Probing the function of RNA methylation through chemical biology

Eukaryotic RNAs contain more than 100 chemical modifications. These RNA modifications were initially thought to be static and unalterable, having a minor role in fine-tuning the structure and function of mRNAs. However, recent studies suggest that some RNA modifications are dynamic and reversible which represents an emerging layer of gene regulation at the RNA level.
One such dynamic mRNA modification is the methylation of adenosine at the N6 position to form N6-methyladenosine (m6A). It is the most abundant base modification known in eukaryotic mRNA, and recent advances in NGS-based approaches have allowed the location of >12,000 m6A sites to be identified within a ~100 nucleotide resolution on a transcription-wide scale, known as the m6A methylome.
The dynamic role of m6A methylation has been emphasised by the identification of RNA methyltransferases (writers) and demethylases (erasers) which make m6A methylation reversible, by catalysing or oxidatively removing the m6A modification in mRNAs. Methyltransferase like 3 (METTL3) and METTL14 have both been shown to have methyltransferase activity forming a stable heterodimer. Wilm's tumour 1-associating protein (WTAP) can also interact with the METTL3/14 complex functioning as a regulatory subunit. In contrast, the human fat mass and obesity (FTO)-associated protein and alkylation repair homolog 5 protein (ALKBH5) oxidatively remove the m6A modification in mRNA.
The functional importance of reversible m6A methylation is yet to be fully elucidated, however aberrant m6A modification has been linked with numerous human diseases. It is thought to control many aspects of RNA processing involving regulating transcription, mRNA transport, splicing, stability, transcript abundance and translation. This is also reinforced by the varied outcomes in numerous cellular processes observed with experimental depletion of the m6A-related enzymes.
This project aims to generate cell-permeable chemical inhibitors of the METTL3/14 heterodimer to enable the functional relevance of the m6A modification to be probed. Building on the work of our external collaborator (crystal structure of METTL3/14), the student will use structure-based design, computational chemistry, biochemical assays and synthetic chemistry approaches.

Overview of the work to be carried out:
-Screen METTL3/14 against in silico chemical library - LifeArc
-Identify and validate hits using biochemical assays -Whitehouse/LifeArc
-Determine crystal structures of hits in complex with METTL3/14 -Bayliss
-Prioritize hits and plan synthetic approaches to optimize inhibitors using computational chemistry - LifeArc/ Nelson
-Optimize inhibitors through Activity Directed Synthesis -Nelson
-Evaluate new inhibitors using biochemical assays and crystallography and iterate with chemical synthesisuntil we have cell-permeable inhibitors with better than 1uM potency against the target - All
-Probe the functional consequences of METTL3/14 inhibition on mRNA transport, processing and tranlsation-Whitehouse


The project will contribute to ongoing efforts in the Bayliss/Nelson groups to accelerate the development of chemical probes for mechanistic biology research, using structural biology and activity directed synthesis. Chemical probes have the advantage over genetic methods that the effects of rapidly and reversibly modulating biochemical events on cellular processes can be studied.
-Mechanistic biology - Chemical inhibitors of the METTL3/14 methyltransferase will enable functional studies on RNA m6A methylation.
-BBSRC strategic priority in technology development for the biosciences Refinement of our approach to chemical probe development will benefit the wider chemical biology and drug discovery community. At present, development of a selective and potent chemical probe is a slow and expensive process - we are using a combination of technologies to make the process more efficient.