Plants coordinate their growth and development through complex regulatory networks involving changes in the expression of thousands of genes. Many developmental pathways are regulated at the level of messenger RNA (mRNA) through alternative choices in mRNA processing. These choices can have consequences for the localization, stability or translatability of mRNAs. One of the key ways in which RNAs are processed is by the methylation of the RNA base adenosine – a modification known as m6A. Even though it was first discovered in the 1970s, the biological significance of m6A marks has only recently become clear. In this feature article, we identify the factors controlling the writing and reading of m6A modifications in plants. We also highlight some of the features of plant development that depend on m6A and explore the recently discovered molecular mechanisms that use m6A to control development or response to environmental stress.
The importance of RNA processing and modification
Messenger RNA (mRNA) is often presented as a copy of the information contained within a gene – an intermediate step in the path to making proteins from DNA. However, during transcription, premature mRNA (pre-mRNA) molecules undergo a range of processing events that shape mature mRNAs. These include splicing of exons and introns, selection of 5′ start and 3′ end sites and the addition of a poly(A) tail that can vary in length. The choices made during pre-mRNA processing can influence the fate of mRNAs. Alternative splicing can change the sequence of the protein that each mRNA codes for, increasing the diversity of proteins that can be produced from any one gene. By adding or removing regulatory elements to which proteins can be bound, the fate of mRNAs can be altered, e.g., they can be stabilized, or targeted for degradation. In plants, RNA processing is crucial in the regulation of key developmental pathways. For example, plants must integrate multiple environmental cues including day length and temperature to choose when to produce flowers. Many of the proteins that tune this carefully balanced process do so by altering the processing of mRNAs encoding core transcription factors.
RNA molecules can also be processed by the covalent addition of chemical modifications to RNA bases. The most common internal modification to mRNAs is the methylation of adenosines at the N6 position, often referred to as m6A. The process by which RNA is methylated appears to be conserved in plants and animals. However, emerging evidence points to key differences between the two kingdoms in the way m6A affects RNA metabolism.
How are plant mRNAs modified?
Methyl groups are added to adenosine ribonucleotides by a group of catalytic and regulatory proteins referred to as the m6A writer complex (Figure 1). The key proteins in this complex are methyltransferases: MTA, the main catalytic component; and MTB, a related protein whose catalytic activity is unconfirmed. Additional proteins co-purifying with MTA and MTB, named VIRILIZER, FIP37 and HAKAI, are required for normal levels of m6A. The exact functions of these regulatory proteins are not yet known. The writer complex appears to recognize, bind and methylate mRNAs whilst transcription is still underway. Recent technological advances, which identified m6A marks dependent on the writer complex component VIRILIZER, showed that in Arabidopsis methylated adenosines are almost exclusively located within 3′ untranslated regions. These regions do not contribute to the protein-coding potential of mRNAs and are instead thought to have regulatory functions in mRNA metabolism.
Writer complex is involved in methylation of adenosines within mRNAs. m6A is formed by adding methyl groups (–CH3) at the N6 position of adenosines located in the 3′ untranslated regions (3′ UTRs) of mRNAs. Methylation is catalysed by the writer complex (green) and might be removed by eraser proteins (red). Adapted from Arribas-Hernández et al. (2020)
Once added to 3′ untranslated regions, m6A might alter the fate of mRNAs by changing the physical properties or structure of the RNA. Alternatively, it could act as a platform for the binding of regulatory proteins known as m6A readers (Figure 2). The majority of known m6A readers contain a specialized YTH domain which is able to bind to m6A directly (Box 1). In Arabidopsis, 13 proteins with YTH domains have been identified. The best characterized of these, ECT2, ECT3 and ECT4, are localized in the cytoplasm and have long disordered tails which might act as scaffolds to recruit other proteins to methylated mRNAs. Plants differ from animals in that a conserved protein in the regulation of cleavage and polyadenylation, CPSF30, has gained a YTH domain (Figure 3). CPSF30 is important for recognizing the motifs that signal the end of an mRNA and recruiting the machinery that cuts it and appends a poly(A) tail. This suggests that m6A, via interaction with CPSF30, could play a role in regulating the formation of mRNA ends in plants.
