Probing the function of RNA methylation through chemical biology

真核RNA包含100多种化学修饰。这些RNA修饰最初被认为是静态的和不可改变的，在微调mRNAs的结构和功能方面起到了很小的作用。然而，最近的研究表明，一些RNA修饰是动态的和可逆的，这代表了RNA水平上新出现的一层基因调控。一种这样的动态mRNA修饰是N6位的腺苷甲基化，形成N6-甲基腺苷(M6A)。这是已知的真核基因中最丰富的碱基修饰，基于NGS的方法的最新进展使&gt；12,000个m6A位点的位置能够在转录范围内的~100个核苷酸分辨率内被识别，称为m6A甲基组。通过鉴定RNA甲基转移酶(WRITS)和去甲基酶(ERASER)，通过催化或氧化去除mRNAs中的m6A修饰，使m6A甲基化可逆，强调了m6A甲基化的动态作用。甲基转移酶样3(METTL3)和甲基转移酶14(METTL14)均具有甲基转移酶活性，形成稳定的异源二聚体。Wilm的肿瘤1相关蛋白(WTAP)也可以作为调节亚单位与METTL3/14复合体相互作用。相比之下，人类脂肪质量和肥胖(FTO)相关蛋白和烷基化修复同源5蛋白(ALKBH5)氧化去除了mRNA中的m6A修饰。可逆性M6A甲基化的功能重要性尚未完全阐明，然而异常的M6A甲基化与许多人类疾病有关。它被认为控制着RNA加工的许多方面，包括调控转录、mRNA运输、剪接、稳定性、转录丰度和翻译。在实验耗尽m6A相关酶的许多细胞过程中观察到的不同结果也加强了这一点。该项目旨在产生METTL3/14异二聚体的细胞渗透性化学抑制剂，以使M6A修饰的功能相关性得以探索。在我们外部合作者的工作基础上(METTL3/14的晶体结构)，学生将使用基于结构的设计、计算化学、生化分析和合成化学方法。将进行的工作概述：-在硅化学文库中筛选METTL3/14-LifeArc-使用生化分析鉴定和验证HITS-Whitehouse/LifeArc-确定HITS的晶体结构与METTL3/14形成复合体-Bayliss-优先考虑HITS并计划合成方法以使用计算化学优化抑制剂-LifeArc/Nelson-通过活性定向合成来优化抑制剂-Nelson-使用生化分析和结晶学评估新的抑制剂并重复化学合成，直到我们得到效力高于1uM的细胞通透性抑制剂为止-全部探测METL3/14对mRNA运输的抑制作用后果加工和翻译-怀特豪斯该项目将有助于贝利斯/纳尔逊集团正在进行的努力，利用结构生物学和活性定向合成，加快开发用于机械生物学研究的化学探针。与遗传方法相比，化学探针的优势在于可以研究快速和可逆地调节生物化学事件对细胞过程的影响。-机械生物学--METTL3/14甲基转移酶的化学抑制剂将使对RNA m6A甲基化的功能研究成为可能。-BBSRC在生物科学技术开发方面的战略优先事项我们化学探针开发方法的改进将使更广泛的化学生物学和药物发现社区受益。目前，开发一种选择性和强大的化学探针是一个缓慢而昂贵的过程-我们正在使用多种技术的组合来使这一过程更有效。

Eukaryotic RNAs contain more than 100 chemical modifications. These RNA modifications were initially thought to be static and unalterable, having a minor role in fine-tuning the structure and function of mRNAs. However, recent studies suggest that some RNA modifications are dynamic and reversible which represents an emerging layer of gene regulation at the RNA level. One such dynamic mRNA modification is the methylation of adenosine at the N6 position to form N6-methyladenosine (m6A). It is the most abundant base modification known in eukaryotic mRNA, and recent advances in NGS-based approaches have allowed the location of >12,000 m6A sites to be identified within a ~100 nucleotide resolution on a transcription-wide scale, known as the m6A methylome. The dynamic role of m6A methylation has been emphasised by the identification of RNA methyltransferases (writers) and demethylases (erasers) which make m6A methylation reversible, by catalysing or oxidatively removing the m6A modification in mRNAs. Methyltransferase like 3 (METTL3) and METTL14 have both been shown to have methyltransferase activity forming a stable heterodimer. Wilm's tumour 1-associating protein (WTAP) can also interact with the METTL3/14 complex functioning as a regulatory subunit. In contrast, the human fat mass and obesity (FTO)-associated protein and alkylation repair homolog 5 protein (ALKBH5) oxidatively remove the m6A modification in mRNA. The functional importance of reversible m6A methylation is yet to be fully elucidated, however aberrant m6A modification has been linked with numerous human diseases. It is thought to control many aspects of RNA processing involving regulating transcription, mRNA transport, splicing, stability, transcript abundance and translation. This is also reinforced by the varied outcomes in numerous cellular processes observed with experimental depletion of the m6A-related enzymes. This project aims to generate cell-permeable chemical inhibitors of the METTL3/14 heterodimer to enable the functional relevance of the m6A modification to be probed. Building on the work of our external collaborator (crystal structure of METTL3/14), the student will use structure-based design, computational chemistry, biochemical assays and synthetic chemistry approaches.Overview of the work to be carried out:-Screen METTL3/14 against in silico chemical library - LifeArc-Identify and validate hits using biochemical assays -Whitehouse/LifeArc-Determine crystal structures of hits in complex with METTL3/14 -Bayliss-Prioritize hits and plan synthetic approaches to optimize inhibitors using computational chemistry - LifeArc/ Nelson-Optimize inhibitors through Activity Directed Synthesis -Nelson -Evaluate new inhibitors using biochemical assays and crystallography and iterate with chemical synthesisuntil we have cell-permeable inhibitors with better than 1uM potency against the target - All-Probe the functional consequences of METTL3/14 inhibition on mRNA transport, processing and tranlsation-WhitehouseThe project will contribute to ongoing efforts in the Bayliss/Nelson groups to accelerate the development of chemical probes for mechanistic biology research, using structural biology and activity directed synthesis. Chemical probes have the advantage over genetic methods that the effects of rapidly and reversibly modulating biochemical events on cellular processes can be studied. -Mechanistic biology - Chemical inhibitors of the METTL3/14 methyltransferase will enable functional studies on RNA m6A methylation. -BBSRC strategic priority in technology development for the biosciences Refinement of our approach to chemical probe development will benefit the wider chemical biology and drug discovery community. At present, development of a selective and potent chemical probe is a slow and expensive process - we are using a combination of technologies to make the process more efficient.