Defining the plant epitranscriptome

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The most prevalent internal modification of eukaryotic mRNA is the methylation of adenosine at the N6 position (m6A) and there are writers, readers and erasers of this epitranscriptome code. The enzyme that writes this code (MTA in Arabidopsis) is essential for life in Arabidopsis, flies and humans, and specific functions for RNA methylation are emerging. Working with Arabidopsis, we used in vivo interaction proteomics to identify a core set of conserved factors that co-purify with MTA. We have shown that these proteins are required for mRNA methylation in Arabidopsis and HeLa cells, and refer to them as the m6A writer-complex. These breakthroughs suggest that Arabidopsis can be a generally usefully model system for understanding the role and impact of RNA methylation.

The aims of this proposal are to define the Arabidopsis epitranscriptome, determine how it is regulated and assess the impact on gene expression of disrupting individual writer-complex components.

We will use Me-RIP-seq to identify sites of mRNA methylation. We will test whether m6A is dynamically controlled by quantifying shifts in m6A in different tissues and in response to stress. We will examine functional conservation of m6A by Me-RIP-seq of the crop plants rice and tomato. We will assess regulatory roles of the writer-complex by identifying in vivo targets (ChIP-Seq), the impact of disrupted writer-complex component function on specific m6A modifications (Me-RIP) and identify in vivo protein partners. We will analyse the consequences for gene expression in these functionally compromised backgrounds by quantitative RNA-seq. Finally we will begin the first characterization of Arabidopsis YTH domain proteins that can bind m6A.

This collaboration combines expertise in RNA methylation (Fray), the molecular and proteomic analysis of RNA processing (Simpson) and quantitative analysis of high throughput sequencing data (Barton).

Public health, Pharmaceutical companies, Taxpayers:
It is now clear that methylation of adenosines in mRNA performs a post-transcriptional gene regulatory role in all Eukaryotes studied. In humans, increased activity of the mRNA de-methylase is associated with increased risk of obesity, diabetes, and Alzheimer's as well as certain cancers. We have shown that MTA, MTB, Fip37, PX1 and a conserved E3 ubiquitin ligase associate together in plants and at least the first four of these are required for mRNA methylation. The same five proteins are also known to associate together in both mammals and Drosophila, although in the case of the last two proteins, a link to methylation has not yet been reported. Thus Arabidopsis, with its ease of transformation, ability to grow even when physiologically compromised and with its lack of welfare issues, is already proving a useful multicellular model organism in which to study fundamentals of the methylation process. Both the E3 ubiquitin ligase and Wilm's Tumor Associated Protein (the human homologue of Fip37) are associated with multiple tumor types and cancer prognosis. In addition, both of the human YTH domain m6A binding proteins have been implicated in various cancers or disease states. Thus scientists and pharmaceutical companies interested in understanding or developing novel therapeutics or screening services for these disease conditions may be users of data and methodologies generated in this research project. Ultimately, improved treatment, diagnosis and understanding of these disease states will benefit the wellbeing and health of the public. It could also reduce the financial timebomb associated with certain age and obesity related conditions. Thus, the taxpayer and UK government would be potential beneficiaries.

Industry and Academia groups using transcriptomics data:
We and others have shown that methylation of mRNA is playing a role in regulating cell differentiation and developmental pathways in plants and yeast and in mammals it has been shown to have a role in stem cell maintenance. The mechanisms involved are currently poorly understood, but any research group in academia or industry with an interest in understanding or interpreting gene expression will be a potential user of this research. mRNA transcript levels are often a poor predictor of the abundance of the encoded protein, and in mice this has been shown to principally be a result of varying translation rates for different transcripts. A clearer understanding of the role of mRNA methylation may thus lead to the ability to use transcriptomics data to build models that more closely match the system being studied.

Following several key publications in the last 18months, the importance of this post-transcriptional regulation is beginning to be appreciated and the research field is poised to expand exponentially in coming years.