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.
Recently, an emerging body of evidence indicates that the function of conding and non-coding RNAs relies not only in their sequence but also on the chemical modifications of RNA bases. Our project addresses a completely novel topic in epitranscriptomics: 5mC deposition in miRNAs. This epitranscriptomic modification has never been reported in miRNAs yet, and deserves investigation since it might affect miRNA processing, miRNA loading onto AGO proteins and affinity for target mRNAs, thus regulating miRNA function at multiple levels.
The first achievement of our project will be to characterize this modification by profiling 5mC in miRNAs from primary human tissue and human cell lines. We will address this point by providing for the first time a profile of 5mC in human cell lines and primary tissues, possibly highlighting differential deposition of this modification in different tissues/cell types. In particular, we will report the miRNA 5mC modification profile in white matter and PBMCs from healthy donors and MS patients. This will allow us to obtain profiles from both immune cells and neural tissues as well as the unprecedented evidence of a disease-associated alteration of miRNA 5mC profile. Coherently with the budget and the time-frame, we choose to extend our investigation to a small cohort of samples. However, we strongly believe that the data collected in this pilot study will help unravel a new topic in the field of epitranscriptomics and will be the starting point for further investigation of more tissues and other diseases. Importantly, 5mC frequency at specific positions of each miRNA might represent a novel biomarker, which, in our opinion deserves to be investigated in different diseases. This project will pose the bases to address these issues.
Secondly, we aim to identify the "writer" enzymes of 5mC in miRNAs. This task also represents a first key achievement toward the understanding of the role of this epitranscriptomic modification and might link it to known causes of disease such as those linked to NSUN2 mutation.
Finally, this project will lead to the development of several innovative tools and resources that will be made available to the scientific community, including a bisulfite-miRNA-seq protocol, an integrated pipeline for miRNA 5mC profiling data analysis across different cell lines and tissues. The software package will be made freely available and will represent an innovative tool allowing the study of miRNA 5mC modification. The outcomes of the data analysis will be published in a peer reviewed international journal and communicated in national and international congresses.