Messenger RNA (mRNA) plays important role in the life process, not only by conveying genetic information from DNA to protein but also by regulating diverse biological processes; the modification on RNA is critical in regulating these processes. RNA modification was first discovered in tRNAs and rRNAs 1. So far, >160 kinds of RNA modifications with different chemical properties have been found, most of which are found in tRNA and noncoding RNA2 in different organisms. N6-methyladenosine (m6A) was first discovered in 1974. Wei et al3,4 found modified nucleotides such as m6A, m7G, N4-acetylcytidine (ac4C), and m6Am on the mRNA of HeLa cells. However, due to the characteristics of the mRNA itself and the technology limitation, the field of mRNA modification was slowly progressed.
m6A is the most abundant internal modification in messenger RNA and is the most well-studied type among all RNA modifications. In 2012, Dominissini et al5 developed a new m6A-seq technology which in turn not only revealed the distribution of m6A modifications at the transcriptome level in both human and mouse but also found >12 000 m6A modification sites on human mRNA. The distribution of the modification sites is highly conserved between human and mouse, mainly distributed near the stop codons, in the 3′-UTR and within internal long exons. Simultaneously, using the similar MeRIP-seq approach, Meyer et al6 also obtained consistent observation that there are m6A modifications on the mRNA of 7676 mammalian genes, which are mainly enriched in the vicinity of the stop codon and in the 3′-UTR region.
The m6A modification can be catalyzed by m6A “writers” including the conventional complex (METTL3/METTL14/WTAP) and other regulators and removed by m6A “erasers” (FTO and ALKBH5). The function of m6A is extensive depends on its “readers” (YTH domain-containing proteins, IGF2BPs and other newly discovered candidates). These proteins play various roles in different cancers (Table1). At the molecular level, m6A can affect almost all aspects of mRNA metabolisms, including splicing, translation, stability, and miRNA maturation7–9. These m6A-mediated mRNA metabolic processes play important roles in regulation of cellular functions, including response to cellular stresses10,11, modulation of differentiation12,13, homeostasis of immune cells14,15, etc. In this review, we systematically introduce the biological functions of m6A RNA modifiers (writers, erasers, and readers) and summarize the current understandings of their roles in human cancers.
The components of m6A methyltransferase complex include METTL3,16 METTL14,17 WTAP,18 RBM15,19 RBM15B,19 and vir-like m6A methyltransferase associated (VIRMA).20,21 So far, among these components, METTL3 is the only protein possessing the m6A catalytic activity. Besides the conventional methyltransferase complex, METTL16 is also reported as an RNA m6A methyltransferase which only methylated a limited number of RNAs with specific RNA structure, such as U6 snRNA.
After the detection of m6A in 1974,22,23 scientists attempted to identify the proteins which can catalyze m6A. To identify these proteins, HeLa cell nuclear extracts and artificial synthetic mRNAs were used for an in vitro methylation assay.21 [3H]-SAM were used as the methyl group donor to detect the formation of new m6As. As a result, the m6A consensus sequence GGACU was confirmed both in natural RNA and synthetic RNA.24 And after the nuclear extract was separated into various fractions by column chromatography, a 70-kDa fraction with SAM-binding motif was isolated, purified, and identified. That was MT-A70, which is called METTL3 nowadays.21 METTL3 turns out to be a member of putative SAM-dependent methyltransferase family, which was highly conserved in yeasts, plants, Drosophila, and mammals25,26. Deletion of METTL3 in mammalian cells results in huge loss of m6A in mature mRNA27. Although METTL3 is the major contributor in the methylation of polyadenylated RNA, the substrates of METTL3 do not include rRNA and snRNA.28
In 2008, Zhong et al found the MTA-FIP37 complex in Arabidopsis; MTA and FIP37 are the METTL3 and WTAP homologs in Arabidopsis, respectively.29 The detailed function of WTAP was then unveiled by Agarwala et al30 in the yeast model. They noticed that the yeast homolog of METTL3, Ime4, interacted with the yeast WTAP homolog mum2 and showed the crucial role of WTAP for catalyzing m6A. Subsequent studies observed the METTL3-WTAP complex in mammalian cells, which indicates this as an evolutionary conserved complex.18 WTAP mainly acts as an adaptor for METTL3 and METTL14. It localizes them to nuclear speckles.18 When WTAP is absent, m6A level in mRNA goes down.18 It helps METTL3 catalyze m6A in nuclear in a more efficient way.
