Reversible 6-methyladenine (m6A) base modification in mRNA: molecular function and role in disease

Chemical modifications in protein and DNA have been studied for decades and the discovery of the reversible potential of such modifications is providing a foundation for our knowledge on dynamic gene regulation. The first dynamic modification in mRNA was recently discovered and preliminary data inducate crucial role of this modification in meiosis, the early embryo and in brain development. In this project we will analyze the role of 6-methyladenine in meiosis and in some selected brain cells in culture. We will also generate novel mutant organisms for studying the role of methylation reader proteins and demethylation. Our ultimate goal is to identify readers and erasers of this modification and to understand the reason that distorted 6-methyladenine cause infertility and neurodegeneration. We have published a novel method for the base-specific identification of 6-methyladenine in mRNA. We now use this method for sequence specific mutagenesis to gain further insight on the reversible nature of 6-methyladenine in cellular homeostasis. More recently we identified a novel modification on another RNA molecule, tRNA, that is also dynamically regulated. In Our last publication (in press) we show the importance of such dynamic regulation in embryo brain Development.

This proposal aims to undertake a systematic analysis of the role of methylation and demethylation at specific adenine bases in mRNA to gain understanding of the cellular and molecular function of m6A, and further to apply the gained knowledge for insight on the involvement of m6A in disease. Our existing mice null for Alkbh5, Fto or both, will be used for assessment of the dynamics of m6A at specific mRNA sites. The line of work that we envisage to unravel the cellular and molecular function of m6A in mR NA is composed of i) identification of mouse mutant specific m6A sites in mRNA by transcriptome-wide mapping, ii) investigating the relation between site specific level of m6A and mRNA splicing, iii) identifying m6A binding proteins and compare the locali zation of such proteins with dynamically methylated m6A sites and mRNA secondary structure, iv) examination of the relationship(s) between mRNA export, metabolism, polyA length and copy number with dynamically methylated m6A sites and protein binding, v) synthesis of top candidate mRNAs for single mRNA functional studies vi) pathway determination and characterization in relation to disease. As an integrated part of this work we will develop a single base resolution method for identification of m6A in RNA to be used as a tool for high resolution validation, for aid in selection of suitable mRNAs for in-detail studies and for highly specific analysis of m6A in single mRNA functional studies.