RNA m⁶A modification and its function in diseases

Abstract

N⁶-methyladenosine (m⁶A) is the most common post-transcriptional RNA modification throughout the transcriptome, affecting fundamental aspects of RNA metabolism. m⁶A modification could be installed by m⁶A “writers” composed of core catalytic components (METTL3/METTL14/WTAP) and newly defined regulators and removed by m⁶A “erasers” (FTO and ALKBH5). The function of m⁶A is executed by m⁶A “readers” that bind to m⁶A directly (YTH domain-containing proteins, eIF3 and IGF2BPs) or indirectly (HNRNPA2B1). In the past few years, advances in m⁶A modulators (“writers,” “erasers,” and “readers”) have remarkably renewed our understanding of the function and regulation of m⁶A in different cells under normal or disease conditions. However, the mechanism and the regulatory network of m⁶A are still largely unknown. Moreover, investigations of the m⁶A physiological roles in human diseases are limited. In this review, we summarize the recent advances in m⁶A research and highlight the functional relevance and importance of m⁶A modification in in vitro cell lines, in physiological contexts, and in cancers.

Keywords

RNA modification / m ⁶A / immunity / cancer / epigenetics

Introduction of m⁶A

To date, more than 100 chemical modifications have been identified in RNA, but most of their functions are still unknown. RNA modifications involve adjusting the RNA structures and functions. Although some RNA modifications, instead of being static, can be dynamically modified by enzymes with opposite modifying activities, indicating that these functional dynamic RNA modifications could be a new layer of gene regulation, termed “RNA epigenetics” [¹]. N⁶-methyladenosine (m⁶A) RNA modification was first discovered in 1974 in purified poly(A) RNA fraction [²]. However, the study of this marker was hampered due to lack of methods for mapping m⁶A within mRNAs.

Using m⁶A specific antibodies, two independent groups performed m⁶A RNA immunoprecipitation, followed by high-throughput RNA sequencing (MeRIP-seq) to map m⁶A throughout the transcriptome [³–⁵]. Their results first revealed that m⁶A was widely distributed in mRNA. More surprisingly, the mapping approaches showed that m⁶A modification was highly enriched near stop codons and in 3′-UTRs in certain mRNAs, and the m⁶A levels varied in different cell contexts, indicating the critical roles of m⁶A in cells. A few months before these two studies, He group described that m⁶A was a major substrate of fat mass and obesity-associated protein FTO [⁶], indicating that m⁶A RNA modification was dynamically regulated, and its dysregulation probably correlated with diseases (Fig.1). These results encouraged the researchers to investigate the function of m⁶A and identify and characterize its modulators (“writers,” “erasers,” and “readers”).

m⁶A writers

METTL3

The first protein responsible for m⁶A formation (m⁶A writer) is the 70-kDa fraction (MT-A70, now referred to METTL3) with S-adenosylmethionine (SAM)-binding activity isolated from the nuclear fractions of HeLa cells by Bokar et al. [⁷]. METTL3 is a critical subunit of (N⁶-adenosine)-methyltransferase [⁸]. It was highly conserved throughout eukaryotes, and METTL3 knockdown in different cells remarkably reduced m⁶A in mRNAs [⁵,⁹–¹¹]. Genetic deletion of METTL3 [¹⁰,¹²,¹³] led to the near-complete loss of m⁶A, suggesting METTL3 as the major catalytic component of m⁶A methyltransferase multiprotein complex. The presence of METTL3 in both cytoplasm and nucleus [¹¹,¹⁴] indicated that m⁶A modification might occur in nucleus and cytoplasm. However, by chromatin fraction coupled with HITS-CLIP [¹⁵], followed by RNA sequencing, Ke et al. [¹⁶] showed that m⁶A was formed on nascent RNAs usually before splicing. Minimal differences were noted on the content and the location of m⁶A in cytoplasmic mRNA and chromatin-associated nascent pre-mRNA or nucleoplasmic mRNA at steady-state [¹⁶], suggesting that m⁶A methylation occurred co-transcriptionally. This idea was supported by recent studies that METTL3 interacted with chromatin and the transcription machinery [¹⁷–¹⁹].

