Biological roles of adenine methylation in RNA

Abstract

N⁶-Methyladenosine (m⁶A) is one of the most abundant modifications of the epitranscriptome and is found in cellular RNAs across all kingdoms of life. Advances in detection and mapping methods have improved our understanding of the effects of m⁶A on mRNA fate and ribosomal RNA function, and have uncovered novel functional roles in virtually every species of RNA. In this Review, we explore the latest studies revealing roles for m⁶A-modified RNAs in chromatin architecture, transcriptional regulation and genome stability. We also summarize m⁶A functions in biological processes such as stem-cell renewal and differentiation, brain function, immunity and cancer progression.

Fig. 1 |. Molecular consequences of m⁶A modification of mRNA.

a | N⁶-Adenosine methylation is a dynamic modification that occurs in the nucleus and is regulated by both a methyltransferase complex (METTL3–METTL14–WTAP) and by demethylases (FTO and ALKBH5). N⁶-Methyladenosine (m⁶A) has been implicated in regulating various nuclear and cytoplasmic processes in the mRNA life cycle, including subcellular localization, splicing (part b), export (part c), stability or degradation (part d) and translation (part e). b | m⁶A regulates pre-mRNA splicing by both direct and indirect mechanisms. In a direct mechanism, YTHDC1 binding to m⁶A leads to exon inclusion by blocking binding of SRSF10 and recruitment of SRSF3. In the absence of N⁶-adenosine methylation, SRSF10 binds resulting in exon skipping. In an indirect mechanism, termed an m⁶A switch, m⁶A induces a conformational change in RNA folding, exposing a heterogeneous nuclear ribonucleoprotein C (HNRNPC) binding motif, which leads to HNRNPC recruitment and exon retention. c | N⁶-Adenosine methylation facilitates mRNA nuclear export. Binding of YTHDC1 to N⁶-adenosine methylated transcripts promotes export by mediating interactions with SRSF3 and nuclear export mediator NXF1. Alternatively, binding of fragile X mental retardation protein (FMRP) to m⁶A facilitates export by mediating a physical association with the nuclear export protein CMR1. d | m⁶A can promote or inhibit mRNA degradation. YTHDF2 promotes the degradation of m⁶A-modified transcripts by recruiting the CCR4–NOT1 deadenylase complex. By contrast, insulin-like growth factor 2 binding proteins (IGF2BPs) bind m⁶A-containing mRNAs and protect them from degradation. e | m⁶A either promotes or slows translation depending on its location within the mRNA. If the methylated adenosine is located within the 5′ untranslated region (UTR), binding of eukaryotic translation initiation factor 3 (eIF3) recruits the 43S translation initiation complex to promote cap-independent translation. If m⁶A occurs in the 3′ UTR, YTHDF1 or YTHDF3 will bind and stimulate translation. However, if m⁶A is localized in the coding region it can slow translation elongation rates by inhibiting efficient tRNA selection. m⁷G, N⁷-methylguanosine; TF, transcription factor.

Fig. 2 |. Molecular consequences of m⁶A modification of non-coding RNAs.

a | N⁶-Adenosine methylation of ribosomal RNAs (rRNAs) can promote translation or specify mRNAs to be translated. ZCCHC4 N⁶-methylates adenosine 4220 in human 28S rRNA resulting in increased translation and inhibition of cell proliferation. METTL5-mediated N⁶-methylation of adenosine 1832 on human 18S rRNA seems to result in the selective increased translation of a unique sets of transcripts. b | N⁶-Adenosine methylation facilitates microRNA (miRNA) processing. N⁶-Methyladenosine (m⁶A) deposition on primary (pri)-miRNAs is recognized by the heterogeneous nuclear ribonucleoprotein HNRNPA2B1, which in turn facilitates their processing to precursor (pre)-miRNAs via the Drosha–DGCR8 microprocessor complex. c | m⁶A regulates circular RNA (circRNA) biogenesis and function. m⁶A induces back-splicing of circRNA through a YTHDC1-dependent mechanism. In addition, YTHDF3 binds to m⁶A-modified circRNAs to enhance cap-independent translation, resulting in protein synthesis from circRNA transcripts. d | m⁶A regulates small nuclear RNAs (snRNAs) to facilitate spliceosome assembly. METTL16-dependent m⁶A deposition on U6 snRNAs regulates spliceosome assembly or 5′ splice site recognition. N⁶,2′-O-Dimethyladenosine (m⁶Am) modification of U2 snRNAs by METTL4 increases splicing whereas FTO demethylation of U2 snRNAs inhibits splicing.

