Viral control of the m6A methylome

Lead Research Organisation: University of Leeds
Department Name: Sch of Molecular & Cellular Biology

Abstract

It has been known for many years that eukaryotic RNAs contain over 100 different types of chemically modified nucleotides. Remarkably, however, the functional significance of these modifications is still poorly defined. Interest in this field has recently been reinvigorated by the identification of enzymes which can make one such RNA modification, m6A methylation, reversible, ie effectively switching this RNA modification on or off. Such reversible modifications in DNA and proteins have important implications for regulating gene expression in many cellular processes, therefore it may be the case that reversible RNA modifications have similar fundamental regulatory functions within the cell.

We have exciting preliminary data suggesting that viruses have evolved ways to control this reversible RNA modification. Specifically, we have demonstrated that during Kaposi's sarcoma-associated herpesvirus (an oncogenic herpesvirus) infection, a specific enzyme, known as FTO, which is responsible for the removal of the m6A methylation modification on mRNAs is redistributed from one site of the nucleus (nuclear speckles) to another site (nucleolus). This is of particular interest as we have previously demonstrated that the nucleolus is involved in viral mRNA processing. In addition, we have further evidence that this virus-mediated redistribution of FTO can result in alterations of m6A modification on both cellular and viral mRNAs.

We now aim to further investigate these observations and identify what effect viral manipulation of this RNA modification has upon all the cellular and viral mRNAs which undergo this modification using a transcriptome-wide next generation sequencing approach called m6A-seq. Moreover, we will investigate what effect altering the m6A modification of both viral and cellular mRNAs have regarding their fate and function during the virus replication cycle. Furthermore, we assess whether altering the m6A methylation pathway is essential for virus replication. If so, this may provide new strategies for the therapeutic intervention of this important pathogen.

Technical Summary

m6A methylation is a common base modification present in eukaryotic mRNA. However, its biological significance is still poorly understood. Recent technological advances have shown that m6A methylation occurs in >7000 transcripts and has also led to the identification of enzymes which make this RNA modification dynamic and reversible. As such, reversible m6A methylation may have critical roles in gene regulation analogous to dynamically regulated DNA and protein modifications. Thus, dynamically reversible RNA modifications, such as m6A methylation, represent an emerging layer of gene regulation at the RNA level, termed RNA epigenetics or epitranscriptomics.

We have exciting preliminary data to suggest Kaposi's sarcoma-associated herpesvirus (KSHV) manipulates pathways which regulate m6A methylation. As such, this provides an excellent model to study the fundamental regulatory aspects of m6A methylation at an RNA epigenetic level. Our preliminary data shows that the KSHV ORF57 protein redistributes the recently identified RNA m6A demethylase, human fat mass and obesity (FTO)-associated protein, from nuclear speckles into the nucleolus. Moreover, this redistribution results in alterations within the m6A methylation status of viral and cellular mRNAs. Specifically, FTO redistribution dramatically reverses viral mRNA m6A methylation while increasing cellular mRNA m6A methylation status.

The aim of this project is to determine what implications virus-mediated FTO redistribution and manipulation of the m6A methylation pathway has upon cellular and viral m6A methylomes. We will examine what effect altering the m6A methylation status has upon the fate and function of viral and cellular mRNAs. In addition, we will test the requirement of FTO redistribution and alterations in the m6A methylome for virus replication and assess the therapeutic potential of inhibiting this virus-based manipulation of the m6A methylation pathway as a novel antiviral strategy.

Planned Impact

The proposal builds upon previous novel work which has focussed on applying quantitative proteomic-based strategies to understanding the interactions between viruses and the host cell. The aim of this current proposal is to test the hypothesis that a herpesvirus protein manipulates pathways which regulate RNA modifications. In particular, we aim to examine the effect of virus infection on the cellular and viral m6A methylomes and what implications this has for the fate and functions of cellular and viral mRNAs.

Whilst this study is fundamental in nature, the impact of the research will be wide reaching. The virus model provides an excellent tool to determine how dynamic RNA modifications regulate gene expression at an RNA epigenetic level and how a virus manipulates these pathways. By analogy to dynamic DNA modifications, such as histone methylation, where epigenetic regulation has offered new insights in to diseases and possible therapies, an understanding the role of enzymes involved in RNA modification and diseases associated with aberrant RNA modification may provide opportunities to identify small molecule inhibitors as potential leads for new therapies. Moreover, aberrant RNA processing and nucleolar function are implicated in a number of human diseases, therefore any clues as to how cellular RNA processing quality control checkpoints are bypassed by virus infection generated from this project will be of interest to the pharmaceutical industry.

A key element of this project is the identification of essential virus-host cell interactions which will provide avenues for novel antiviral strategies. For example, the natural product Rhein, has recently been identified as a specific RNA m6A demethylase inhibitor and may have potential as a novel KSHV antiviral agent. Moreover, as a number of virus-host cell interactions are conserved in herpesviruses this approach may have generic applications for the treatment of a variety of additional human and animal diseases caused by this large family of viruses. Therefore, these discoveries may foster new collaborations with the pharmaceutical and other commercial industries to exploit these findings for new therapeutic strategies.

In the longer term, exploitation of these findings by the commercial sector may lead to new treatments for a wide range of diseases and virus infections, and this will provide benefits to the quality of life of the general public. Moreover, exploitation of the research findings by the commercial sector is also likely to have a direct impact on the prosperity of the general public of the UK, through increased investment and employment opportunities that will arise from new therapeutic drugs.

