FUNZIONE DELLA METILAZIONE DEL RNA N6-METILADENOSINA NELLA REGOLAZIONE DELL'ALLUNGAMENTO DELLA TRASCRIZIONE

Transcription and mRNA processing are highly coordinated. The mRNAs are marked cotranscriptionally by several modifications which have significant effects on mRNA biology, gene expression, and cellular metabolism as well as on the survival and development of the whole organism. N6-methyladenosine (m6A) is the most common internal modification of mRNAs that is catalyzed by the methyltransferase complex (MTC) which contains Methyltransferase-like 3 (METTL3), Methyltransferase-like 14 (METTL14) and Wilms’ tumour associated protein (WTAP). m6A had been shown to regulate mRNA translation and other biological processes including cell cycle progression, cell proliferation, cell apoptosis, cell migration and invasion, cell differentiation and inflammatory response. Here we evaluated the role of m6A in the regulation of transcription, focusing on RNA polymerase II (RNAPII) pausing and its rate in human cells. The m6A methyltransferase complex (MTC) with its catalytic subunit METTL3 was recruited to gene promoter. Downregulation of METTL3 led to changes in mRNA expression levels of Immediate-Early Genes. RNAPII levels at the pausing site were reduced under METTL3 downregulation and RNAPII signal at the coding region was increased in such conditions. Decreasing METTL3 negatively affected Immediately Early genes leading to a reduction in pre-mRNA expression levels. Although decreasing in premRNA expression, basal expression levels resulted in increased after METTL3 downregulation. We exploited a simple and efficient method to study rates of transcriptional elongation, described by Singh and Padgett, in 2009 to study the RNAPII transcription rate. We analysed RNAPII transcription rate along gene body and we observed that pre-mRNA levels decreased after 20 minutes from the treatment in METTL3 downregulation conditions. Collectively, our data highlighted a correlation between RNA transcription, the rate of RNAPII during transcription and m6A modification.