Mechanistic basis of m6A-mediated mRNA regulation

人体的复杂组织及其对挑战的反应需要在不同的细胞和不同的时间对基因密码转化为蛋白质的精细控制。信使RNA （mRNA）分子的动态修饰在这一过程中起着重要作用，因为这种修饰被调节蛋白读取，从而为基因表达的调节增加了一层。n6 -甲基腺苷（m6A）的甲基化是最常见的mRNA修饰。m6A甲基化水平是动态的，由一组“书写者”和“擦除者”蛋白质控制。反过来，由修改后的m6A产生的代码被一组“读取器”蛋白质读取。这些蛋白质的功能失调或错误表达与重要的全身性疾病有关，如糖尿病和多种癌症。虽然m6A甲基化的书写、删除和阅读的规范途径已经得到澄清，但最近的工作表明，存在更多的非规范阅读器。这些读取器是已知的结合和调节mRNA的蛋白质，该发现将m6A调节与一般RNA调节过程（如mRNA剪接和mRNA转换增强）联系起来。然而，对于大多数非规范读者来说，我们不理解分子水平上的这种关系。我们研究的是一种高度保守的rna结合蛋白IMP1，它调节mRNA的代谢、转运和翻译。最近有研究表明，癌细胞中一组IMP1靶点的调控依赖于m6A甲基化，而这种甲基化调控对癌症蛋白c-myc的合成很重要。我们要问的问题是i) IMP1蛋白如何识别m6A甲基化的靶标，ii)癌细胞中m6A甲基化调控IMP1的范围是什么。为了回答这些问题，我们将首先确定与甲基化RNA靶标复合物中的IMP1蛋白的结构，并将其与我们在2017年发表的非甲基化RNA的结构进行比较。然后，使用该结构，我们将设计突变以创建仅识别非甲基化RNA的IMP1变体。最后，我们将使用这些变体来确定，在细胞中，哪些mrna以甲基依赖的方式与IMP1结合，即定义m6A甲基化的选择性。此外，我们将创建生物物理模型来解释甲基化和非甲基化RNA识别之间的差异，以及这些差异如何与蛋白质和RNA浓度相关。我们希望这项工作将在分子水平上阐明IMP1如何识别m6A甲基化的靶mrna，以调节其在癌细胞中的稳定性。此外，我们期望识别的分子特征将是越来越多的相关非规范m6A甲基读取器的共同特征，并且本研究提供的概念和工具将允许研究m6A在这些蛋白质中的作用。总之，这项工作将为m6A调控如何整合到癌细胞的全球RNA调控网络中提供第一个范例分子洞察力。

The complex organisation of the human body and its response to challenges requires a fine control of the translation of the gene code into proteins in different cells and at different times. The dynamic modification of the messenger RNA (mRNA) molecules play an important part in this process, as this modification is read by regulatory proteins that add a layer to the regulation of gene expression. Methylation of the Adenosine in position 6, N6-Methyladenosine (m6A) is the most common mRNA modification. The level of m6A methylation is dynamic and is controlled by a set of 'writer' and 'eraser' proteins. In turn, the code created by the modified m6A is read by a set of 'reader' proteins. Mis-function or mis-expression of these proteins is linked to important systemic illnesses, such as diabetes and many forms of cancer. While the canonical pathways for the writing, erasing and reading of m6A methylation have been clarified, recent work has shown that a larger number of non-canonical readers exist. These readers are proteins known to bind and regulate mRNA and the discovery relates m6A regulation to general RNA regulation processes, such as mRNA splicing and enhanced mRNA turnover. However, for most non-canonical readers, we do not understand this relation at the molecular level.We work on IMP1, a highly conserved RNA-binding protein that regulates mRNA metabolism, transport and translation. It has been recently shown that the regulation of a set of IMP1 targets in cancers cells is dependent on m6A methylation, and this methyl-regulation is important for the synthesis of the cancer protein c-myc. The questions we are asking are i) how the IMP1 protein recognises m6A methylated targets and ii) which is the reach of m6A methyl-regulation of IMP1 in cancer cells.To answer these questions we will first determine the structure of the IMP1 protein in complex with the methylated RNA target and compared it with the structure with a non-methylated RNA, which we have published in 2017. Then, using the structure, we will design mutations to create IMP1 variants that recognise only non-methylated RNA. Finally we will use these variants to determine, in the cell, which mRNAs are bound by IMP1 in a methyl-dependent fashion, i.e. to define the selectivity of m6A methylation. In addition we will create biophysical models that explain the differences between the recognition of methylated and non-methylated RNAs and how these are differences are related to protein and RNA concentration.We expect this work will clarify, at the molecular level, how IMP1 recognises the m6A methylated target mRNAs to regulate their stability in cancer cells. Further, we expect the molecular features of recognition will be common to a growing class of related non-canonical m6A methyl readers and that the concepts and tools provided by this study will allow the investigation of the role of m6A in these proteins. Together the work will provide a first paradigmatic molecular insight into how m6A regulation is integrated in global RNA regulation networks in cancer cells.