Viral control of the m6A methylome

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.

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.

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.