The epitranscriptome in Drosophila melanogaster

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通路的主要成分，并证明其在神经发生和性别决定的关键作用。虽然这项工作大大提高了我们对m6 A生物起源和功能的理解，但它也提出了新的问题，同时揭示了一些局限性。例如，甲基转移酶复合物（m6 A writer）如何以及通过哪些组分识别推定的m6 A甲基化位点以及为什么这些位点中只有少数被甲基化仍然不清楚。到目前为止，五种不同的因子被证明属于甲基转移酶复合物;然而，它们的确切功能仍然不清楚，目前还没有一种令人信服地解释复合物的特异性是如何实现的。此外，我们通过抗体进行的m6 A分析强调了开发更稳健和更少偏倚的方法以高置信度精确定位mRNA上m6 A的需要。在这项合作提案中，我们希望i）深入了解m6 A甲基转移酶复合物的组成和作用模式，ii）开发新的m6 A残基单分辨率作图技术，最后iii）解决果蝇mRNA上是否存在其他RNA修饰以及它们如何被调控。我们的初步数据显示，其他因素可能属于甲基转移酶复合物，特别是尚未表征的蛋白质CG 7358，我们建议研究其在复合物中的功能。此外，我们已经产生了有希望的结果方面的识别m6 A网站使用基于小分子化学的方法。这种方法必须在转录组水平上开发。最后，我们未发表的工作表明，2-3个额外的修饰可能存在于果蝇的mRNA上。我们计划进一步描述这些修改，并确定其监管参与者。建议的合作研究有可能极大地影响我们对mRNA修饰的新生领域的理解，并为可能是复杂的调控网络提供重要的见解。

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 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 neurogenesis and sex determination. While this work substantially advanced our understanding on m6A biogenesis and function, it also opened new questions while revealing several limitations. For instance it remains unclear how, and through which components the methyltransferase complex (m6A writer) recognizes the putative m6A methylation sites and why only few of these sites are methylated. So far, five different factors were shown to belong to the methyltransferase complex; however their exact function is still unclear and currently none of them convincingly explains how the specificity of the complex is achieved. In addition, our m6A profiling via antibodies highlighted the need for the development of more robust and less biased approaches to precisely map m6A on mRNA with high confidence. In this collaborative proposal we want i) to obtain insights into the composition and mode of action of the m6A methyltransferase complex, ii) to develop new single resolution mapping techniques for m6A residues, and lastly iii) to address whether other RNA modifications are present on mRNA in Drosophila and how they might be regulated. Our preliminary data revealed that additional factors likely belong to the methyltransferase complex, in particular the not yet characterized protein CG7358, and we propose to study its function within the complex. Furthermore, we have generated promising results in regards to the identification of m6A sites using an approach based on small molecule chemistry. This approach must be developed for the transcriptome wide level. Lastly our unpublished work suggests that 2-3 additional modifications could be present on mRNA in Drosophila. We plan to further characterize these modifications as well as to identify their regulatory players. The suggested cooperative study has the potential to greatly impact our understanding of the nascent field of mRNA modifications and to provide important insights into what is likely to be a sophisticated regulatory network.