Publications

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Manners O (2018) Contribution of the KSHV and EBV lytic cycles to tumourigenesis in Current Opinion in Virology

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Manners O (2019) m6A: Widespread regulatory control in virus replication. in Biochimica et biophysica acta. Gene regulatory mechanisms

 
Description We made good progress and identified novel sites of m6a methylation on viral mRNAs using m6A-seq.
We identified a novel m6a reader protein - SND1- which binds viral mRNA
We showed that SND1 binding is required to stabilise viral mRNAs
Exploitation Route Novel antiviral targets and fundemental understanding of mechanisms which regulate gene expression
Sectors Pharmaceuticals and Medical Biotechnology

URL https://elifesciences.org/articles/47261
 
Description It is demonstrating that RNA modifications are important in the fate and function of viral mRNAs - it could provide a new therapeutic target for human tumour viruses
First Year Of Impact 2017
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Societal

 
Description Targeting the RNA helicase, UAP56: understanding KSHV RNA processing mechanisms to novel antiviral approaches
Amount £622,319 (GBP)
Funding ID MR/R010145/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 08/2018 
End 08/2021
 
Title New software for data analysis 
Description m6aViewer: software for the detection, analysis, and visualization of N6-methyladenosine peaks from m6A-seq/ME-RIP sequencing data 
Type Of Material Data analysis technique 
Year Produced 2017 
Provided To Others? Yes  
Impact New technique 
URL http://dna2.leeds.ac.uk/m6a
 
Description International collaboration - Whitehouse Schneekloth 
Organisation National Cancer Institute
Country Lithuania 
Sector Hospitals 
PI Contribution We have identified specific viral mRNAs that are m6A modified resulting in structural changes which implies that inhibitory small molecules may be able to differentiate between the normal and modified forms.
Collaborator Contribution To identify inhibitory RNA-binding small molecules which selectively bind the ORF50 stem loop, in its normal and modified forms, Prof Schneekloth has performed a Small Molecule Microarray Screen (SMMS), encompassing 22,828 drug-like molecules. This identified the following small molecules: 14-bind 'N' only; 40-bind 'M' only and 13-bind both 'N' and 'M' forms. Excitingly, we show that addition of these compounds, at non-cytotoxic concentrations, can effectively reduce KSHV lytic replication and infectious virion production. These results suggest that an RNA-binding small molecule which selectively binds the m6A-modified ORF50 stem loop can inhibit KSHV lytic replication
Impact None to date Multi-disiplinary - virology and RNA biology, medicinal chemistry
Start Year 2020
 
Description International collaboration whitehouse Pasqual 
Organisation University of Paris
Country France 
Sector Academic/University 
PI Contribution Prof. Pasqual, Paris - molecular modeller. Collaboration has allowed us to investigate how m6A methylation affects the structure of viral mRNAs
Collaborator Contribution Generated in silico 3D structures of the KSHV ORF50 RNA stem loop in its native 'N' or m6A-modified 'M' forms detailed in our recent BioRxiv preprint. Results using free energy landscapes and secondary structure representations show that m6A modification allows structural transitions that are highly distinct from the Native form in viral mRNAs
Impact Multi-disiplinary bringing together virology and molecular modellers. Röder, K. et al. bioRxiv (2021)
Start Year 2020
 
Description Be curious Leeds Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Be Curious is the University's annual, family-friendly, research open day, an event which is usually held on campus, but in 2020 we went virtual. Over 100 of our brilliant researchers were involved in creating lots of fantastic content - from 2 minute demonstrations to try at home activities, research-related digital jigsaws and cartoon strips - to help you explore just some of the exciting things we get up to at the University of Leeds.
Year(s) Of Engagement Activity 2020,2021,2022
URL http://www.leeds.ac.uk/info/4000/around_campus/460/be_curious_festival-about_leeds_and_yorkshire
 
Description Invited plenary talk to celebrate 20 years of the Astbury Centre, University of Leeds 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Plenary talks to publicise the Astbury Centre at University of leeds
Year(s) Of Engagement Activity 2019
URL http://www.astbury.leeds.ac.uk/
 
Description Leeds Discovery Zone 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Discovery zone is large exhibition of biological science related activities to get children interested in sceince. My lab runs a stall on extracting DNA from fruit and explaining what DNA is. School participants are varied from affulent or very poor areas of Leeds. The children really enjoy this activity.
Year(s) Of Engagement Activity 2017,2018,2019,2020,2021,2022
URL http://www.fbs.leeds.ac.uk/outreach/schools/lfos.php
 
Description Leeds Philosophical and Literary Society Public Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Open public lecture hosted by the Leeds Philosophical and Literary Society
Year(s) Of Engagement Activity 2018
URL https://www.leedsphilandlit.org.uk/
 
Description Presentation at Beaver camp 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Talk and experiment to extract DNA from fruit as local scout and beavers group in Leeds
Year(s) Of Engagement Activity 2017,2018,2019,2020,2022
 
Description School visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact I do a talk about DNA and viruses and run an practical (extraction of DNA from fruit) and imaging of cheek cells to KS1 and KS2 (years 3,4,5,6) pupils at Bramhope primary school. Once a year as part of science week
Year(s) Of Engagement Activity 2016,2017,2018,2019,2020,2022