Development of diagnostic tools for detection and quantifications of mRNA methylation

DNA的信息量主要存在于碱基G、A、T和C沿其长度出现的顺序中。额外的信息可以通过甲基的存在作为碱基之一的“标记”(在多细胞生物体中，Cs几乎是唯一的靶标)。这种“标签”或碱基修饰可能会影响哪段DNA被复制到信使RNA(信使RNA)中。DNA甲基化可以对基因表达产生深远影响，而甲基化的程序化模式有助于调节正常发育。然而，在转录过程中，甲基化的C以正常的方式用作G的模板，DNA中甲基化的存在不会改变所合成蛋白质的氨基酸序列。在将基因复制到信使核糖核酸后，可以对信使核糖核酸本身的碱基进行特定的改变。动植物和酵母菌的mRNA中最常见的修饰是在腺苷(M6A)上添加一个甲基“标签”。M6A在mRNA中的频率往往高达核苷酸的0.2%。这相当于每个典型消息平均一到两次，但我们知道一些消息包含几个m6A站点，而另一些消息则不包含。M6A“标签”的存在不会改变翻译过程中结合的氨基酸，它的功能30多年来一直是个谜。然而，在人类中，一种从mRNA中去除这种甲基化的酶活性增加的人容易患肥胖症、糖尿病和阿尔茨海默氏症。此外，我们还证明了甲基化是植物和酵母正常发育程序所必需的。这种甲基化调节基因表达的机制尚不清楚，但最可能的作用是通过改变RNA蛋白质相互作用-潜在地影响mRNA的翻译、周转或亚细胞位置。与DNA甲基化不同，腺苷甲基化的潜在位点不能用限制性内切酶或亚硫酸氢盐测序来分析，缺乏同等的技术是这一研究领域进展的主要限制因素。我们已经产生了一种抗m6A的单抗，我们和其他人最近使用免疫沉淀来鉴定超过7000个候选甲基化转录本。然而，目前还不存在直接测定特定位置的甲基化或准确测量这种甲基化水平的方法。该项目将开发含有标记的苯硒胸腺胺的修饰DNA寡核苷酸探针，该探针将在紫外光激活时与靶标的腺苷/甲基腺苷交联。用适当的核酸酶消化后，可以很容易地检测到标记的A=dT或m6A=dT二聚体，这些产物相互之间的比例将给出在该特定位点甲基化的信息的比例。直接检测特定腺苷是否存在甲基化并测量这种甲基化如何随着环境和发育条件的不同而变化的能力将从根本上改变研究这一现象的方式。显然，mRNA甲基化起源于古老的进化，在所有真核生物王国中发挥着重要的调节作用。最近，人类的主要疾病都与信使核糖核酸甲基化有关，开发分析这些甲基化转录本的工具将具有直接的相关性和影响。

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.