From A to m6A: The Emerging Viral Epitranscriptome

Abstract

There are over 100 different chemical RNA modifications, collectively known as the epitranscriptome. N⁶-methyladenosine (m⁶A) is the most commonly found internal RNA modification in cellular mRNAs where it plays important roles in the regulation of the mRNA structure, stability, translation and nuclear export. This modification is also found in viral RNA genomes and in viral mRNAs derived from both RNA and DNA viruses. A growing body of evidence indicates that m⁶A modifications play important roles in regulating viral replication by interacting with the cellular m⁶A machinery. In this review, we will exhaustively detail the current knowledge on m⁶A modification, with an emphasis on its function in virus biology.

Keywords: RNA modification; epitranscriptomics; m6A; viral infection.

Figure 1

The m⁶A RNA modification and its cellular interactome. The N⁶-methyladenosine (m⁶A) modification is installed co-transcriptionally to mRNA transcripts by the METTL3-METTL14-WTAP writer complex and can be removed by the FTO and ALKBH5 erasers. The family of YTH proteins comprises the major readers that specifically recognise and directly bind m⁶A modifications. YTHDC1 is a nuclear m⁶A reader and facilitates mRNA splicing and nuclear export. In the cytoplasm, YTHDF1, YTHDF2, YTHDF3 and YTHDC2 bind mRNAs to enhance their translation or induce their degradation in P-bodies. Other multiple secondary readers that also target m⁶A modifications have been recently elucidated. Some of these bind to m⁶A through structural RNA switches in the mRNA transcript induced by m⁶A itself.

Figure 2

The most common transcriptome-wide m⁶A profiling techniques used to locate m⁶A in viral RNAs. (A) The first step of the m⁶A-seq technique consists of the chemical fragmentation of the RNA into 100–200 nucleotides-long RNA molecules. An input control (which does not undergo m⁶A-immunoprecipitation) and an m⁶A-immunoprecipitated (IP) sample are required to build cDNA libraries for deep-sequencing and subsequent bioinformatic analysis. m⁶A-seq offers a low-resolution approach to map m⁶A modifications because of the relatively large size of the starting fragmented RNAs. (B) Photo-crosslinking-assisted m⁶A-sequencing (PA-m⁶A-seq) requires incubating cells with 4-thiouridine (4SU), which is incorporated into mRNAs. Full length mRNAs are then immunoprecipitated with m⁶A antibodies and UV-crosslinked. Next, crosslinked mRNAs are digested into around 30 nucleotides-long molecules that are isolated to prepare the cDNA libraries. Crosslinked 4SU is read as C during reverse transcription, therefore T to C transitions can be identified when compared to the reference genome. (C) In miCLiP-seq, RNA containing m⁶A-modifications is fragmented, immunoprecipitated (IP) using an anti-m⁶A antibody and UV-crosslinked to the IP antibody. As similarly to the previous techniques, during library preparation adapters are ligated to the precipitated RNA but in this case the 5′-end of the RNA is radioactively labeled. After purification of the RNA-antibody complexes using SDS-PAGE and membrane transfer, the RNA fragments are then reverse-transcribed leading to either a transition of C to T or truncations in cDNA. Following library construction and high-throughput sequencing localization of m⁶A modifications at single nucleotide resolution is achieved. (D) Nanopore sequencing of m⁶A modifications takes advantage of changes in electric current when a nucleotide containing a modification traverses a nanopore. Using specialised software, m⁶A modifications can then be identified. This allows for fast, accurate and sensitive detection of long sequencing reads reaching up to 2 Mb.

Figure 3

How viral m⁶A modifications and the host m⁶A machinery affect viral replication. Both DNA and RNA viruses are subjected to extensive m⁶A modification. Importantly, m⁶A modifications differentially impact the steps of the viral life cycles depending on the virus. (1) m⁶A modifications enhance the translation of some viral mRNAs. (2) m⁶A modifications prevent replication through the inhibition of reverse transcription in retroviruses and enhance replication of HBV through the facilitation of reverse transcription. (3) m⁶A modifications reduce or increase viral mRNA stability. (4 and 5) m⁶A modifications affect the splicing and nuclear export of some viral RNAs that replicate in the nucleus. (6) The encapsidation of genomic RNA or assembly of viral components can be regulated by m⁶A modifications. (7) m⁶A modifications reduce or increase infectious viral titers. (8) Viral m⁶A decorations can prevent virus-sensing by RIG-I or MDA5 receptors and consequently inhibit type I IFN induction and anti-viral immunity.