Defining the plant epitranscriptome
Lead Research Organisation:
University of Nottingham
Department Name: Sch of Biosciences
Abstract
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Technical Summary
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).
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).
Planned Impact
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.
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.
People |
ORCID iD |
Rupert Fray (Principal Investigator) |
Publications
Zhang M
(2022)
Two zinc finger proteins with functions in m6A writing interact with HAKAI.
in Nature communications
Bhat SS
(2020)
mRNA adenosine methylase (MTA) deposits m6A on pri-miRNAs to modulate miRNA biogenesis in Arabidopsis thaliana.
in Proceedings of the National Academy of Sciences of the United States of America
Ružicka K
(2017)
Identification of factors required for m6 A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI.
in The New phytologist
Haussmann IU
(2016)
m6A potentiates Sxl alternative pre-mRNA splicing for robust Drosophila sex determination.
in Nature
Fray RG
(2015)
The Arabidopsis epitranscriptome.
in Current opinion in plant biology
Description | As a result of this grant, we reported the identification of a plant complex, which is required for mRNA methylation in Arabidopsis. We found three additional factors required of mRNA methylation. Two of them were reported to be involved in in the same process in mammals (though not in yeast) whilst our manuscript was in preparation. The third protein we identified was a homologue of the E3 ubiquitin ligase HAKAI. The mammalian and Drosophila Hakai homologues were subsequently also shown to be involved in m6A formation in metazoans in later publications. Aberrant expression of human Hakai has been associated with tumorigenesis, and this has been suggested to be due to it altering cell-cell connections at the plasma membrane. However, Arabidopsis hakai can not act in this way as plants lack the proposed membrane target cadherin proteins. The conservation of hakai as a member of the m6A "writer" complex since the last common ancestor of plants and animals, has caused a reassessment of the role of this protein in human disease and in plant gene regulation. Knock out of MTA, FIP37, VIRILIZER or MTB (METTL14) in Arabidopsis results in embryo lethality, but we were able to generate a set of hypomorphic and RNAi lines in which functional expression and m6A levels were dramatically reduced. These hypomorphic mutants show a range of similar pleiotropic developmental defects, notably altered vascular development. The latter defect is also seen in the hakai knockout plants, although these plants are viable and have a more modest reduction in mRNA methylation levels. Thus, it may be that the hakai is acting to tune and regulate the activity and targets of the methylation complex. |
Exploitation Route | In the past few years, mRNA methylation has emerged as an important factor in various human diseases, and several companies are competing to develop therapeutic interventions (https://cen.acs.org/business/start-ups/Epitranscriptomics-new-RNA-code-race/97/i7 and https://www.nature.com/articles/nbt1218-1123). Thus, understanding the conserved and any divergent aspect of the methylation process is likely to support these activities. |
Sectors | Agriculture Food and Drink Healthcare |
Description | In the past few years, mRNA methylation has emerged as an important factor in various human diseases, and at least five companies are competing to develop therapeutic interventions with reported investment in epitranscriptomic therapies totalling $189 million (https://www.nature.com/articles/nbt1218-1123). The work of the Nottingham group has been acknowledged by some of these startups (https://cen.acs.org/business/start-ups/Epitranscriptomics-new-RNA-code-race/97/i7), and the identification of core conserved mediators of m6A writing is likely informing these activities. |
Description | Industrial Studentship |
Amount | £55,000 (GBP) |
Organisation | Syngenta International AG |
Sector | Private |
Country | Switzerland |
Start | 01/2017 |
End | 01/2021 |
Description | Project de Recherche collaborative (PRC) appel a project générique 2017 (collaborative research project, call 2017) |
Amount | € 508,572 (EUR) |
Funding ID | ANR-Heat-EpiRNA: ANR-CE20-0007-04 |
Organisation | National Agency for Research |
Sector | Public |
Country | France |
Start | 02/2018 |
End | 01/2022 |
Title | Improvement of MeRIPSeq protocol |
Description | Improved method for immuno precipitation and sequencing for detection of m6A methylated sites in mRNA. |
Type Of Material | Technology assay or reagent |
Year Produced | 2016 |
Provided To Others? | No |
Impact | This method allows the more efficient detection and mapping of m6A methylated sites in mRNA. |
Description | Brian Gregory |
Organisation | University of Pennsylvania |
Country | United States |
Sector | Academic/University |
PI Contribution | Supply of low methylation plant material for collaborative RNA processing and structure analysis. |
Collaborator Contribution | Partners have undertaken detailed analysis of mRNA secondary structures and processing changes that take place as a result of reduced RNA methylation. |
Impact | Submitted joint publication. |
Start Year | 2015 |
Description | Poznan - m6A mapping |
Organisation | Adam Mickiewicz University in Poznan |
Country | Poland |
Sector | Academic/University |
PI Contribution | Training given to visiting researcher in methodologies for detecting m6A methylation in primary micro RNAs. |
Collaborator Contribution | Partner provided RNA samples from control plants and from our low methylation lines. Partner also carried out quantification of specific transcripts after m6A pulldown in Nottingham. |
Impact | Generation of data for future joint publication. |
Start Year | 2017 |
Description | Talk on GM to growers |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | Talk to G's vegetable growers and retailers. Guest dinner speaker and chair of GM debate held by their graduate training programme. |
Year(s) Of Engagement Activity | 2014 |
Description | Wallaton Lecture Club |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | Public lecture describing my work to a local group of mostly retired individuals. |
Year(s) Of Engagement Activity | 2019 |