Mechanistic basis of m6A-mediated mRNA regulation

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The complex organisation of the human body and its response to challenges requires a fine control of the translation of the gene code into proteins in different cells and at different times. The dynamic modification of the messenger RNA (mRNA) molecules play an important part in this process, as this modification is read by regulatory proteins that add a layer to the regulation of gene expression. Methylation of the Adenosine in position 6, N6-Methyladenosine (m6A) is the most common mRNA modification. The level of m6A methylation is dynamic and is controlled by a set of 'writer' and 'eraser' proteins. In turn, the code created by the modified m6A is read by a set of 'reader' proteins. Mis-function or mis-expression of these proteins is linked to important systemic illnesses, such as diabetes and many forms of cancer. While the canonical pathways for the writing, erasing and reading of m6A methylation have been clarified, recent work has shown that a larger number of non-canonical readers exist. These readers are proteins known to bind and regulate mRNA and the discovery relates m6A regulation to general RNA regulation processes, such as mRNA splicing and enhanced mRNA turnover. However, for most non-canonical readers, we do not understand this relation at the molecular level.We work on IMP1, a highly conserved RNA-binding protein that regulates mRNA metabolism, transport and translation. It has been recently shown that the regulation of a set of IMP1 targets in cancers cells is dependent on m6A methylation, and this methyl-regulation is important for the synthesis of the cancer protein c-myc. The questions we are asking are i) how the IMP1 protein recognises m6A methylated targets and ii) which is the reach of m6A methyl-regulation of IMP1 in cancer cells.To answer these questions we will first determine the structure of the IMP1 protein in complex with the methylated RNA target and compared it with the structure with a non-methylated RNA, which we have published in 2017. Then, using the structure, we will design mutations to create IMP1 variants that recognise only non-methylated RNA. Finally we will use these variants to determine, in the cell, which mRNAs are bound by IMP1 in a methyl-dependent fashion, i.e. to define the selectivity of m6A methylation. In addition we will create biophysical models that explain the differences between the recognition of methylated and non-methylated RNAs and how these are differences are related to protein and RNA concentration.We expect this work will clarify, at the molecular level, how IMP1 recognises the m6A methylated target mRNAs to regulate their stability in cancer cells. Further, we expect the molecular features of recognition will be common to a growing class of related non-canonical m6A methyl readers and that the concepts and tools provided by this study will allow the investigation of the role of m6A in these proteins. Together the work will provide a first paradigmatic molecular insight into how m6A regulation is integrated in global RNA regulation networks in cancer cells.