Titolo della tesi: Identification and profiling of 5-methyl-cytosine and 5-hydroxy-methyl-cytosine in human microRNAs.
Epitranscriptomics is the field that studies the post-transcriptional modifications of RNA. To date, one hundred and seventy-two epitranscriptomics modifications have been unveiled in all classes of RNA (including microRNAs) and in all domains of life. The most common and best-characterised epitranscriptomic modifications include N6-methyl-Adenosine (m6A), pseudoUridine (Ψ), Adenosine-to-Inosine (A-to-I) editing and 5-methyl-Cytidine (m5C). In the last ten years, a plethora of studies has highlighted how they can regulate gene expression affecting fundamental processes such as splicing, export to the cytoplasm, translation and RNA decay. Therefore, it is not surprising the emerging role of epitranscriptomic modifications in physiological processes such as cell differentiation and development as well as in diseases such as cancer, neurodevelopmental and neuronal disorders, stroke, diabetes and cardiovascular disease. These new insights confirm that epitranscriptomic modifications represent a new layer in the regulation of gene expression.
MicroRNAs (miRNAs) are small (18-24 nt) non coding RNAs acting as regulators of gene expression at the post-transcriptional level in metazoa, playing key roles in several physiological and pathological processes. Recent reports showed that human miRNAs undergo cytosine methylation.
In this thesis, I describe the first high-throughput NGS-based method (BS-miRNA-seq) and an analysis pipeline (MAmBA) to attain high-resolution mapping of (hydroxy)-methyl-5-cytosine ((h)m5C) modifications in human miRNAs. Thanks to this protocol, my colleagues and I have demonstrated that miRNAs undergo widespread cytosine modification in various sequence contexts. Furthermore, we have validated our data with specific antibodies revealing both m5C and hm5C residues in human mature miRNAs. BS-miRNA-seq and MAmBA may contribute to the precise mapping of (h)m5C on miRNAs in various cell types and tissues, a key achievement towards the understanding of the functional implications of this modification in miRNAs.