Acetylation Of AGO2 Promotes Cancer Progression By Facilitating MiR-19b Maturation

The microRNA(miRNA)pathway plays important role in regulating gene expression.Abnormal expression and function of miRNAs may induce various human diseases,including cancer.In mammal,mature miRNAs are generally generated by a two-step processing.First,miRNA genes are transcribed by RNA polymerase?/? into primary miRNAs(pri-miRNAs),which are cleaved into precursor miRNAs(pre-miRNAs)by a microprocessor complex composed of ribonuclease? DROSHA and double-stranded RNA-binding(dsRBD)protein DGCR8.Then pre-miRNAs are recognized and transported from nucleus to cytoplasm by RNA binding protein Exportin-5/Ran-GTP complex.Second,cytoplasmic pre-miRNAs are bound with RNase ? DICER,TARBP2 and AGO2 for the formation of miRISC(miRNA-induced silencing complex)loading complex(miRLC),and then trimmed into miRNA/miRNA* duplexes.The miRNA/miRNA* duplexes are then loaded onto AGO2 and unwound into two single strand miRNAs,one strand as the guide strand is incorporated into AGO2 to form the functional center of the miRISC,while the other as the passenger strand is degraded.Targeted messenger RNAs(mRNAs)are recognized and associated with mature miRNAs for gene silencing through translational repression or degradation.AGO2 is an effector of small RNA-mediated gene silencing by acting as a core of the miRISC.AGO2 has two main functions,one is AGO2 modulates miRNA/siRNA mediated gene silencing;and the other is AGO2 regulates certain miRNAs maturation.It has also been reported that some pre-miRNAs directly interact with AGO2 for the formation of miRNA precursor deposit complexes(miPDC),and subsequently associate with DICER and TARBP2 to form miRLCs.However,the mechanism details of AGO2 recruiting these pre-miRNAs for miPDC formation is still unclear.Known protein post-translational modifications(PTMs)of AGO2 including prolyl-4-hydroxylation,phosphorylation,ubiquitination,SUMOylation and PARylation(poly-ADP-ribosylation)play key roles in either miRNA biogenesis or miRNA-guided gene silencing.Until now,whether AGO2 is acetylated and the specific function of AGO2 acetylation is unveiled.In this study,we identified that AGO2 is mainly acetylated at K355,K493 and K720 in vivo and in vitro.P300/CBP and HDAC7 are the acetyltransferases and deacetylase of AGO2,respectively.By using miRNA high-throughput deep sequencing with A549 stable cell lines and a series of biochemical experiments,we found that AGO2 acetylation at K493 and K720,but not at K355,specifically increased mature miR-19 b biogenesis.RNA immmunoprecipitation(RIP)assays showed that acetylation of AGO2 increased miR-19 b maturation by enhancing the AGO2 recruitment of pre-miR-19b1,which contains a specific motif UGUGUG in its terminal loop,to form the AGO2-pre-miR-19b1 complex(miPDC).More importantly,analyses of clinical database data,clinical lung cancer tissue microarray and xenograft tumor models,confirmed our hypothesis of that AGO2 acetylation promoted cancer progression through facilitating miR-19 b biogenesis.Our study revealed a novel molecular mechanism of that high acetylation level of AGO2 may lead to the formation of malignant tumors,which may provide a theory guide for tumor diagnosis and therapy.