Reading m6A marks in mRNA: A potent mechanism of gene regulation in plants

Abstract

Modifications to RNA have recently been recognized as a pivotal regulator of gene expression in living organisms. More than 170 chemical modifications have been identified in RNAs, with N⁶-methyladenosine (m⁶A) being the most abundant modification in eukaryotic mRNAs. The addition and removal of m⁶A marks are catalyzed by methyltransferases (referred to as "writers") and demethylases (referred to as "erasers"), respectively. In addition, the m⁶A marks in mRNAs are recognized and interpreted by m⁶A-binding proteins (referred to as "readers"), which regulate the fate of mRNAs, including stability, splicing, transport, and translation. Therefore, exploring the mechanism underlying the m⁶A reader-mediated modulation of RNA metabolism is essential for a much deeper understanding of the epigenetic role of RNA modification in plants. Recent discoveries have improved our understanding of the functions of m⁶A readers in plant growth and development, stress response, and disease resistance. This review highlights the latest developments in m⁶A reader research, emphasizing the diverse RNA-binding domains crucial for m⁶A reader function and the biological and cellular roles of m⁶A readers in the plant response to developmental and environmental signals. Moreover, we propose and discuss the potential future research directions and challenges in identifying novel m⁶A readers and elucidating the cellular and mechanistic role of m⁶A readers in plants.

Keywords: RNA metabolism; YTH; epitranscriptomics; m6A modification; m6A reader.

Figure 1

The domain structures of m⁶A reader proteins and the m⁶A motifs recognized by m⁶A readers Schematic domain structures of ECT, YTP, CPSF30, and FLK in plants and YTH, IGF2BP, FMR1, and HNRNP in animals: YTH, YT521‐B homology; KH, K‐homology; RRM, RNA‐recognition motif; ZF, zinc finger; PrLD, prion‐like domain. The RRACH motifs are found in both plant and animal mRNAs, whereas the URUAH motifs are found only in plant mRNAs. R and H represent G/A and A/C/U, respectively. The underlined As represents the m⁶A modification sites.

Figure 2

The molecular and cellular roles of m⁶A readers regulating mRNA stability, alternative polyadenylation, and alternative splicing In the cytoplasm, ECT8 regulates salt response through DECAPPING 5 (DCP5)‐mediated mRNA decay in processing bodies. The ECT2/ECT3/ECT4 complex stabilizes target genes by interacting with poly(A)‐binding proteins (PABs), leading to an ABA response. ECT1 regulates the salicylic acid (SA) and pathogen response through liquid–liquid phase separation (LLPS) to control mRNA decay and storage. ECT2 enhances the stability of m⁶A‐modified mRNAs, thereby controlling trichome morphology. The apple MhYTP2 promotes the degradation of MdMLO19 mRNAs and enhances powdery mildew resistance. In the nucleus, CPSF30‐L recognizes m⁶A‐modified far‐upstream elements (FUEs) to control alternative polyadenylation by forming nuclear bodies, which modulate floral transition and ABA response. FLK binds to the m⁶A‐modified FLC to reduce splicing, leading to early flowering. ECT12 plays a role in the salt and drought stress response by regulating the stability of the m⁶A‐modified salt‐ and drought‐responsive genes.

Figure 3

The molecular and cellular roles of m⁶A readers regulating translation and phase separation In the cytoplasm, ECT1 undergoes liquid–liquid phase separation (LLPS) to regulate mRNA decay and storage, leading to salicylic acid (SA) and pathogen response. ECT8 regulates salt and ABA response through LLPS to sequester PYL7, an ABA receptor, and salt‐responsive genes. The rice YTH07‒early heading date 6 (EHD6) complex binds to and sequesters OsCOL4 mRNA to ribonucleotide protein (RNP) granules through LLPS to promote flowering. The tomato SlYTH2 binds to SlHPL and SlCCD1B transcripts and forms cytosolic condensates with translational regulators SlelF3C and SlelF4B to inhibit aroma production. The apple MhYTP2 promotes the translation of antioxidant genes and enhances powdery mildew resistance. In the nucleus, CPSF30‐L enhances the formation of liquid‐like nuclear bodies to control alternative polyadenylation, which modulates floral transition and ABA response.

Figure 4

Techniques commonly used for identifying and characterizing m⁶A readers (A) Electrophoretic mobility shift assay (EMSA): The purified recombinant protein and the synthetic target RNA are mixed together, and the RNA‒protein complexes are separated on a non‐denaturing polyacrylamide gel and detected by an image analyzer. (B) UV/FA‐CLIP: The RNA‒protein complexes are fixed using UV or formaldehyde (FA) cross‐linking and immunoprecipitated using the antibody specific to the m⁶A reader protein. The RNAs bound to the m⁶A reader are recovered and subsequently analyzed by qRT‐PCR or sequencing. (C) HyperTRIBE: The m⁶A reader is fused to the Drosophila adenosine deaminase acting on the RNA (dADAR) domain, and this fusion protein is expressed in target plants, allowing ADAR to edit adenosine to guanosine in RNAs bound by the m⁶A reader. These edits are subsequently detected by RNA sequencing. (D) DART‐seq: The cytidine deaminase APOBEC1 is fused with the m⁶A reader to direct C‐to‐U editing at cytidine residues adjacent to m⁶A sites. APOBEC1‐m⁶A reader fusion protein is expressed, after which total RNA is extracted and analyzed using RNA sequencing. The presence of C‐to‐U mutations is used to pinpoint the locations of m⁶A mark at m⁶A reader binding site. (E) RIP‐LC–MS/MS: The tissues are homogenized, and the RNA‐m⁶A reader complex is immunoprecipitated using m⁶A reader‐specific antibody. The bound RNA is recovered, digested into nucleotide fragments, and analyzed using LC–MS/MS to identify and quantify RNA‐m⁶A reader interactions. (F) CRISPR/dCas13‐based m⁶A reader targeting: The m⁶A reader protein is fused with a catalytically inactive dCas13b protein, which can target the m⁶A reader to a specific m⁶A site within an mRNA using guide RNA complementarity. The binding of m⁶A reader to specific m⁶A mark can alter the stability or translation of the mRNA, leading to phenotype changes of the transgenic plants.