METTL14 is also a member of methyltransferase-like (METTL) proteins, but the SAM-binding domain of it loses its function. As a result, METTL14 does not have the enzymatic activity of m6A generation. METTL14 has strong interaction with METTL3 and crystal structures of the METTL3-METTL14 complex show that it is not METTL14 but METTL3 which transfers a methyl group to RNA.31–33 It binds to RNA substrate and interacts with METTL3 at the same time, which not only enhances the enzymatic activity of the complex but also positions the methyl group to the targeted site.
VIR-Like m6A Methyltransferase Associated (VIRMA/KIAA1429)
VIRMA is another component of methylation complex. It mainly mediates preferential m6A mRNA methylation in 3′-UTR and near stop codon. Also, it seems to correlate with alternative polyadenylation34.
RBM15 and its paralog, RBM15B, were first noticed through proteomics analysis of WTAP.19,35 Then it was reported that RBM15 and RBM15B can interact with METTL3 through WTAP.19 Also, severe loss of m6A was observed in RBM15 and RBM15B knocked down cells, which showed their significance in m6A formation. Further iCLIP studies show that RBM15 and RBM15B bind to U-rich regions in mRNA which are close to m6A sites. Thus, RBM15 and RBM15B seem to work as adaptors which set near m6A motifs and then recruit WTAP/METTL3 complexes to generate new m6As. In 2018, it is revealed that Flacc(Zc3h13) serves as an adaptor between Nito(RBM15B) and Fl(2)d(WTAP). It can stabilize the complex and promote the deposition of m6A on mRNA36.
METTL16 is confirmed as a new m6A “writer” in 2017.37 It is found to methylate hairpin structures in pre-mRNA of gene MAT2A, which encodes a SAM forming enzyme.37 Since the sequence of the MAT2A pre-mRNA hairpin is similar to that in U6 snRNA, Conrad et al37 did the in vitro methylation assay and confirmed that METTL16 is also the enzyme which catalyzes m6A formation in U6 RNA. In addition, METTL16 is also reported to associate with non-coding RNAs and pre-mRNAs.38 A large, positively charged groove is found in METTL16’s 3D structure and it is likely to represent the RNA-binding site of this protein.39
3. THE ROLE OF m6A WRITERS IN HUMAN CANCERS
METTL3 is mis-regulated in many kinds of cancers such as glioblastoma,40,41 lung cancer,42 liver cancer,43 colon cancer,42 acute myeloid leukemia,44,45 pancreatic cancer,46 bladder cancer,47 and breast cancer.48 Its roles differ from cancer to cancer. In glioblastoma, METTL3 can increase SOX2 mRNA stability and expression to enhance the growth of the tumor.41 But according to Cui et al,40 knocking down METTL3 and METTL14 in glioblastoma stem cell (GSC) promotes human GSC growth, self-renewal, and tumorigenesis. In liver cancer, METTL3 can reduce SOCS2 mRNA expression and increase tumor cell proliferation, migration, tumorigenicity, and lung metastasis.43 In lung cancer and colon cancer, METTL3 promotes oncogene translation and leads to cancer cell growth and invasion. In acute myeloid leukemia (AML), METTL3 enhances MYC and BCL2 mRNA translation, inhibits cell differentiation, and accelerates leukemia progression in mice.42 And in breast cancer, METTL3 increases m6A level in HBXIP mRNA which promotes breast cancer cells proliferation and survival.48 In endometrial cancer, downregulation of METTL3 causes reduction of m6A methylation and leads to the decreased expression of PHLPP2, the negative AKT regulator and the increased expression of mTORC2, the positive AKT regulator, which results in the promotion in tumor cell proliferation.49
