGO2 protein consists of four core domains which are N,piwi/Argonaute/zwille(PAZ),MID and PIWI.The PAZ and Mid domains are functionally related to the selective small RNA(sRNA)binding.Based on the crystal structure,the 5’ end of an sRNA binds to the Mid domain,while its 3’ end is anchored in the PAZ domain.The PIWI domain possesses RNaseH-like endonuclease activity.Thus,this domain is responsible for target RNA cleavages through the small interfering RNA(siRNA)or microRNA(miRNA)-guided RNAi pathway.However,it should be emphasized that in the mRNA silencing reaction,each domain of Ago2 is necessary.The silencing function of Ago2 is regulated by translational modifications,in which phosphorylation is an important mode of regulation.The multiple C-terminal serine sites of Ago2 receive phosphorylation of casein kinase and negatively regulate the loading of certain mRNA into RISC,but have no effect on the entry of miRNA into RISC,thus regulating the silencing of certain genes.Phosphorylation of the Ago2 387 serine site has also been reported in RISC by many laboratory experiments using mass spectrometry.But its biological function has not been fully revealed,Using mass spectrometry after Ago2 in vitro kinase assay,it was found that Akt can mediate the phosphorylation of Ago2 S387 in vitro.However,there is no evidence that Akt also phosphorylates Ago2 S387 in vivo.Moreover,the Ago2 S387 site does not harbor the amino acid recognition sequence of the traditional Akt phosphorylation substrate of RxRxxS.Therefore,which protein kinase mediates the phosphorylation of Ago2 S387 and the function of the phosphorylation of this site needs further study.mTORC1 is the most important protein kinase that positively regulates mRNA translation,while mi RNA is an important mechanism that negatively regulates mRNA translation.How does the cell as a whole coordinate the two mechanisms of mRNA translation? Does mTORC1 negatively regulate mRNA silencing? If so,what is its molecular mechanism? These are the objects of this study.Research methods:In this study,co-immunoprecipitation(Co-IP)and GST fusion protein pulldown were used to study whether there is a physical interaction between Ago2 and Raptor or other components of mTORC1.By using phospho-Ago2 S387 site-specific antibody,we investigated the possibility of whether mTORC1 mediates the phosphorylation of Ago2 at S387.After obtaining Ago2 S387 A and S387 E mutants with DNA site-directed mutagenesis,we further studied the effects of Ago2 S387 phosphorylation on the binding of Ago2 to RNA with Ago2-mediated RNA immunoprecipitation(RIP).Research results:1.Co-IP and Pulldown experiments revealed the physical interaction between Ago2 and mTORC1.2.Under various cell culture conditions to regulate mTORC1 activities,we observed that changes in the phosphorylation of Ago2 S387 were associated with that in mTORC1 activities but not with Akt activity,indicating that phosphorylation of Ago2 S387 site is carried out by mTORC1 but not Akt.3.RIP study demonstrated that co-precipitated RNA with Ago2 was significantly reduced in TSC1 knockout cells which mTORC1 highly activation,RNA bound to Ago2 was increased after inhibition of mTORC1 activity by rapamycin;Ago2 S387 A mutant RNA binding capacity increased,S387 E mutant RNA binding capacity decreased,respectively.Results:mTORC1 regulates the RNA binding capacity of Ago2 by phosphorylating the S387 site of Ago2 to regulate miRNA silence function.The higher the mTORC1 activity,the lower the amount of RNA bound to Ago2 and vice versa.