Mechanistic basis of m6A-mediated mRNA regulation

m6A介导的mRNA调节的机制基础

• 批准号: BB/S014438/1
• 负责人: Andres Ramos
• 金额: $ 40.98万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FS014438%2F1
• 关键词: Mechanistic; basis; m6A; mediated; mRNA

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.

人体的复杂组织及其对挑战的反应需要在不同细胞和不同时间对基因代码翻译成蛋白质的精细控制。信使 RNA (mRNA) 分子的动态修饰在此过程中发挥着重要作用，因为这种修饰是由调节蛋白读取的，从而为基因表达的调节添加了一层。 6 位腺苷的甲基化 N6-甲基腺苷 (m6A) 是最常见的 mRNA 修饰。 m6A 甲基化水平是动态的，由一组“写入”和“擦除”蛋白控制。反过来，由修改后的 m6A 创建的代码被一组“读取器”蛋白质读取。这些蛋白质的功能错误或表达错误与重要的全身性疾病有关，例如糖尿病和多种癌症。虽然 m6A 甲基化的写入、擦除和读取的规范途径已被阐明，但最近的研究表明，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，这一发现将 m6A 调节与一般 RNA 调节过程（例如 mRNA 剪接和增强的 mRNA 周转）联系起来。然而，对于大多数非规范读者来说，我们并不了解分子水平上的这种关系。我们研究的是 IMP1，一种高度保守的 RNA 结合蛋白，可调节 mRNA 代谢、运输和翻译。最近的研究表明，癌细胞中一组 IMP1 靶标的调节依赖于 m6A 甲基化，这种甲基化调节对于癌症蛋白 c-myc 的合成非常重要。我们要问的问题是 i) IMP1 蛋白如何识别 m6A 甲基化靶标，以及 ii) 癌细胞中 IMP1 的 m6A 甲基调节范围。为了回答这些问题，我们首先确定复合物中 IMP1 蛋白的结构与甲基化RNA靶标，并将其与我们于2017年发表的非甲基化RNA的结构进行比较。然后，利用该结构，我们将设计突变以创建仅识别的IMP1变体非甲基化RNA。最后，我们将使用这些变体来确定细胞中哪些 mRNA 以甲基依赖性方式与 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。我们预计这项工作将在分子水平上阐明 I​​MP1 如何识别甲基化 RNA m6A 甲基化靶标 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越来越多的相关非规范 m6A 甲基读取器所共有的，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。这项工作将为 m6A 调控如何整合到癌细胞的全局 RNA 调控网络中提供第一个范例性分子见解。

项目成果

Direct m6A recognition by IMP1 underlays an alternative model of target selection for non-canonical methyl-readers.

• DOI: 10.1093/nar/gkad534
• 发表时间: 2023-09-08
• 期刊: Nucleic acids research
• 影响因子: 14.9