METTL14

METTL14, a homolog of METTL3, was initially discovered as another putative m⁶A methyltransferase by different approaches from independent groups [¹⁰,²⁰,²¹]. METTL3 interacts with METTL14, and knockdown of METTL14 reduces the m⁶A content in vitro and in vivo. In addition, purified METTL14 manifested methyltransferase activities [¹⁰,²⁰], suggesting that METTL14 is a distinct m⁶A writer. However, recent independent crystallization studies [²²–²⁴] demonstrated that METTL3, not METT14, bound to SAM and catalyzed m⁶A formation. Based on crystal structure analysis of METTL3–METTL14 complex, METTL14 possessed a degenerated SAM-binding domain, which was not functional. As an alternative, METTL14 was critical for substrate RNA binding and the methyl group positioning to facilitate the catalytic activities of METTL3. Thus, METTL14 was an essential adaptor for METTL3 activities. METTL3 and METTL14 constitute the core m⁶A methylation complex. However, METTL3 or METTL14 functions or activities, other than m⁶A methylation, need further investigation.

Wilms tumor suppressor-1-associated protein (WTAP)

WTAP was found to interact with METTL3 in 2008 by Zhong et al. [¹²] via two-hybrid screening. In the interaction between mum2 and Ime4 [²⁵], the WTAP and METTL3 homologs , were required to form m⁶A in yeast. Furthermore, the WTAP–METTL3 interaction was observed both in vitro and in vivo [²⁰,²¹,²⁶]. Although WTAP alone did not exhibit any methyltransferase activity, WTAP knockdown led to METTL3 and METTL14 degradation and reduced the m⁶A levels remarkably [²⁰]. Detailed characterization of WTAP revealed the importance of WTAP that guided and localized the METTL3–METTL14 complex into nuclear speckles to efficiently methylate target RNAs [²⁶]. Furthermore, Schwartz et al. [²¹] revealed the WTAP-dependent and WTAP-independent m⁶A modification sites upon WTAP depletion. WTAP-dependent sites were located at internal positions and were topologically static, whereas the WTAP-independent sites were found in the cap structure of the transcripts [²¹], indicating the complexity of co-transcriptional regulation.

KIAA1429

In a proteomics screening performed by Schwartz et al. to isolate METTL3-associated proteins, KIAA1429 was among the top candidates [²¹]. KIAA1429 knockdown by siRNA led to a considerably more striking reduction of m⁶A peaks in mRNA than knockdown of either METTL3 or METTL14, suggesting that KIAA1429 was required for the intact catalytic activity of the methyltransferase complex. Meanwhile, virilizer interacted with Fl (2) d [²⁷,²⁸], the Drosophila homologs of KIAA1429 and WTAP, respectively, which were involved in controlling the sex determination in females by alternative splicing of Sxl transcripts.

RBM15/15B and METTL16

m⁶A is highly enriched in certain mRNA species [⁴] and specific regions of a transcript [⁵]. However, the mechanism of this selection is still poorly understood. RBM15 and RBM15B, two other WTAP interactors identified by coimmunoprecipitation, bound to U-rich regions in transcripts that were closely adjacent to DRACH, the consensus sequence of m⁶A [²⁹], suggesting that RMB15 and RMB15B could recruit methyltransferase complex to DRACH consensus sequence sites for m⁶A methylation. Furthermore, RBM15/15B targets lncRNA XIST [²⁹], which was involved in mediating X-chromosome inactivation and gene silencing during development, for methylation. Therefore, the formation of specific m⁶A sites in specific mRNA might require distinct components of methyltransferase complex. This idea was further supported by the findings of METTL16 [³⁰], which was shown to specifically form m⁶A in U6 snRNA and U6-like hairpins of MAT2AmRNA in a C-m⁶A-G context.

m⁶A erasers and the dynamics of m⁶A modification

FTO

FTO belongs to the non-heme Fe(II)- and α-KG-dependent dioxygenase AlkB family of proteins and demethylated m3T and m3U in single-stranded DNAs and single-stranded RNAs [³¹]. The demethylase activity of FTO against m⁶A [⁶] was further characterized, and for the first time, m⁶A residues in RNAs were found to be real substrates of FTO in vitro. This study was first to indicate that the ubiquitously distributed m⁶A modifications on mRNA might be reversely regulated by methyltransferases and demethylases, thereby serving as a previously unappreciated layer of regulation on mRNAs. Furthermore, FTO-mediated m⁶A demethylation was involved in adipogenesis [³²–³⁴], acute myeloid leukemia (AML) cell transformation, leukemogenesis [³⁵,³⁶], and ultraviolet-induced DNA damage response [¹⁹], emphasizing the importance of m⁶A modification in different cell contexts. Although FTO catalyzed the demethylation of m⁶A residues both in vitro and in vivo [⁶,³⁷], FTO was instead the genuine demethylase of m⁶Am, which was highly similar to m⁶A and enriched in the 5′-UTR adjacent to the N⁷-methylguanosine (m⁷G) cap [³⁸]. In this study, FTO manifested a catalytic activity to m⁶Am nearly 10 times greater than m⁶A, despite the cap m⁶Am levels were considerably lower (less than 1/20) than those of m⁶A [³⁹].