In endometrial cancer and GBM, downregulation of METTL14 and METTL3 resulted to increased tumorigenicity.49 The upregulation of METTL14 in AML can stabilize MYB and MYC through m6A modification, which leads to the development of AML.50
WTAP is highly expressed in high-grade serous ovarian carcinoma, and this high expression predicts a shorter overall survival.51 In liver cancer, WTAP is also upregulated and promotes liver cancer development by guiding the m6A modification on ETS1 mRNA.52
YTH domain proteins
YTHDF2 and YTHDF3 were first identified by Dominissini et al5 in an m6A pull down assay and were named because of their YTH domain. The affinity of YTH domain and m6A methylated mRNA is 10–50 times higher than that of unmethylated mRNA.53–55 Mammals have two YTH protein families; one family consists of YTHDC1 and YTHDC2, and the other family has three members (YTHDF1/2/3). These genes are conserved across species.
YTHDF1 was originally found to bind to the stop codon and 3′UTRs of m6A-modified transcripts, and its overall distribution is highly consistent with the m6A site distribution. In addition, YTHDF1 binds directly to the eukaryotic translation initiation factor eIF3, promoting the translation efficiency of m6A-modified RNA substrates.56
YTHDF2 mediates the degradation of m6A-modified mRNA. Under normal conditions, YTHDF2 co-localizes with the deadenylase complex and the decapping complex proteins and carries the transcripts of its targeted genes into the degrading body.57 Subsequent studies further showed that YTHDF2 accelerated the degradation of m6A-modified transcripts by recruiting CCR4-NOT adenosine complexes58.
Based on the PAR-CLIP-seq results, YTHDF3 and YTHDF1 bind to the similar RNA motifs, and the binding sites are mainly located in the 3′UTR. YTHDF3 can promote the translation efficiency of consensus target genes of YTHDF1, suggesting that YTHDF3 and YTHDF1 synergistically regulate mRNA translation efficiency. In addition, YTHDF3 can also mediate mRNA degradation by directly interacting with YTHDF256,59.
YTHDC1, locating in the nucleus, interacts with the splicing factors SRSF3 and SRSF10, which suggests that m6A regulates mRNA alternative splicing via YTHDC160.
YTHDC2 has the largest molecular weight in the YTH family and also tends to bind to the conserved m6A-modified motifs. It regulates the stability of m6A-modified mRNAs and make interactions between m6A-modified RNAs and the ribosomes to facilitate translation efficiency.61,62 In addition, YTHDC2 also participates in mouse spermatogenesis.63,64
Human IGF2BPs were first cloned in 1999 by Nielsen et al65 who found three of high affinity proteins to IGF-II mRNAs, and these proteins were named IGF-II mRNA binding proteins (IMPs). IGF-II mRNA binding proteins are highly conserved from fish to human66 and play an important role in regulating translation, stability, splicing, and intracellular localization of targeted RNAs.67 IGF2BPs were identified associating with m6A by RNA pull down assays in 2018. They recognize m6A by the KH domains and play oncogenic roles in cancers as m6A readers68.
