Development of diagnostic tools for detection and quantifications of mRNA methylation

Lead Research Organisation: University of Nottingham
Department Name: Sch of Biosciences

Abstract

The information content of DNA resides primarily in the order in which the bases G, A, T and C occur along its length. Additional information may be given by the presence of a methyl group as a "tag" to one of the bases (in multicellular organisms Cs are almost exclusively the target). This "tag" or base modification may influence which stretch of DNA is copied into messenger RNA (mRNA). DNA methylation can have profound effects on gene expression, and programmed patterns of methylation help regulate normal development. However, a methylated C is used as a template for G in the normal way during transcription, and the presence of methylation within DNA does not change the amino acid sequence of the protein that is made.

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 animals, plants and yeast is the addition of a methyl "tag" to adenosines (m6A). The frequency of m6A in mRNA is often as high as 0.2% of nucleotides. 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 has remained a mystery for more than 30 years. However, in humans individuals with increased activity of an enzyme that removes this methylation from mRNA are susceptible to obesity, diabetes and Alzheimer's. In addition, we have also shown that methylation is required for normal developmental programmes in both plants and yeast.

The mechanism by which this methylation regulates gene expression is not known, but most probably acts through altering RNA protein interactions - potentially influencing mRNA translation, turnover or sub-cellular location. Unlike DNA methylation, potential sites of adenosine methylation cannot be assayed using restriction enzymes or bisulphite sequencing, and the lack of equivalent technologies is the major limiting factor in the progression of this research field.

We have generated a monoclonal antibody against m6A and we and others have recently used immunoprecipitation to identify over 7000 candidate methylated transcripts. However, currently no method exists to directly assay for metylation at a specific site or to accurately measure levels of such methylation.

This project will develop modified DNA oligonucleotide probes containing a labelled phenylselenothymine that will crosslink to a target adenosine/methyladenosine upon UV photoactivation. After digestion with appropriate nucleases, the labelled A=dT or m6A=dT dimmer can be readily detected and the ratio of these products to each other will give the proportion of messages that are methylated at that particular site.

The ability to directly assay specific adenosines for the presence of methylation and to measure how this methylation changes with different environmental and developmental conditions will fundamentally change the way in which the phenomena can be studied.

Clearly mRNA methylation is of ancient evolutionary origin and is playing an important regulatory role in all eukaryote Kingdoms. Major human diseases have recently been associated with mRNA methylation and developing tools to analyse these methylated transcripts will have immediate relevance and impact.

Technical Summary

The post-transcriptional methylation of adenosine (m6A) is a common modification found in mRNA from all Eukaryotes. The frequency of methylation varies according to tissue and environmental cues, but in mRNA from some tissues approximately 1 in every 500 nucleotides is m6A. This is equivalent to an average of 2-3 modified adenosines per message, however, some messages contain no methylation and some more than this. De-methylation of mRNA is associated with risk of obesity, diabetes and Alzheimer's in humans, and reduced methylation causes homeotic changes in plants and blocks starvation responses in yeast.
We have developed a monoclonal anti-m6A antibody, and this can be used for identifying methylated messages. However, the research field requires a simple technique to allow the methylation status of specific adenosines in a transcript to be determined and methylation levels quantified. For studying DNA methylation, this can be achieved by using bisulfite sequencing or methylation sensitive restriction enzymes, but these techniques can not be used for m6A analysis. In an interdisciplinary collaboration, we will develop a novel system in which an oligonucleotide containing labelled phenylselenothymine (PhSeT) can be hybridised and crosslinked to any target adenosine. The resulting A=dT or m6A=dT products can then be detected and quantified to determine the presence and degree of methylation in the mRNA of interest.
We anticipate that such a technique will have as profound an effect on the study of mRNA methylation as have those methods routinely used for DNA epigenetic studies.

Planned Impact

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 "Fat Mass and Obesity " (FTO) de-methylase reduces levels of mRNA methylation and is associated with increased risk of obesity, diabetes, and Alzheimer's as well as certain cancers. The methylated mRNA targets of this de-methylase are currently not known, but the ability to assay single sites for the presence of N6-methyladenosine (m6A) will clearly be key to furthering this research field. Thus scientists and pharmaceutical companies interested in understanding or developing novel therapeutics or screening services for these disease conditions may become users of the technology. 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 these age and obesity related conditions. Thus, the taxpayer and UK government would benefit from the application of this enabling technology.
The methylation of mRNA is also playing a role in regulating cell differentiation and developmental pathways in plants and yeast. 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 technology. Following several key publications in the last 12 months, the importance of this post-transcriptional regulation is beginning to be appreciated and the research field is poised to expand exponentially in coming years.
 