ALKBH5

As a member of the ALKB family, ALKBH5 is another m⁶A demethylase. ALKHB5 depletion was related to the increase of m⁶A, whereas its overexpression reduced m⁶A in mRNAs of human cell lines [⁴⁰]. ncRNAs, which are small nucleolar RNAs, and snRNAs could also be the targets of ALKBH5 [⁴¹]. In addition, a specific m⁶A site in FOXMI mRNA was demethylated by ALKBH5 in an RNA–RNA interaction-dependent manner [⁴²]. ALKBH5 knockout mice bore defects in spermatogenesis because of elevated apoptosis of meiotic metaphase-stage spermatocytes [⁴⁰], emphasizing the fundamental significance of m⁶A in mammalian cells.

Dynamics of m⁶A modification

The broad distribution of m⁶A throughout the transcriptome and the discovery of FTO as m⁶A “eraser” spurred the hypothesis that in parallel to reversible functional modifications of DNA and histones, reversible m⁶A RNA modifications might represent an unappreciated layer of regulation, affecting fundamental aspects of mRNA metabolism. This hypothesis became noteworthy in the RNA modification field.

However, FTO showed greater enzyme activity against m⁶Am in 5′-UTR than against m⁶A in the internal of mRNA [⁴³]. In addition, in HeLa cells, m⁶A formation primarily occurred co-transcriptionally and maintained in nucleoplasmic and cytoplasmic mRNAs, whereas no mRNA methylated and demethylated in the cytoplasm [¹⁶]. Thus, m⁶A might not be subjected to reversible “epigenetic” changes.

Nevertheless, m⁶A methylation and demethylation were proven by human AML studies [³⁵], showing that FTO knockdown remarkably increased m⁶A abundance of m⁶A peaks containing transcripts. Specifically, FTO targeted the internal m⁶A sites of the ASB2 and RARA mRNAs to regulate the stability and expression of these two transcripts [³⁵]. Importantly, FTO was present and functional both in the nucleus and cytoplasm in this study. FTO also targeted the m⁶A of several oncogenes (ADAM19, EPHA3, and KLF4) to promote the growth of glioblastoma stem cell (GSC) and tumor progression; ALKBH5 demethylated 3′-UTR m⁶A sites in NANOG and FOXM1 mRNAs, thereby increasing their expression in breast cancer stem cells and GSC, respectively. In addition, m⁶A was also demethylated during stress responses. Zhou et al. [⁴⁴] demonstrated that the nuclear localization of YTHDF2 induced by heat shock stress could prevent the m⁶A in the 5′-UTR of stress-induced transcripts from demethylation by FTO. Moreover, Xiang et al. showed that in response to ultraviolet-induced DNA damage, METTL3/METTL14 was recruited to damaged sites to install m⁶A on RNAs that can be removed by FTO in nucleus [¹⁹]. Hence, m⁶A demethylation could occur both in the nucleus and cytoplasm with substantial functional outcomes.

m⁶A readers

YTH domain-containing proteins

The manner on how m⁶A executes its effects remains to be unknown. Biochemical approaches have been used to screen the m⁶A-binding proteins (“readers”). Primarily, YTHDF2 and YTHDF3 bound to m⁶A in an RNA pulldown experiment performed by Dominissini et al. [⁵]. This observation was further confirmed by using gel shift assays and crystallography [⁴⁵–⁴⁷]. The YTH domain-containing proteins have five members in mammalian genomes—YTHDC1, YTHDC2, YTHDF1, YTHDF2, and YTHDF3. By using the individual-nucleotide-resolution UV crosslinking and immunoprecipitation (iCLIP) method [²⁹,⁴⁸], the binding sites of the individual proteins in an endogenous context were mapped and revealed that all the YTH domains containing proteins bound to m⁶A in specific contexts, except YTHDC2, which showed primarily noncoding RNA-binding activities. Although YTHDF proteins closely resembled each other and were predominantly cytoplasmic, their function could be diverse. YTHDF2 mediated the decay of target mRNAs, evidenced by the interactions between YTHDF2 and P-bodies [⁴⁷]. In contrast, the half-lives of mRNAs were not affected by YTHDF1 depletion, whereas its interaction with eIF3 and other translation initiation factors suggested that it might affect translation [⁴⁹].