5. THE ROLE OF m6A READERS IN HUMAN CANCERS
RNA m6A readers play important but diverse roles in human cancers. For instance, YTHDF1 is reported to be overexpressed in colorectal cancer tissues, and in vitro studies shows that knockdown of YTHDF1 can inhibit the spread of cancer and enhance the sensitivity of anticancer drug exposure.69 YTHDF2 was upregulated in clinical hepatocellular carcinoma (HCC) tissues as an oncogene, and miR145 modulates m6A levels by targeting the 3′-UTR of YTHDF2 mRNA in HCC cells.70 Interestingly, YTHDF2 has been reported to have dual effects in pancreatic cancer. YTHDF2 is upregulated in mRNA and protein level in pancreatic cancer tissues in which YTHDF2 promotes tumor proliferation by activating AKT/GSK3beta/cyclin D1 but inhibits metastasis by degrading YAP mRNA.71 RNA helicase YTHDC2 promotes colon tumor metastasis by promoting translation of hypoxia-inducible factor 1α (HIF-1α).72
IGF2BPs are shown to be highly expressed in various human cancers. When each of the IGF2BPs is knocked down in HeLa and HepG2 cells, MYC expression is significantly repressed and cell proliferation, colony formation, and migration ability are repressed68. Yisraeli et al73 also demonstrated that in mice and human lung carcinomas, VICKZ1(IGF2BP1) can enhance tumor progression by synergizing with Kras.
FTO and ALKBH5
FTO is the first mammalian RNA m6A demethylase.74 Besides m6A, FTO was also reported to have ability to catalyze the demethylation of the mRNA 5′ cap m6Am.75 FTO belongs to the AlkB family of nonheme Fe(II)/α-ketoglutarate-dependent dioxygenases.76 Other members of this family include ALKBH1-8. In mammals, m6A also can be recovered to adenosine through ALKBH5. It has been found that the high expression of ALKBH5 in mouse testis is indispensable for spermatogenesis and mouse reproduction. The level of mRNA m6A increases in the male mice with ALKBH5 deficiency, which affects the apoptosis of spermatocyte in the metaphase of meiosis and consequently leads to impaired fertility.76
7. THE ROLE OF m6A ERASERS IN HUMAN CANCERS
In AML, FTO promotes tumorigenesis by reducing m6A on ASB2 and RARA at UTRs and promotes AML carcinogenesis by enhancing the stability of MYC and CEBPA mRNA.77,78 In cervical cancer, FTO enhances the chemoradiotherapy-resistance by reducing the m6A modification of β-catenin.79 Treatment with MA2, a chemical inhibitor of FTO, inhibited GSC growth and self-renewal considerably.40 ALKBH5 also could promote GSC proliferation and tumor progression by reversing m6A methylation on FOXM1 nascent transcripts.80 Unlike the above studies, the demethylase activity of ALKBH5 acts an anti-oncogene role in pancreatic cancer by reducing lncRNA KCNK15-AS1 methylation81.
The m6A modification not only modulates multiple RNA metabolic processes such as RNA splicing, nuclear export, localization, translation, and stability, but also plays crucial roles in many physiological processes including tumorigenesis, self-renewing, and so on. The functions of m6A modification in these processes are mainly determined by the m6A modifiers (writers, erasers, readers). However, in cancer development, the role of these proteins displayed unexpected complexity; the same m6A modifier may have completely opposite effects in different cancers. For example, the high expression of METTL14 contributes to the development of AML50 but can repress the development of liver cancer82 due to the different downstream targets of METTL14 in these two cancers. Mechanistically, METTL14 stabilizes ontogenetic mRNA of MYB and MYC in AML50 but also enhances the level of miR126, which acts as a tumor suppressor in liver cancer.82 This demonstrated that both overall level and distribution of m6A are important for the regulation of cellular processes and development of cancer.
In addition, the field of Epitranscriptomics is blooming during the recent years. Besides m6A, many new modifications are discovered and investigated continuously, such as RNA 5-methylcytosine (m5C), 2′-O-methylation modification (2′OMe), ac4C, etc. m5C is mostly studied in tRNAs, it helps tRNA to generate the L-shaped three-dimensional structures83. 2′OMe was found nearly at the same time as m6A, but its function is still not well-studied22. The function of ac4C on mRNA was first described in 2018. Oberdoerffer et al84 reported that ac4C enhanced substrate translation in vitro and in vivo. However, the detailed biological functions of these modifications are still unclear and have to be further investigated.
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