Description Technique were developed for the chemical synthesis of a modified DNA base, phenylselenyl-thymidine (PhSeT) and this was successfully incorporated into DNA oligonucleotides.
Treatment of PhSeT with certain chemicals can induce a free radical that will result in crosslinking of the thymidine with any adenosine with which it is paired.
A modified, base methylated adenosine can be found in the messenger RNA of many eukaryotes. The function of this base modification is poorly understood, but it is believed to be required for stem cell differentiation, and reduced levels of the modification are associated with certain diseases related to metabolic syndrome. However, detecting which adenosines in an mRNA are methylated and quantifying how frequently a particular adenosine is modified is technically difficult. Crosslinking of a labelled thymine to a potentially modified adenosine would allow the dinucleotide pair to be assayed for the modification state of the target adenosine.
Techniques were also developed for the chemical synthesis of a range of base modified adenosines, and these were incorporated into RNA oligonucleotides at defined positions.
We showed that, following activation, the PhSeT has differential crosslinking activity towards the methylated and the non-methylated adenosines.
It should be possible to use this finding to develop a purely chemical detection system for methylated adenosine. In addition, the ability to make N6 modified adenosine nucleotides at defined sites is likely to be of interest to molecular biotechnology companies.

In addition, we further characterised a monoclonal antibody raised against methylated adenosine (initially developed under grant BB/C513369/1). As a consequence of this, the antibody is now being marketed by Millipore and Ximbio.
Exploitation Route The modified nucleotide tagging approach could be taken forward by molecular diagnostic companies interested in the area of epitranscriptomics.
Sectors Agriculture, Food and Drink,Chemicals,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Agri-Food Technology Seedling Catalyst
Amount £25,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2017 
End 02/2018
 
Description Doctoral training programme PhD studentship
Amount £120,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2015 
End 09/2019
 
Title Identification of methylated mRNAs by antibody pulldown 
Description Use of polyclonal and monoclonal antibody raised against N6 methyladenosine for pull down and identification of methylated transcripts. 
Type Of Material Biological samples 
Year Produced 2011 
Provided To Others? Yes  
Impact Method combined with RNA seq and Cell and Nature papers resulted. 
 
Description Kamil 
Organisation Central European Institute of Technology (CEITEC)
Country Czech Republic 
Sector Charity/Non Profit 
PI Contribution Generation of CRISPR lines. Measurement of mRNA methylation levels. Assistance with manuscript preparation.
Collaborator Contribution Identification of new methylase complex interaction partners. Plant phenotyping. Manuscript preparation.
Impact Manuscript submitted.
Start Year 2012
 
Description Perpignon group 
Organisation The Plant Genome and Development Laboratory (LGDP)
Country France 
Sector Academic/University 
PI Contribution Supply of transgenic plant lines and and analysis of methylation levels of mRNA from heat stressed plants grown by our collaborator.
Collaborator Contribution Preparation of mRNA from heat treated Arabidopsis lines.
Impact Ongoing application grant application to French government by Perpignan Group "Reading the plant epitranscriptome: molecular and physiological functions of m6A readers in Arabidopsis thaliana". We are named as partners in this application. Conference invitation (Summer 2016)
Start Year 2014
 
Description Yka 
Organisation University of Cambridge
Department The Sainsbury Laboratory
Country United Kingdom 
Sector Academic/University 
PI Contribution Measurement of mRNA methylation levels in various transgenic and mutant lines. Generation of CRISPR mutant plants. Assistance with manuscript preparation.
Collaborator Contribution Identification of novel interacting partners of the mRNA methylation complex.
Impact Submission of manuscript.
Start Year 2012
 
Title Licensing of a monoclonal antibody against N6 methyladenosine 
Description We developed and tested a monoclonal antibody which we raised against N6 methyladenosine. Following use of this in one of our publications, we were approached by several companies and Nottingham University has now licensed this to Millipore Ltd. 
IP Reference  
Protection Protection not required
Year Protection Granted 2014
Licensed Yes
Impact This is newly licensed, but we anticipate extensive use by other researchers in this
 
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