Development of diagnostic tools for detection and quantifications of mRNA methylation

开发用于检测和定量 mRNA 甲基化的诊断工具

• 批准号: BB/K013637/1
• 负责人: Rupert Fray
• 金额: $ 15.26万
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FK013637%2F1
• 关键词: Development; diagnostic; tools; detection; quantifications

The information content of DNA resides primarily in the order in which the bases G, A, T and C occur along its length. Additional information may be given by the presence of a methyl group as a "tag" to one of the bases (in multicellular organisms Cs are almost exclusively the target). This "tag" or base modification may influence which stretch of DNA is copied into messenger RNA (mRNA). DNA methylation can have profound effects on gene expression, and programmed patterns of methylation help regulate normal development. However, a methylated C is used as a template for G in the normal way during transcription, and the presence of methylation within DNA does not change the amino acid sequence of the protein that is made.After a gene has been copied into mRNA, specific changes can be made to the bases of the mRNA itself. The most common modification within mRNA of animals, plants and yeast is the addition of a methyl "tag" to adenosines (m6A). The frequency of m6A in mRNA is often as high as 0.2% of nucleotides. This would correspond to an average of roughly once or twice per typical message, but we know that some messages contain several m6A sites whilst others contain none. The presence of the m6A "tag" does not change which amino acids are incorporated during translation and its function has remained a mystery for more than 30 years. However, in humans individuals with increased activity of an enzyme that removes this methylation from mRNA are susceptible to obesity, diabetes and Alzheimer's. In addition, we have also shown that methylation is required for normal developmental programmes in both plants and yeast.The mechanism by which this methylation regulates gene expression is not known, but most probably acts through altering RNA protein interactions - potentially influencing mRNA translation, turnover or sub-cellular location. Unlike DNA methylation, potential sites of adenosine methylation cannot be assayed using restriction enzymes or bisulphite sequencing, and the lack of equivalent technologies is the major limiting factor in the progression of this research field. We have generated a monoclonal antibody against m6A and we and others have recently used immunoprecipitation to identify over 7000 candidate methylated transcripts. However, currently no method exists to directly assay for metylation at a specific site or to accurately measure levels of such methylation. This project will develop modified DNA oligonucleotide probes containing a labelled phenylselenothymine that will crosslink to a target adenosine/methyladenosine upon UV photoactivation. After digestion with appropriate nucleases, the labelled A=dT or m6A=dT dimmer can be readily detected and the ratio of these products to each other will give the proportion of messages that are methylated at that particular site. The ability to directly assay specific adenosines for the presence of methylation and to measure how this methylation changes with different environmental and developmental conditions will fundamentally change the way in which the phenomena can be studied.Clearly mRNA methylation is of ancient evolutionary origin and is playing an important regulatory role in all eukaryote Kingdoms. Major human diseases have recently been associated with mRNA methylation and developing tools to analyse these methylated transcripts will have immediate relevance and impact.

DNA的信息含量主要取决于碱基G、A、T和C沿其长度排列的顺序。一个甲基作为一个碱基的“标签”（在多细胞生物中，c几乎完全是靶标）的存在可以提供额外的信息。这种“标签”或碱基修饰可能影响哪段DNA被复制成信使RNA （mRNA）。DNA甲基化可以对基因表达产生深远的影响，甲基化的程序化模式有助于调节正常的发育。然而，在转录过程中，甲基化的C以正常的方式被用作G的模板，并且DNA中甲基化的存在不会改变所合成的蛋白质的氨基酸序列。基因被复制成mRNA后，mRNA本身的碱基就会发生特定的变化。在动物、植物和酵母的mRNA中最常见的修饰是在腺苷（m6A）上添加甲基“标签”。mRNA中m6A的频率通常高达核苷酸的0.2%。这对应于每个典型消息平均大约一到两次，但我们知道有些消息包含几个m6A站点，而其他消息则不包含。m6A“标签”的存在并没有改变翻译过程中纳入的氨基酸，其功能30多年来一直是一个谜。然而，在人类中，去除mRNA甲基化的酶活性增加的个体容易患肥胖症、糖尿病和阿尔茨海默氏症。此外，我们还表明甲基化是植物和酵母正常发育程序所必需的。甲基化调节基因表达的机制尚不清楚，但最有可能通过改变RNA蛋白相互作用起作用——潜在地影响mRNA翻译、周转或亚细胞定位。与DNA甲基化不同，腺苷甲基化的潜在位点无法通过限制性内切酶或亚硫酸盐测序来测定，缺乏相应的技术是该研究领域进展的主要限制因素。我们已经产生了一种针对m6A的单克隆抗体，我们和其他人最近使用免疫沉淀技术鉴定了7000多种候选甲基化转录本。然而，目前还没有方法可以直接测定特定位点的甲基化或准确测量这种甲基化水平。该项目将开发含有标记苯硒腺苷的修饰DNA寡核苷酸探针，该探针将在紫外线光激活下与目标腺苷/甲基腺苷交联。用适当的核酸酶消化后，标记的A=dT或m6A=dT二聚体可以很容易地检测到，这些产物彼此的比例将给出在特定位点甲基化的信息的比例。直接测定特定腺苷甲基化存在的能力，以及测量甲基化如何随不同环境和发育条件而变化的能力，将从根本上改变研究这种现象的方式。显然，mRNA甲基化具有古老的进化起源，并在所有真核生物王国中发挥着重要的调节作用。最近，主要的人类疾病与mRNA甲基化有关，开发工具来分析这些甲基化转录本将具有直接的相关性和影响。

项目成果

The importance of m 6 A topology in chicken embryo mRNA; a precise mapping of m 6 A at the conserved chicken ß-actin zipcode

m 6 A拓扑结构在鸡胚mRNA中的重要性；

• DOI: 10.1101/2022.03.04.483006
• 发表时间: 2022
• 作者: Baron F