Interestingly, tethering experiment performed by Kennedy et al. showed that all YTHDF proteins promoted translation [⁵⁰], whereas those performed by Du et al. [⁵¹] showed that all three YTHDF proteins triggered mRNA degradation and deadenylation. Consistently, studies from viruses showed that all YTHDF proteins promoted degradation of virus-encoded m⁶A transcripts [⁵²–⁵⁴].

YTHDC1 was initially critical for alternative splicing events when its overexpression or depletion caused splicing abnormalities. Studies of YTHDC2 function were limited. YTHDC2 bound to noncoding RNAs and promoted translation of HIF1a via its helicase function [²⁹,⁵⁵].

eIF3

Eukaryotic initiation factor 3 (eIF3), a component of the translation pre-initiation complex, directly bound to m⁶A in 5′-UTR and mediated an eIF4E-independent translation initiation [⁴³]. Importantly, in another study, eIF3 was recruited to 5′-UTR by YTHDF1, which bound to m⁶A near stop codon [⁴⁹].

HNRNPA2B1

HNRNPA2B1 was an m⁶A reader that interacted with the microRNA microprocessor complex protein DGCR8 and promoted miRNA biogenesis. Loss of HNRNPA2B1 showed similar processing and alternative splicing defects as the deletion of METTL3. However, the poor overlap of HNRNPA2B1 binding sites with m⁶A suggested that HNRNPA2B1 might not directly bind to m⁶A [⁵⁶]. Indeed, m⁶A could affect the structure of a region in the RNAs to make it more single-stranded or accessible, which in turn, facilitated the binding of heterogeneous nuclear ribonucleoprotein [⁵⁷].

IGF2BPs

Insulin-like growth factor 2 mRNA binding proteins (IGF2BPs, IGF2BP1/2/3) were previously known as highly conserved oncofetal RNA-binding proteins that contained two RNA recognition motif (RRM) domains and four K homology (KH) domains [⁵⁸]. IGF2BP expression was often correlated with malignant transformation and poor prognosis of various cancer types. IGF2BP affected multiple aspects of target RNAs, including localization, translation, and stability [⁵⁸]. However, the mechanism of how IGF2BPs function was obscure. Huang et al. [⁵⁹] re-identified IGF2BPs as a new class of m⁶A readers by m⁶A RNA bait pulldown experiment combined with a computational screening of m⁶A-binding proteins. In their study, IGF2BPs recognized the consensus GG(m⁶A)C sequence of mRNA targets through their K homology domains. Interestingly, in contrast to YTHDF2, which promoted the decay of target mRNAs, IGF2BPs promoted the translation of target mRNAs (MYC, for example) by increasing their stability and storage [⁵⁹]. The findings of m⁶A readers with distinct regulatory functions suggested the diversity of m⁶A functions. Interestingly, m⁶A readers may function in context-specific manners.

m⁶A and mRNA metabolism

The function of m⁶A in RNAs was first described in 1978, showing that m⁶A in mRNAs were remarkably reduced when RNA synthesis was blocked by either actinomycin D or 5,6-dichloro-I-b-d-ribofuranosyl-benzimidazole ⁶⁰. Thus, m⁶A possibly promoted RNA decay. Correspondingly, m⁶A-containing mRNAs had reduced half-lives, compared with mRNAs lacking m⁶A in ES cell [⁹,¹³]. In addition, by conditional deletion of METTL3 in T cells [⁶¹,⁶²], we could investigate the in vivo physiological functions of m⁶A in adult mammalian cells. We found that METTL3 depletion resulted in disrupted naïve T cell homeostasis [⁶¹]. Specifically, overexpression of SOCS family proteins prevented the activation of IL-7-STAT5 signaling, which was critical for naïve T cell homeostasis, due to the increased half-lives of m⁶A-containing mRNAs of SOCS family genes [⁶¹]. Similarly, Socs mRNA levels were also elevated in METTL3 depleting Tregs [⁶²]. Furthermore, m⁶A was recognized by YTHDF2, which delivered m⁶A-target mRNAs to P-bodies for degradation. The half-lives of the YTHDF2-target mRNAs increased upon its depletion [⁴⁷].