Possible functions for m6A in mRNA metabolism. Experimentally verified (bold) and potential functions for m6A in mRNA metabolism. These functions may depend on the m6A recognition and binding by reader proteins. Adapted from Arribas-Hernández et al. (2020)
First identified in 2002 in the mammalian splicing factor YT521-B.
Examples are found across the eukaryotic domain.
Interact with mRNAs using conserved aromatic residues which form an m6A-binding pocket (Figure 3).
Plant genomes tend to contain large and variable numbers of YTH domain proteins, some of which may be functionally redundant.
The crystal structure of the
Arabidopsis CPSF30 YTH domain (blue), in complex with an m6A-modified RNA (yellow). Image created using Mol* using structural data deposited by Wu, B.X., Nie, H.B., Li, S.S., Patel, D.J. (2019). RCSB PDB, ID: 5ZUU. DOI: 10.2210/pdb5ZUU/pdb
The discovery of a class of proteins which are able to remove m6A from mRNAs (referred to as erasers) has led to the idea that m6A might be added to or removed from mRNAs in response to different cellular and environmental stimuli. However, to date, only two mRNA m6A demethylases, ALKBH9B and ALKBH10B, have been described in Arabidopsis.
What is the biological relevance of m6A modification?
The importance of m6A is highlighted by the fact that Arabidopsis mutants defective in the writer MTA exhibit abortion of embryos early in development. Only mutant alleles that retain at least a low level of m6A can develop further. Most viable m6A writer mutants, e.g. mta, virilizer and fip37, are therefore weak alleles with 5%–15% of the m6A detected in normal plants. These residual m6A levels allow plants to grow, albeit with severe defects, enabling the study of the effects of low methylation. Interestingly, complete loss of function of HAKAI is not embryonic lethal. In these mutants, the m6A level is reduced by only 35% compared to normal plants, with no significant plant growth defects detected.
m6A is not only required for the growing embryo, but is also important for later stages of plant development. Plants with mutations in genes encoding m6A writers have stunted growth and defective organ definition. Issues with cell differentiation lead to a bushier appearance and abnormal formation of leaves. Root growth, architecture and vascular system development are also affected. Loss of m6A also seems to cause problems with measuring day length: in virilizer mutants, the circadian period is prolonged from 24 to 25 hours. Mutations in individual m6A reader proteins generally lead to less severe defects than mutations disrupting m6A writing. This suggests that reader proteins share some redundant functions. When multiple readers are mutated at the same time, however, problems start to emerge. Double mutants of the m6A reader proteins ECT2 and ECT3 exhibit defects in the morphogenesis of leaf trichomes (specialized structures on leaf surfaces which are important for reducing water loss and providing defence against insects), as well as delayed and aberrant leaf formation. These deformities are even more visible in triple mutants lacking ECT2, ECT3 and ECT4, and begin to resemble the appearance of a weak m6A writer mutant mta. This suggests that the defects displayed in m6A writer mutants may be caused by a loss of reader binding sites.
How does m6A affect the fate of plant mRNAs?
The huge impact that loss of m6A has on plant development raises questions regarding the molecular mechanisms which utilize m6A. High throughput sequencing of the global mRNA pools extracted from mta, fip37 and virilizer mutants has identified a reduction in the relative abundance of mRNAs which would be methylated under normal conditions. This indicates that in plants, m6A might control the gene expression by stabilizing mRNAs, in contrast to mammals, where it appears to speed up their degradation. Similar observations have been made for some m6A readers – complete inactivation of ECT2 also causes destabilization of methylated mRNAs. This finding demonstrates that the stabilizing effect of m6A can be explained by the binding of m6A by reader proteins.