Fig. 3 |. Genomic consequences of m⁶A.

a | Crosstalk between N⁶-methyladenosine (m⁶A) and chromatin modifications reinforces epigenetic signatures. YTHDC1 can bind to both m⁶A-modified transcripts and to the histone H3 lysine 9 (H3K9) demethylase KDM3B to induce histone demethylation to reinforce chromatin accessibility in regions being actively transcribed. Moreover, methylation of H3 lysine 36 (H3K36) can recruit the METTL3–METTL14 N⁶-adenosine methylation complex to m⁶A-methylate nascent transcripts in regions of active chromatin. b | m⁶A regulates activity and folding of long non-coding RNAs (lncRNAs). Xist lncRNA is N⁶-adenosine methylated at many locations and m⁶A promotes Xist-mediated gene silencing and X chromosome inactivation in a YTHDC1-dependent manner. c | N⁶-Adenosine methylation of MALAT1 lncRNA induces a conformational change, which leads to heterogeneous nuclear ribonucleoprotein C (HNRNPC) binding and alterations in nuclear organization and tumorigenesis. d | N⁶-Adenosine methylation of chromosome-associated regulatory RNAs (carRNAs) causes their degradation via the nuclear exosome targeting complex (NEXT) complex, resulting in gene repression. e | m⁶A facilitates endogenous retrovirus RNA (ERV) suppression by multiple mechanisms to maintain genome stability. m⁶A-modified intracisternal A-particle (IAP) mRNA is bound by YTHDC1, which recruits the histone H3K9 methyltransferase SETDB1, resulting in the deposition of repressive chromatin marks. In addition, YTHDF2 binds to m⁶A-modified IAP transcripts to induce degradation of IAP mRNAs. f | m⁶A prevents genome instability caused by RNA:DNA hybrids (R-loops). N⁶-Adenosine methylation of RNA in R-loops results in YTHDF2-dependent RNA degradation, which suppresses the formation of R-loops and prevents genomic instability. m⁷G, N⁷-methylguanosine; TF, transcription factor.

Fig. 4 |. m⁶A is a critical modification during early development and embryogenesis.

a | N⁶-Methyladenosine (m⁶A) modulates embryonic stem (ES) cell renewal and differentiation. Transcripts encoding ES cell pluripotency factors, such as SOX2 and NANOG, are selectively m⁶A-modified in the nucleus of naive ES cells by the METTL3–METTL14 methyltransferase complex in conjunction with the zinc-finger protein ZC3H13. m⁶A-dependent degradation of pluripotency factor mRNAs drives ES cell differentiation to the primed state, whereas new m⁶A deposition is inhibited by sequestration of the METTL3–METTL14 complex to the cytoplasm by the zinc-finger protein ZFP217. b | m⁶A facilitates clearance of maternal mRNAs during the earliest developmental stages of zebrafish and mice. During zebrafish maternal-to-zygotic transition (MZT), YTHDF2 binds to m⁶A-modified transcripts to direct degradation via the CCR4–NOT deadenylase complex. Binding of the microRNA mir-430 to maternal transcripts further promotes their clearance. By contrast, C5-cytosine methylation (m⁵C) of maternal transcripts promotes stabilization. c | m⁶A promotes haematopoietic stem cell (HSC) differentiation at several points in the differentiation pathway. m⁶A deposition on Myc mRNA stimulates MYC protein synthesis, which promotes HSC differentiation to haematopoietic progenitor cells. Subsequently, m⁶A-dependent degradation of Myc and Myb transcripts drives haematopoietic progenitor cell (HPC) differentiation to the myeloid cell lineage.

Fig. 5 |. m⁶A function in adult cell homeostasis in the nervous system and immune reactions.

a | In neurons, N⁶-methyladenosine (m⁶A) is proposed to regulate local translation of selected dendritic transcripts encoding proteins that facilitating memory consolidation during learning and fear conditioning, such as ARC, a synaptic protein important for fast excitatory neurotransmission. The N⁶-adenosine methyltransferase METTL3, demethylase FTO and binding proteins YTHDF1–YTHDF3, as well as m⁶A-modified transcripts, are enriched near synapses, raising the possibility of localized regulation of these transcripts. b | m⁶A facilitates degradation of specific transcripts in immune cells to allow a rapid response to viral infection and a return to basal states once the virus has been inhibited. Viral infection induces type I interferon activation to inhibit additional viral infection and, subsequently, N⁶-adenosine methylation of mRNA encoding interferon-β (IFNβ) elicits its decay, facilitating a return to basal IFNβ levels. In macrophages, following vesicular stomatitis virus (VSV) infection, mRNA encoding 2-oxyglutarate dehydrogenase (OGDH) are N⁶-adenosine methylated, which causes their degradation and blocks synthesis of the naturally produced itaconate, which is an activator of VSV replication.