The enrichment of m⁶A in long exons and transcripts [⁵] with alternative splicing variants clearly indicated that m⁶A might regulate the process of splicing. m⁶A facilitated the binding of a splicing regulator, HNRNPC, to its targets by m⁶A-induced structural switch [⁶³]. Similarly, the m⁶A, specifically catalyzed by METTL16 in the hairpins of MAT2A pre-mRNAs, was required for the splicing of this intron [³⁰]. Furthermore, depletion of m⁶A reader protein YTHDC1 caused splicing abnormalities, which could only be resolved by YTH domain-containing YTHDC1 proteins [⁶⁴]. In flies, the splicing of Sxl transcripts was critical in sex determination in females [²⁷,²⁸]. This process depended on m⁶A formation on Sxl transcripts formed by m⁶A methyltransferase complexes, namely, virilizer, Fl (2) d, and IME4 [⁶⁵–⁶⁷]. m⁶A also affected mRNA translation by the interaction of m⁶A readers and translation machinery proteins. In one study, YTHDF1 bound to m⁶A near the stop codon to facilitate the binding of eIF3 to the 5′-UTR for translation initiation [⁴⁹]. eIF3 was also recruited and bound to m⁶A in 5′-UTR to promote translation [⁴³].

m⁶A in cancers

m⁶A modification affects fundamental aspects of mRNA metabolism. Given that deregulated m⁶A modulators result in dysregulation of gene expression related to cell differentiation, homeostasis, and response to stresses, m⁶A is indeed involved in cancer.

In a recent study, METTL3 was overexpressed in AML cells [⁶⁸]. METTL3 depletion induced differentiation and apoptosis of AML cell lines and delayed leukemia progression [⁶⁸]. m⁶A promoted the translation of c-MYC, BCL2, and PTEN mRNAs in the human AML MOLM-13 cell line [⁶⁸]. In another study, METTL3 promoted the maintenance of a leukemic state by binding to the promoter of active genes, installing m⁶A modification within the target mRNA transcripts and resulting in enhanced translation of those transcripts by relieving ribosome stalling [⁶⁹]. METTL3 overexpression was also observed in human hepatocellular carcinoma (HCC) and associated with poor prognosis of patients with HCC [⁷⁰]. Experimentally, the proliferation, migration, and colony formation of HCC cell were inhibited by METTL3 knockdown in vitro. Knockout of METTL3 in vivo suppressed HCC tumorigenicity and lung metastasis [⁷⁰].

m⁶A demethylases also play essential roles in cancer. Both FTO and ALKBH5 could promote tumorigenesis of GSC [⁴²,⁷¹]. Under hypoxia conditions, ALKBH5 mediated m⁶A demethylation of pluripotency factors, such as NANOG and KLF4, thereby promoting the breast cancer stem cell (BCSC) phenotype [⁴²,⁶⁰]. FTO was also overexpressed in certain AML cells [³⁵,³⁶]. Similar to the effects of ALKBH5 in GSC tumorigenesis, FTO enhanced oncogene-mediated cell transformation and leukemogenesis of AML cells by reducing m⁶A levels in ASB2 and RARA mRNA transcripts, resulting in enhanced leukemic activity of AML cells [³⁵]. R-2-hydroxyglutarate (R-2HG) inhibited the demethylation activity of FTO, thereby stabilizing the MYC/CEBPA transcripts to exert a broad anti-leukemic activity [³⁶].

Conclusions

In 1974, m⁶A modification was discovered initially in the poly(A) RNA fractions and was predicted to be functional in mRNA processing [²]. The discovery of the m⁶A demethylase FTO [⁶,³¹] suggested that dynamic regulation of m⁶A modification on target RNAs might represent a previously undefined layer of reversible regulation in parallel to reversible DNA and histone modifications. Its importance has already been appreciated for decades. This idea sequentially triggered the “golden rush”on the screening and characterization of possible m⁶A “writers,” “erasers,” and “readers.”

Given that m⁶A affects multiple aspects of the mRNA metabolism, which in turn, regulates the expression of genes, dysregulation of key modulators of m⁶A will be certainly implicated in cancer. The overexpression levels of m⁶A modulators, such as METTL3 [⁶⁸–⁷⁰], FTO [³⁵,³⁶], and ALKBH5 [⁷²], in different cases of cancers emphasize the potential therapeutic importance of targeting m⁶A modulators. To date, the in vivo functions of m⁶A system in different cell types and in different microenvironments are gradually known and will give us accumulating evidence to further define the concepts of RNA epigenetics. Further investigations are needed to broaden our views regarding the principles of the m⁶A biological function and its disease relevance.

Over 100 different modifications have been identified on histones; how those different modifications coordinate to regulate the transcription prompted the well-known concepts of histone code hypothesis. Similarly, over 100 different RNA modifications are known; how those different RNA modifications regulate RNA metabolism may constitute the “RNA code” (Fig.2). Acquiring experimental evidence on the communication between different RNA modifications in the same RNA transcripts of the same cells in response to various stimulations is an exciting research goal.

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