The RNA Binding Protein EWS Is Broadly Involved In Regulating MicroRNA Biogenesis In Mammalian Cells

MicroRNA is a class of small non-coding RNA with?22 nt(nucleotides)in length,which function in post-transcriptionally regulating the expression of many genes by targeting 3'UTR or coding regions.They participate in regulation of diverse biological processes such as proliferation,differentiation and apoptosis.The dysfunction of microRNA can cause various diseases including tumor genesis.MicroRNA is transcribed by RNA polymerase II and processed by Microprocessor which is composed by Drosha and its partner DGCR8 to generate?70 nt hairpin structure called microRNA precursor.The precursor binds RAN-GTP and is transported by Exportin5 to the cytoplasm where it is processed to mature microRNA.Many RNA binding proteins involve in regulating the biogenesis and processing of microRNAs.The Ewing Sarcoma protein(EWS)is a multifaceted RNA binding protein(RBP)with established roles in transcription,pre-mRNA processing,and DNA damage response.EWS protein belongs to the TET(TLS/TAF15/EWS)family.This family usually connects with neuron disease and cancer.The proteins in this family can bind DNA and RNA in regulating transcription.It has been reported that EWS may interact with Drosha,but the function of EWS in regulation of microRNA processing is not clear yet.In this thesis we try to investigate the function and mechanism of EWS in microRNA processing.By generating high quality RNA interactome of EWS,we uncovered its specific and prevalent interaction with a large subset of primary microRNAs(pri-miRNA).Depletion of EWS reduced,while overexpression of EWS enhanced,the expression of its target miRNAs.Biochemical analysis revealed that EWS binds the sequences flanking the stem-loop of its target pri-miRNAs and sequence swap between target and non-target demonstrated that the flanking sequences provide the specificity for enhanced pri-miRNA processing by the Microprocessor Drosha/DGCR8.Interestingly,as previously reported and we here confirmed,EWS depletion also causes elevated expression of Drosha.Together,these findings suggest that EWS may positively and negatively regulate miRNA biogenesis,but via distinct mechanisms,thus providing a new foundation to understand the function of EWS in development and disease.