Development of diagnostic tools for detection and quantifications of mRNA methylation

点击翻译按钮获取中文摘要

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.