The Arabidopsis Epitranscriptome

Working with pea plants in his monastery garden, the Austrian monk Gregor Mendel discovered that they inherit from their parents, what we now know to be genes, which control how they grow. Like peas, the genes in the DNA of our chromosomes have the code for life. But what is that code exactly? DNA is comprised of long chains of chemicals of four different types: A, C, G and T. The genetic code is copied into a related molecule called RNA that is the messenger of this code. RNA is comprised of almost the same chemicals, A, C, and G, but U replaces T. Cellular machines called ribosomes, take the message and use it to build proteins corresponding to this code.

Interestingly, the RNA chemicals can be altered, and by far the most common modification within the messenger RNA chain is m6A. Consequently, messenger RNA is effectively comprised of five different chemicals: A, C, G, U and m6A. You never heard of it? It is surprising how little attention it has had because if humans, flies or plants don't have it, they die. Recently, a human gene called FTO, which is linked to several human diseases, was found to encode a protein able to convert m6A back to A. This revealed that m6A levels in RNA could be controlled, and if this was disrupted, disease could result. It seems that m6A doesn't change the genetic code itself, but it does affect the message and so affects how the code is used in everyday life. This project is all about m6A in plants, but based on what we have done so far, it should tell us about animals and people as well.

Like Mendel, our project results from discoveries we have made with plants. While studying a protein that naturally helps plants flower, Gordon Simpson's team discovered it controlled where messages end. Using a specially developed technique, they discovered that this protein is found close together with enzymes that make m6A. This made some sense because Rupert Fray, an RNA methylation expert, had previously shown that m6A is mostly found near the end of messages. So, using the same techniques to see what proteins were closely associated with the enzymes that make m6A, Gordon Simpson worked with Rupert Fray, and together, they discovered several proteins that were highly related across lots of different plants and animals, that helped these enzymes make m6A not only in plants but in humans as well.

The aim of this project is to understand m6A a lot better by using plants. Plants are vital to our food and energy security so it is important that we know how they work. Because we can make mutant plants in the lab that still live but have altered levels of m6A, we can study them more simply and use that knowledge to try to understand why plants and animals use m6A in the message of their genetic code.

First, we want to know which messages have m6A and where in the message is this found. We want to know if this changes in different situations such as in flowers compared to leaves or when the plant is stressed. Second, we want to know how m6A is made by the factors that help the enzymes we have found. Do they do it to all genes, or only some and only in specific parts of some messages? How do they talk about what they are doing to all the other parts of the cell that are making and reading the code as well? Third, we want to understand exactly what goes wrong when m6A is changed. What happens to individual messages? Finally, we'd like to begin to understand how the m6A code is read. Proteins with YTH domains apparently bind m6A, they are found in plants but we don't know what they do.

We form a hugely experienced team in this area and we hope to learn very basic knowledge about the message of our genetic code. This work will provide state-of-the-art training for early career scientists working as a team on plants, genetics, RNA, proteins and computational analysis of large sequencing datasets - assembling the skills modern plant science needs to ensure future food and energy security.

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).

1. Cultural Life. Our work defines a new area of science connected to the very nature of the genetic code. This curiosity-led discovery of new knowledge is a feature that the UK public expect of their scientists as GGS experienced when he spoke about non-coding antisense RNAs at a BBSRC organized public engagement event at the Edinburgh International Science Festival.

2. Agricultural Industry. Our work benefits the development of world agriculture in several distinct ways. First GGS and RGF are training a new generation of plant scientists familiar with working with genetics, making crosses and phenotyping plants. Second, we are training biologists used to working in multi-disciplinary teams, combining the iterative interaction of bench scientists with computational biologists. Third, through analysis of huge sequencing datasets we are drawing into plant biology, scientists from mathematical and physics backgrounds, who bring with them quite different skill-sets and insight that can be highly beneficial to understanding plant biology and hence crop science. By establishing cross-link based in vivo interaction proteomics, we are optimizing a relatively neglected research tool for the plant biology community. In light of the relative challenges in extracting proteins from living plants compared to human cell lines, this development may be particularly beneficial to plant biologists. Finally, the link between mRNA methylation and translatability under stress conditions identified by RGF's group may benefit the agricultural industry by aiding selection/breeding programmes. For example, knowledge of how plants survive hypoxic conditions could lead to crops better able to withstand flooding and waterlogging.

3. World Economy. Dundee takes the training of PhD and Post-Doctoral scientists particularly seriously and has a specific department called "OPD" that delivers "non-bench" training in, for example, public speaking and public engagement. Both Dundee and Nottingham provide a highly international working environment with staff from over 60 different nationalities. Dundee houses the 3rd largest biotech cluster in the UK, whilst Nottingham University is a founding partner in BioCity Nottingham - one of Europe's largest bioscience incubators. Together, these aspects of research life provide rounded, highly skilled and educated employees to the international work-force. Among recent alumni from GGS's and GJB's BBSRC-funded work who came to Dundee from overseas, C. Hornyik has gone on to hold a P.I. position in crop science in the UK, L. Terzi a managerial position in a Swiss pharmaceutical company and Alexander Sherstnev analyses RNA-Seq data for Glaxo Smith Kline in Hertfordshire. Alumni from RGF's lab include University faculty (G. Kahka and S. Zhong) and staff scientists in industry (J. Button, MedImmune).

4. Society Through Public Engagement. This proposal relates to fundamental understanding of the genetic code. The GM controversy highlights the importance of public understanding and support for the research we do. GGS became responsible for Dundee Plant Sciences impact activities in 2010 and since then the Division has successfully developed valuable links with Dundee's Botanic Garden, a hands-on DNA extraction activity to communicate information about plants having genes and a sustainable "Genetics Garden". In this proposal we describe a computer generated animation of the epitranscriptome and social media tools that we will use to communicate our research interests in gene expression to the general public. The work of our groups in public engagement is not unique, but part of the culture of Dundee University College of Life Sciences, reflected by the fact that Dundee won the inaugural BBSRC "Excellence with Impact" competition and are participants in the current competition.