Epitranscriptomic modifications of coding and non-coding RNAs are emerging as a further layer of gene expression regulation. In this project I will focus on the role of 5-methyl-cytosine (5mC) and 5-hydroxy-methyl-cytosine (5hmC) modifications in mature human microRNAs. I have collected preliminary results by bisulfite NGS analysis of microRNAs in different human cell lines and primary tissues which highlight widespread 5(h)mC modification of mature microRNAs. Furthermore, I have confirmed such modification by dot-blot and RNA immunoprecipitation followed by qPCR using specific antibodies raised against 5mC and 5hmC. I will assess the role of these modifications on microRNA function by assessing subcellular localization, pre-miRNA processing into mature miRNA, mature miRNA stability and target mRNA repression by 5mC and 5hmC modified microRNAs. I will focus this analysis on two 5(h)mC modified, cancer related microRNAs (mir-16-5p and miR-34a-5p). Furthermore, I will identify writer enzymes of these modifications by looking at global microRNA 5(h)mC profile by bisulfite NGS and at single miRNA modifications by RIP in loss-of-function cellular models. This project will yield molecular characterization of the mechanisms through which these modifications are deposed and a mechanistic explanation of their effect on microRNA activity possibly unraveling links with pathological and physiological processes. Furthermore, these data will set the basis for the therapeutic use of 5(h)mC modified miRNA mimics.