Probing the function of RNA methylation through chemical biology

真核RNA含有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mRNA的结构和功能方面起着次要作用。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水平上基因调控的一个新兴层。一种这样的动态mRNA修饰是腺苷在N6位置的甲基化以形成N6-甲基腺苷（m6 A）。它是真核生物mRNA中已知的最丰富的碱基修饰，并且基于NGS的方法的最新进展已经允许在转录范围内的约100个核苷酸分辨率内鉴定> 12，000个m6 A位点的位置，称为m6 A甲基化组。m6 A甲基化的动态作用已经通过鉴定RNA甲基转移酶（writers）和脱甲基酶（erasers）而得到强调，所述RNA甲基转移酶（writers）和脱甲基酶（erasers）通过催化或氧化去除mRNA中的m6 A修饰而使m6 A甲基化可逆。甲基转移酶样3（胃L3）和胃L14均显示具有甲基转移酶活性，形成稳定的异源二聚体。Wilm's tumor 1-associated protein（WTAP）也可以与胃L3/14复合物相互作用，作为调节亚基发挥作用。相比之下，人脂肪量和肥胖（FTO）相关蛋白和烷基化修复同源物5蛋白（ALKBH 5）氧化去除mRNA中的m6 A修饰。可逆m6 A甲基化的功能重要性尚未完全阐明，然而异常m6 A修饰与许多人类疾病有关。它被认为控制RNA加工的许多方面，包括调节转录、mRNA转运、剪接、稳定性、转录本丰度和翻译。这也通过在m6 A相关酶的实验性消耗中观察到的许多细胞过程中的不同结果得到加强。该项目旨在产生胃L3/14异二聚体的细胞可渗透化学抑制剂，以使m6 A修饰的功能相关性得以探测。在我们外部合作者工作的基础上（L3/14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。- 根据计算机模拟化学库筛选L3/14-LifeArc-使用生物化学测定法鉴定和验证命中-怀特豪斯/LifeArc-确定与L3/14复合的命中的晶体结构-Bayliss-使用计算化学对命中进行优先级排序并计划合成方法以优化抑制剂- LifeArc/纳尔逊-通过活性导向合成优化抑制剂-纳尔逊-使用生物化学测定和晶体学评估新抑制剂，并使用化学合成法进行筛选，直到我们获得对靶点的效力优于1 μ M的细胞渗透性抑制剂- All-Probe研究胃L3/14抑制mRNA转运的功能性后果，该项目将有助于Bayliss/纳尔逊小组正在进行的努力，以加速用于机械生物学研究的化学探针的开发，使用结构生物学和活性导向合成。与遗传学方法相比，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-胃L3/14甲基转移酶的化学抑制剂将使RNA m6 A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对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，开发一种选择性和有效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，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.