Molecular mechanisms of m6A mRNA modification in Drosophila neurogenesis

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The field of RNA modifications, now termed Epitranscriptomics, has made major strides to the central stage of gene expression control in the recent years. Many modified nucleotides were first discovered in transfer RNAs and ribosomal RNAs in all studied organisms. Few of the same nucleotide modifications were later identified in messenger RNAs (mRNA) of eukaryotes, where they were recently revealed as an entire new layer of regulation of gene expression. The most prevalent and best-studied internal modification on mRNA is N6-methyladenosine (m6A). This modification was shown to regulate several physiological processes in various organisms via posttranscriptional control of gene expression. We recently characterized the m6A pathway in Drosophila melanogaster by identifying its main components and demonstrating its critical role during brain development. Accumulating evidences suggest that this function in the nervous system is conserved in vertebrates. However, due to the difficulty of studying m6A (and RNA modifications in general) in vivo, the molecular mechanisms by which m6A controls neurogenesis remains elusive. In this collaborative proposal, we want i) to develop new tools to map m6A RNA modification in vivo by a non-invasive RNA labeling approach (TRIBE) and by low input immunoprecipitation protocols and in quantitative manner ii) to explore the effect of m6A on gene expression in sub-populations of neuronal cells and iii) to identify and molecularly characterize m6A reader proteins, which are relevant to neuronal functions. We have generated promising results in regards to the identification of methylated transcripts in vivo using a genetic approach combined with high throughput sequencing and sophisticated computational analysis. Furthermore, we have delineated the cell types that are sensitive to the loss of m6A in the nervous system. Lastly our unpublished work suggests that additional m6A readers exist. We plan to characterize their physiological functions as well as their cross talks within the m6A pathway. While our work will be initially focused on m6A, we plan to later adapt the developed tools to additional modifications. The suggested cooperative study has the potential to foster several collaborations within the SPP, and to lead to a better understanding of the nascent field of mRNA modifications.