Molecular mechanisms of m6A mRNA modification in Drosophila neurogenesis

近年来，RNA修饰领域（现称为表位转录组学）已取得重大进展，进入基因表达调控的中心阶段。许多修饰的核苷酸首先在所有研究的生物中的转移RNA和核糖体RNA中发现。 后来在真核生物的信使RNA（mRNA）中发现了一些相同的核苷酸修饰，它们最近被揭示为基因表达调控的全新层。mRNA上最普遍和研究最多的内部修饰是N6-甲基腺苷（m6 A）。这种修饰被证明通过基因表达的转录后控制来调节各种生物体中的几种生理过程。我们最近的特点是在果蝇m6 A通路的主要组成部分，并证明其在大脑发育过程中的关键作用。越来越多的证据表明，这种功能在神经系统中是保守的脊椎动物。然而，由于在体内研究m6 A（和一般的RNA修饰）的困难，m6 A控制神经发生的分子机制仍然难以捉摸。在这项合作提案中，我们希望i）开发新的工具，通过非侵入性RNA标记方法（TRIBE）和低输入免疫沉淀方案，以定量方式在体内绘制m6 A RNA修饰，ii）探索m6 A对神经元细胞亚群中基因表达的影响，iii）鉴定和分子表征与神经元功能相关的m6 A阅读器蛋白。我们已经产生了有希望的结果，在使用结合高通量测序和复杂的计算分析的遗传方法在体内鉴定甲基化转录本。此外，我们还描述了神经系统中对m6 A丢失敏感的细胞类型。最后，我们未发表的工作表明存在额外的m6 A阅读器。我们计划表征它们的生理功能以及它们在m6 A通路中的交叉作用。虽然我们的工作最初将集中在m6 A上，但我们计划稍后对开发的工具进行额外的修改。建议的合作研究有可能促进SPP内的几个合作，并导致更好地了解mRNA修饰的新生领域。

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.