m6A reader proteins: the executive factors in modulating viral replication and host immune response

Abstract

N⁶-Methyladenosine (m⁶A) modification is the most abundant covalent modification of RNA. It is a reversible and dynamic process induced by various cellular stresses including viral infection. Many m⁶A methylations have been discovered, including on the genome of RNA viruses and on RNA transcripts of DNA viruses, and these methylations play a positive or negative role on the viral life cycle depending on the viral species. The m⁶A machinery, including the writer, eraser, and reader proteins, achieves its gene regulatory role by functioning in an orchestrated manner. Notably, data suggest that the biological effects of m⁶A on target mRNAs predominantly depend on the recognition and binding of different m⁶A readers. These readers include, but are not limited to, the YT521-B homology (YTH) domain family, heterogeneous nuclear ribonucleoproteins (HNRNPs), insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs), and many others discovered recently. Indeed, m⁶A readers have been recognized not only as regulators of RNA metabolism but also as participants in a variety of biological processes, although some of these reported roles are still controversial. Here, we will summarize the recent advances in the discovery, classification, and functional characterization of m⁶A reader proteins, particularly focusing on their roles and mechanisms of action in RNA metabolism, gene expression, and viral replication. In addition, we also briefly discuss the m⁶A-associated host immune responses in viral infection.

Keywords: N6-methyl adenosine; epitranscriptomics; immune response; m6A reader; viral replication.

Figure 1

Functions of m⁶A reader proteins. RNA transcripts are m⁶A-methylated by a m⁶A writer complex composed of core subunits METTL3 and METT14 and additional accessory factors. METTL16 installs m⁶A on U6 snRNA and MAT2A mRNA. The m⁶A can be removed by demethylase FTO or ALKBH5. The reader proteins (purple) play diverse roles in RNA metabolism and gene expression. In nucleus, DC1 plays a role in alterative splicing, polyadenylation and carRNA decay (1) as well as m⁶A RNA export (2). The other readers hnRNPC/G or hnRNP2AB1 mediates alternative splicing (3) and pri-miRNA processing when drasha and pasha present (4). After export of the m⁶A RNA, three classes of readers in cytoplasm bind the m⁶A RNA in different mechanisms: (I) YTH readers bind m⁶A directly and regulate translation and RNA stability; (II) m⁶A switch-mediated binding. A local hairpin structure disrupted by m⁶A methylation favors binding events of a group of readers to regulate RNA structural changes and stability; (III) binding through tandem common RNA binding domain (RBD), such as KH, RGG and GRE domain. These readers as indicated regulate translation, splicing and RNA stability. During cellular stress, certain multiple m⁶A-containing RNAs and readers facilitate phase-separated compartment formation to produce stress granules for storage or transfer to P-body, leading to degradation.

Figure 2

The 5'UTR m⁶A promotes cap-independent mRNA translation initiation. Under normal condition, the process of mRNA translation initiation occurs through a cap-dependent mechanism. When cells are subjected to stress, the m⁶A methylation machinery is activated. This leads to the destabilization of the mRNA secondary structure, thereby promoting the binding of reader protein eIF3 and/or METTL3 to the 5' UTR m⁶A site, and consequently initiating translation. This process is independent of the m⁷G-cap, as well as cap-binding proteins eIF4E and eIF4A. Furthermore, the reader DF-1 promotes translation by interacting with eIF3.