Functional Consequences Of The Plant Epitranscriptome

DNA is comprised of long chains of "bases" of four different types: A, C, G and T. The information content of DNA resides primarily in the order in which these occur along its length. The genetic code is copied into a related molecule called RNA that is the messenger of this code. This messenger RNA (mRNA) moves out from the cell nucleus and is used by cellular machinery called ribosomes as a template on which to build proteins. RNA is comprised of almost the same bases, A, C, and G, but U replaces T. After a gene has been copied into mRNA, specific changes can be made to the bases of the mRNA itself. The most common modification within mRNA of both plants and animals is the addition of a small chemical "tag" to adenosines to make m6A in a process referred to as methylation. The frequency of m6A in mRNA is about 0.1-0.2%. This would correspond to an average of roughly once or twice per typical message, but we know that some messages contain several m6A sites whilst others contain none. The presence of the m6A "tag" does not change which amino acids are incorporated during translation and its function remained a mystery for more than 30 years. Ten years ago we showed that this modification was needed for normal developmental programmes in plants and yeast and other groups subsequently showed that this was also the case in animals. The reason for this is that the presence of m6A can affect how a mRNA is processed, when it is degraded and how many times it is used as a template for protein synthesis. However, the mechanisms by which m6A achieves this are still poorly understood.

The methylation process is itself dynamic. The human fat mass and obesity associated gene, FTO, has been shown to encode a protein that can remove the "tag" and convert m6A back to A. FTO is linked to several serious human diseases, so understanding the function of m6A at a molecular level has become important for drug companies and researchers worldwide.

Using plants as a model system, we have identified the group of enzymes that act together to put the "tag" on the mRNA and we have shown that the tag is usually placed near the end of mRNA. These enzymes and features of mRNA methylation were subsequently found to be highly conserved between plants and animals. Thus, experiments in plants that are much simpler to perform than in mammals can reveal fundamental principles that are likely to operate in humans also.

Plants that we have engineered to have low levels of m6A are small, weak and have specific developmental defects. By randomly mutating thousands of seeds from these plants, we found some plants that grew normally, even though they still had very low m6A levels. One of the mutations that restores normal growth is in a gene that encodes a protein that forms part of the ribosome. This ribosomal protein is needed to restart or continue translation of mRNAs with certain structures, both in plants and in some human viruses.

The aim of this project is to understand the way in which m6A regulates how some mRNAs are translated by ribosomes. This is important because it is likely a conserved process that underlies m6A function in human processes too. We will also identify the mutations in other genes that allow our low m6A plants to grow normally, we anticipate that similar gene functions will also be present in human and that insights gained from our plant model system will continue to inform the research field.

Eukaryotic mRNAs can be modified by the methylation of adenosine (m6A) and there are writers, readers and erasers of this epitranscriptome mark. Genes encoding the protein complex that "writes" the methylation, as well as some of the proteins that "read" m6A, have remained conserved since the last common ancestor of plants and metazoans nearly 1.5 billion years ago. m6A is essential for mammals and plants and null mutants die early in embryogenesis. However, using a range of genetic approaches, we have created a set of hypomorphic m6A writer proteins in which m6A levels are reduced by 90%.
Methylation of mRNA has been shown to regulate gene expression at multiple levels including alternative splicing, alternative poladenylation, nuclear export, translation and decay. However, it is not clear which of these are more recently evolved "special cases" and which represent the ancient "core" function. Using a hypomorphic low m6A writer line in a genetic screen for suppressor mutations, we obtained more than 20 independent events that restored or partially restored normal plant growth. In some cases this was associated with m6A levels closer to wild type levels, but in other cases m6A levels remained low. Conceptually suppressors could result from altered writer, eraser or reader/interpreter functions, but only reader/interpreter mutations would be expected to suppress the low m6A phenotype without changing m6A levels. We have identified one suppressor mutant as encoding a ribosomal protein that is required for re-initiation after an upstream open reading frame (uORF) and we have shown that some uORF containing transcripts are mistranslated in low m6A plants. This proposal will identify and test the features and mechanisms by which m6A in 3' sequences interacts with translation machinery to regulate expression of a specific mRNA subset. It will also characterise other suppressor mutants to identify regulators of reader, writer and eraser activity.

1. Cultural Life. The field of epitranscriptomics is a new area of science connected to the very nature of the genetic code. The UK public has an expectation that their scientists undertake fundamental, curiosity driven research and they have enthusiasm when such fundamental finding are explained in layman's terms. This was evident from a recent public talk on RNA modification given by RGF at the University.

2. Disease, Medicine and Health. The m6A demethylase, FTO, is implicated as a risk factor in obesity, diabetes and metabolic syndrome and an FTO variant has also been linked Alzheimer risk. A better fundamental understanding of m6A action in gene regulation could lead to improved diagnostics as well as drug development and treatment opportunities.

3. Agricultural Industry. Our work benefits the development of UK and world agriculture in several ways. Firstly, a better understanding of how epitranscriptomic modifications alter gene expression and ultimately protein levels will help to bridge the "genotype to phenotype gap". Plant breeders are keen for a clearer understanding of how specific genotypes and environment interact to impact crop performance. uORFs can participate in the integrated stress response in many organisms, any knowledge that led to better predictions of how a crop plant would respond to a biotic or abiotic stress would be of considerable importance to the industry. Secondly, through training of plant scientists familiar with working with genetics, making crosses, phenotyping plants and also analysing large sequencing data sets.