Studies On RNA N6-methyladenosine (m~6A) Site Selective Mechanism And Its Functions

N6-methyladenosine (m6A) has been identified as the most prevalent epitranscriptomic modification on eukaryotic mRNAs. RNA m A modification is catalyzed by a multicomponent methytransferase complex composed of at least three subunits, METTL3, METTL14 and WTAP, where METTL3 and METTL14 serve as catalytic subunits, while WTAP serves as regulatory subunit. Dioxygenases FTO and ALKBH5 are the two known m6A demethylases, catalyzing the removal of methyl group from m6A. m6A modification is recongnized by YTH domain containing family of proteins namely YTHDF1, YTHDF2 and YTHDC1. Cytoplasmic m6A readers YTHDF1 and YTHDF2 are implicated in the regulation of mRNA translation and stability respectively in m6A dependent manner whereas nuclear m6A reader YTHDC1 regulates alternative splicing. RNA m6A modification occurs in RRACH consensus motif where R represents purine and H is usually A, C or U. Despite the abundance of RRACH motifs along the mRNA only few of them undergo m6A modification. Given the prevalence and dynamic nature of RNA m6A modification and its implication in various aspects of RNA metabolism it is not yet clear how the sequence specificity for RNA m6A modification is determined.Our current study found that microRNAs regulate the formation of m6A modification via a sequence pairing mechanism. m6A modification and microRNAs have some physical correlation on their common RNA substrates. A subset of microRNAs reversely complementing the RRACH motif in mRNA can mediate m6A formation demonstrating the ability of microRNAs to induce de novo m6A formation in a sequence dependent manner.We investigated the effect of expression of microRNA processing enzyme Dicer and microRNAs themselves on global m6A modification levels by m6A dot blot. Interestingly we found that Dicer overexpression can significantly increase mRNA m6A level while its depletion can remarkably decrease mRNA m6A level both in human HeLa cells and mouse NSCs. It was also revealed that microRNAs overexpression increases the m6A level while their repression by antogomirs decrease the m6A level of their mRNA targets.We further investigated how the alteration in microRNAs level affect the m6A level by immunofluorescence and PAR-CLIP assays. We found that depletion of Dicer reduces the nuclear staining density of METTL3 and its nuclear speckle localization. We also observed that depletion of Dicer or the microRNAs affect the RNA binding ability of METTL3 to mRNAs targeted by microRNA for m6A modification. These observations suggest that microRNAs modulate the binding ability of METTL3 to a subset of mRNAs targeted by microRNAs.We also investigated role of m6A modification on cell reprogramming and found that m6A regulates the expression level of Oct4, Sox2 and Nanog, promoting the reprogramming of mouse embryonic fibroblasts (MEFs) to pluripotent stem cells. RNA m6A methyltransferase METTL3 overexpression significantly increased the reprogramming efficiency while its depletion severely compromised the reprogramming efficiency which could be rescued by reconstitution with METTL3.The thesis also investigated the biological significance of m6A by studying the effect of demethylase FTO on metabolism signaling pathway related to obesity and diabetes. So far, FTO substrates has not been reported yet, because of its ability to remove methyl group from RNA m6A swiftly. We employed PAR-CLIP and PAR-CLIP-seq analysis to investigate the FTO substrate and found that FTO mutant is capable of pulling down its potential RNA substrates. PAR-CLIP-seq analysis in combination with KEGG pathways and GO functional studies of FTO substrates further revealed that these RNAs are enriched in mRNA processing, translation, cell cycle, etc.Collectively this study uncovered the role of microRNAs in regulating m6A formation in mRNAs by modulating the binding of METTL3 to mRNAs, also showed the involvement of m6A in promoting cell reprogramming and identified the FTO substrates. These findings provide a foundation for future functional studies of m6A modification and serve as a new brand in modulating m6A as a new strategy to regulate normal and abnormal life activity. The FTO substrates investigation will provide important molecular clues for the further studies of FTO inhibitors and its role in metabolic diseases like obesity and diabetes.