Characterization of the 5mC profile of miRNAs in human cell lines and primary cells from healthy donors and Multiple Sclerosis patients
This project aims to describe the pattern of 5-methyl Cytosine (5mC) in miRNAs in human cell lines as well as in primary human tissues. Preliminary data obtained in my lab show that human and murine miRNAs contain 5mC residues. This modification might be a key determinant of miRNA function as well as a novel biomarker. In fact, it has been observed that 5mC stabilizes nucleic acid duplex molecules, suggesting that it might facilitate miRNA-mediated recruitment of AGO proteins on their targets. Notably, deposition of 5mC nearby AGO binding sites has been reported on mRNAs extracted from mouse brain. Furthermore, 5mC might affect miRNA processing and regulate their expression at post-transcriptional level.
Within this project we aim to:
1) Assess the 5mC profile of miRNAs in human cell lines (HeLa, HEK293T, HCT116, MCF7, HOG). We will also assess the 5mC profile of miRNAs in human primary tissues to confirm that this epitranscriptomic modification is also deposed in vivo. In particular, we will analyze miRNAs in primary human tissue samples from multiple sclerosis patients and matched controls, including brain white matter and peripheral blood mono-nucleated cells.
2) Develop a dedicated software pipeline to analyze 5mC profile in microRNAs, allowing identification of differentially methylated cytosines across different samples.
3) Validate our findings and identify the writer enzymes of 5mC in miRNAs by loss of function approaches in 293T and HeLa cell lines.
These achievements will represent an important proof of principle to demonstrate that vertebrate miRNAs contain 5mC and a starting point to investigate the biological role of this epitranscriptomic modification.