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.
 
Description This grant started in Sept 2015. However, we have made good progress and identified novel site of m6a methylation on viral mRNAs using m6A-seq. We are now assessing the role of these modifications for the fate and function of viral mRNAs.
Exploitation Route Novel antiviral targets and fundemental understanding of mechanisms which regulate gene expression
Sectors Pharmaceuticals and Medical Biotechnology

 
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

 
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 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 zonme 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 2010,2011,2012,2013,2014
URL http://www.fbs.leeds.ac.uk/outreach/schools/lfos.php
 
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 2015,2016,2017,2018
 
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 cell 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 2013,2014,2015,2016,2017