Only slight changes in mRNA splicing have been observed in mutants of the writer complex. However, in virilizer and fip37 mutants, problems with the proper formation of mRNA 3′ ends have been detected. In these mutants, loss of m6A causes a global switch to mRNAs with shorter 3′ untranslated regions. The mechanism for this is still unclear, but one hypothesis is that it involves the recognition of m6A by the RNA processing factor CPSF30. Why plants would want to control mRNA length in this way is also not yet known. However, untranslated regions often contain regulatory sequences which might be included or excluded by changes in length. This may impact the repertoire of regulatory proteins binding to mRNAs, potentially affecting their stability, localization, translatability or folding.
m6A in a changing environment
Plants adapt to changes in their environment by reprogramming gene expression. m6A is a potential candidate for the control of stress response, since recognition of m6A marks by readers could target specific mRNAs for rapid post-transcriptional control. However, there is currently only a small amount of work to support this hypothesis. It has been shown that the expression of genes encoding m6A writers and readers is affected by abiotic (e.g., heat and salinity) and biotic (pathogen-triggered) stresses. For example, in tobacco plants which have been infected by the tomato mosaic virus, the abundance of mRNAs coding for m6A writers decreases, whilst the abundance of potential erasers increases. There are also indications that m6A within mRNAs encoding salt stress response proteins increases the levels of these mRNAs upon salinity stress treatment.
In general, m6A appears to be a feature of mRNAs which are highly abundant and efficiently translated into proteins. These proteins are involved in translation and key growth and development processes such as photosynthesis and respiration. In stress conditions, a large proportion of such mRNAs are rapidly targeted for degradation or translational silencing as cells halt their normal growth. During heat stress, Arabidopsis ECT2 relocalizes from the cytosol to cytoplasmic foci called ‘stress granules’. These are dense aggregations composed of proteins and RNA molecules, which are prevented from being translated into new proteins. Similar relocalization events have also been identified for ECT2, ECT4 and ECT3 under osmotic stress. This suggests that the selective deposition of m6A could have a role in controlling which mRNAs are relocalized during stress. However, the lack of experiments to determine how mutants lacking m6A function in stress conditions means that there is not yet an explanation as to how this affects plant stress adaptation.
The missing pieces of the puzzle
Despite the work that has been done to identify m6A writers, readers and erasers, it is far from clear if all the factors which play a role in m6A biology have been identified. We understand little of how the regulatory proteins which are associated with the writer complex function, nor whether there are other RNA methyltransferases targeting different RNA species. Characterization of m6A readers lags further behind. Most have only been identified by the presence of a YTH domain, and so their ability to bind m6A needs to be experimentally verified. It is also possible that other domains can bind m6A, and that the repertoire of m6A readers is broader than currently thought. The least explored class of m6A regulating proteins are the erasers. It is not clear how many m6A erasers exist in plants, whether they are able to efficiently remove m6A from mRNAs, or what their biological importance is. Getting answers to these questions would help to resolve the controversial question of whether m6A is a dynamic mark.
In addition to m6A, more than 150 different modifications have been identified in eukaryotic and prokaryotic RNAs. It is unclear which of these occur within plant mRNAs – studies have so far only described 5-methylcytosine (m5C) in Arabidopsis and rice. Technologies such as nanopore direct RNA sequencing, which has been recently used to identify Arabidopsis m6A, could in future help us to recognize all plant mRNA modifications and map their positions in the transcriptome.
From the current evidence, it is clear that m6A modifications are important for regulating the processing and stability of plant mRNAs. In future, a clearer understanding of the mechanisms behind this regulation might help us to improve the design and productivity of plant transgenes, with applications in pharmaceutical or vaccine production. m6A is also an interesting candidate for the regulation of plant stress responses, and there may be further applications in adapting plants to environmental change. This overlooked layer of gene regulation promises to yield new insight into the life of plants.
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Matthew Parker is a bioinformatician working as a post-doctoral researcher at the University of Dundee. He specializes in the analysis of mRNA sequencing and functional genomics datasets, in particular, nanopore direct RNA sequencing, which he has used to map m6A in Arabidopsis thaliana. Email: email@example.com
Katarzyna is an RNA biologist working as a post-doctoral researcher at the University of Dundee, funded by the Marie Sklodowska-Curie fellowship. She has pioneered use of nanopore direct RNA sequencing to understand the complexity of gene expression regulation in plants and optimized it to detect full-length mRNA transcripts and RNA modifications. E-mail: firstname.lastname@example.org
Gordon is a Professor of Molecular Genetics and the Deputy Head of Plant Sciences at the School of Life Sciences, University of Dundee. His current research focuses on the link between Arabidopsis mRNA 3’ end processing and RNA methylation, as well as the annotation of mRNAs in understudied crop species. E-mail: email@example.com