Where, When, and How: Context-Dependent Functions of RNA Methylation Writers, Readers, and Erasers

Abstract

Cellular RNAs are naturally decorated with a variety of chemical modifications. The structural diversity of the modified nucleosides provides regulatory potential to sort groups of RNAs for organized metabolism and functions, thus affecting gene expression. Recent years have witnessed a burst of interest in and understanding of RNA modification biology, thanks to the emerging transcriptome-wide sequencing methods for mapping modified sites, highly sensitive mass spectrometry for precise modification detection and quantification, and extensive characterization of the modification "effectors," including enzymes ("writers" and "erasers") that alter the modification level and binding proteins ("readers") that recognize the chemical marks. However, challenges remain due to the vast heterogeneity in expression abundance of different RNA species, further complicated by divergent cell-type-specific and tissue-specific expression and localization of the effectors as well as modifications. In this review, we highlight recent progress in understanding the function of N⁶-methyladenosine (m⁶A), the most abundant internal mark on eukaryotic mRNA, in light of the specific biological contexts of m⁶A effectors. We emphasize the importance of context for RNA modification regulation and function.

Keywords: FTO; METTL14; METTL3; N(6)-methyladenosine; RNA modifications; YTHDF proteins; context-dependent functions; epitranscriptome; gene expression regulation; m(6)A.

Figure 1.. m⁶A effectors: writers, erasers, and readers.

Writers: Majority of m⁶A methylation on mRNA is installed by a writer complex ① composed of core subunits METTL3 and METTL14 and additional adaptors proteins including WTAP, VIRMA, ZC3H13, HAKAI, and RBM15/15B in a sequence context of RRACH (R = A or G; H = A, C, or U). The other known writer METTL16 ② installs m⁶A in a sequence context of UAC(m⁶A)GAGAA on top of a hairpin structure in transcript MAT2A. Erasers: two erasers have been characterized for m⁶A methylation on mRNA, including FTO and ALKBH5. Readers: Three classes of reader proteins utilize different mechanisms to prefer binding m⁶A-containing RNAs. ① YTH-domain containing proteins (YTHDF1–3, YTHDC1–2) use a well-characterized YTH domain to direct recognize m⁶A methylation. ② A local structure disrupted by the presence of m⁶A could favor RNA-binding events of HNRNPC/G and HNRNPA2B1. ③ RNA binding proteins including IGF2BP1–3 and FMR1 prefer m⁶A-containing RNAs through their tandem common RNA binding domains (RBDs) via a mechanism yet to be characterized.

Figure 2.. A model of cellular-localization dependent functions of m⁶A writer proteins.

(A) m⁶A writer complex installs m⁶A co-transcriptionally in the nucleus. The recruitment of the writer complex to specific genomic loci by transcription factors (TFs) or histone marks may contribute to the gene- or region-specificity in m⁶A installation. Examples include ① TF SMAD2/3 interacts with m⁶A writer complex in response to TGFβ signaling; ② In acute myeloid leukemia (AML) cells, METTL3 is recruited to TSS (transcription start sites) regions with dependence on TF CEBPZ, which subsequently mediates methylation of transcripts important for cancer maintenance; ③ Gene-body enriched histone mark H3K36me3 recruits the m⁶A writer complex by interacting with METTL14, a process contributing to preferential m⁶A installation at CDS and 3’UTR of nascent transcripts. (B) In the cytoplasm, METTL3 itself recognizes 3’UTR m⁶A sites on mRNA and promotes protein translation from the transcript by facilitating translation loop formation through interaction with eIF3h.

Figure 3.. Region-, reader-, and stimulation-dependent roles of m⁶A methylation on mRNA.

Multiple layers of contexts could substantially affect how m⁶A methylation regulates the fate of modified mRNA. Region-dependent regulation: 5’UTR m⁶A is linked to cap-independent translation, especially during stress response when cap-dependent translation is repressed (①). Reader-dependent regulation: YTHDF2 and IGF2BPs can affect mRNA stability in opposite directions. YTHDF2 recruits CCR4-NOT complex via its N-terminal domain to promote mRNA decay (②) while IGF2BPs stabilizes mRNA likely through co-factors HuR and MATR3 (③). They may recognize distinct m⁶A sites and exhibit differential binding density in CDS and 3’UTR. Stimulation-dependent regulation exemplified by YTHDF1 (④): YTHDF1 preferentially binds 3’UTR m⁶A and promotes translation through interaction with translation initiation factors. While it constitutively promotes translation in cancer cells including HeLa cells and endometrial cancer cells, in post-mitotic cells the translation promotion effect only significantly manifests when induced by stimulations such as recovery/repair processes or learning signals.

Figure 4.. Distinct effectors and functions of cap N⁶, 2-O-dimethyladenosine (m⁶A_m) versus internal m⁶A in mRNA.

While FTO exhibits catalytic demethylation activity towards both cap m⁶A_m and internal m⁶A in mRNA, in the cell nucleus, m⁶A is the main substrate of FTO since cap m⁶A_m is most likely masked by cap-binding complex (CBC) in mRNA (①). The methyl group on the N⁶ position of m⁶A_m is installed by a cap-specific methyltransferase PCIF1 while internal m⁶A is installed by METTL3-METTL14 in a multi-component writer complex (②). The role of m⁶A on mRNA stability through the m⁶A readers have been established. However, recent characterization of the m⁶A_m writer PCIF1 showed that depletion of the writer had minimum effect on the abundance/stability of the cap-m⁶A_m-modified mRNA in cell lines tested(③), refusing the role of the FTO-mediated mRNA cap m⁶A_m demethylation in affecting transcript stability.