RNA methylation, surveillance and the plant immune response

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 as a template on which to build proteins. RNA is comprised of almost the same bases, A, C, and G, but U replaces T. As a gene is being copied into mRNA, specific changes can be made to the bases of the RNA 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 m6A is usually added near the end of the mRNA, close to the site were translation in to protein ends, but we know that some messages contain several m6A sites whilst others contain none. Fifteen 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. Plants that we engineered to have low levels of m6A are stunted, have specific leaf and developmental defects and constitutively activate pathogen defence responses. 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 is "read" by nuclear and cytoplasmic m6A binding "YTH" proteins. In the nucleus, YTH proteins can be involved in telling the cell where to stop copying the DNA into RNA, and in the cytoplasm, different YTH proteins tell the cell whether to degrade, translate or store an mRNA. In both plants and animals, cytoplasmic YTH proteins interact with UPF1 (UP-Frameshift-1), a protein involved in "quality control" by determining if the correct end to translation has been reached. Mutation of UPF1 also constitutively activates the immune response and gives stunted plants with similar leaf defects. By mutating thousands of seeds in a "suppressor screen", and by carrying out targeted genetic crosses, we have shown that inactivation of defence signalling pathways restores normal plant stature and leaf shape in low m6A plants. Similar crosses also restore normal growth and leaf defects to UPF1 mutants.
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, m6A "writers" and YTH "readers" are all 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 enzyme writers 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 with low m6A are more resistant to certain bacterial pathogens, thus understanding the function of writing, reading and integration with UPF1 "quality control" will inform marker assisted breeding programmes for developing higher yielding disease resistant crops.
The aim of this project is to understand the way in which m6A regulates how some mRNAs are degraded or translationally suppressed by the UPF1-dependent surveillance pathway. This is important because it is likely a conserved process that underlies m6A function across eukaryotes.

Many eukaryote mRNAs contain N6-methyladenosine (m6A), and there are writers, readers and erasers of this mark. The protein complex that "writes" the methylation, as well as some the proteins that "read" m6A, have remained conserved since the last common ancestor of plants and metazoans nearly 1.5 billion years ago. Mammals and plants lacking m6A die early in embryogenesis. However, using a range of genetic approaches, we have created a set of stable hypomorphic m6A writer mutants with m6A reduced by up to 90%. Low m6A plants show severe developmental defects including loss of apical dominance, a serrate leaf phenotype, extreme dwarfism, insensitivity to the plant hormone auxin, defects in organ formation and a constitutive immune response, and these phenotypes become progressively more severe as the m6A level is reduced. The modification is "read" by cytoplasmic (YTHDF type) and nuclear (YTHDC type) proteins that bind m6A. Arabidopsis has 11 YTHDF genes, and triple mutants in which three of the most highly expressed members are knocked out phenocopy the defects seen in the low m6A lines.
In both plants and animals, cytoplasmic YTH proteins interact with UPF1 (UP-Frameshift-1), a component of the RNA surveillance nonsense mediated decay (NMD) pathway. Mutation of UPF1 also constitutively activates the immune response and gives stunted plants with similar leaf defects seen in the m6A writer and reader mutants. Using a low m6A writer line in a screen for suppressor mutations, and by targeted genetic crosses, we identified genes in the defence signalling pathway that restore near normal growth to low m6A lines. The same mutations also rescue similar growth defects in upf1 mutants. This grant will use a combination of molecular and genetic approaches to test whether the YTH-UPF interaction potentiates NMD, and will determine the role of m6A in generating immune receptor mRNA and other NMD substrates whose expression is naturally restricted by the NMD pathway.