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编码翻译成蛋白质进行精细控制。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。6位腺苷的甲基化，N6-甲基腺苷（m6 A）是最常见的mRNA修饰。m6 A甲基化的水平是动态的，并由一组“写入器”和“擦除器”蛋白控制。反过来，由修饰的m6 A创建的代码被一组“阅读器”蛋白质读取。这些蛋白质的错误功能或错误表达与重要的全身性疾病有关，如糖尿病和许多形式的癌症。虽然m6 A甲基化的写入、擦除和阅读的规范途径已经阐明，但最近的工作表明存在大量的非规范读取器。这些阅读器是已知结合和调节mRNA的蛋白质，并且该发现将m6 A调节与一般RNA调节过程（例如mRNA剪接和增强的mRNA周转）相关。然而，对于大多数非经典读者来说，我们不了解分子水平上的这种关系。我们研究IMP 1，一种高度保守的RNA结合蛋白，调节mRNA的代谢，运输和翻译。最近的研究表明，癌细胞中一组IMP 1靶点的调节依赖于m6 A甲基化，这种甲基化调节对于癌蛋白c-myc的合成很重要。我们要问的问题是：i）IMP 1蛋白如何识别m6 A甲基化靶点; ii）IMP 1在癌细胞中的m6 A甲基调控范围。为了回答这些问题，我们将首先确定IMP 1蛋白与甲基化RNA靶点复合的结构，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IMP 1变体。最后，我们将使用这些变体来确定在细胞中哪些mRNA以甲基依赖性方式与IMP 1结合，即定义m6 A甲基化的选择性。此外，我们还将建立生物物理模型，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IMP 1如何识别m6 A甲基化靶mRNA，以调节其在癌细胞中的稳定性。此外，我们预计识别的分子特征将是常见的一类相关的非典型的m6 A甲基读者和本研究提供的概念和工具，将允许调查的m6 A在这些蛋白质中的作用。这项工作将为m6 A调控如何整合到癌细胞的全球RNA调控网络中提供第一个典型的分子见解。

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.