Sequencing, Physiological Regulation, and Representative Disease Research Progress of RNA m⁶A Modification

Abstract

To date, more than 150 chemical modifications have been disclosed in different RNA species, which are employed to diversify the structure and function of RNA in living organisms. The N ⁶-methyladenosine (m⁶A) modification, which is found in the adenosine N ⁶ site of RNA, has been demonstrated to be the most heavy modification in the mRNA in cells. Moreover, the m⁶A modification in mRNAs of mammalian and other eukaryotic cells is highly conserved and mandatorily encoded. Increasing evidence indicates that the m⁶A modification plays a pivotal role in gene-expression regulation and cell-fate decisions. Here, we summarize the most recent m⁶A-sequencing technology, as well as the molecular mechanism underlying its occurrence, development, and potential use as a target for the treatment of human diseases. Furthermore, our review highlights other newly discovered chemical modifications of RNA that are associated with human disease, as well as their underlying molecular mechanisms. Thus, significant advancements have been made in qualitative/quantitative m⁶A detection and high-throughput sequencing, and research linking this RNA modification to disease. Efforts toward simplified and more accessible chemical/biological technologies that contribute to precision medicine are ongoing, to benefit society and patients alike.

Keywords

RNA modification - N ⁶-methyladenosine - methyltransferase - binding protein - cancer

Introduction

The decades-long development of RNA innovations, from the discovery of the new functions of RNA (serving as a catalyst and regulator of many biochemical reactions) to its conventional role as a genetic-information carrier, linker in protein molecule synthesis, and structural scaffold of subcellular organelles, has contributed to the progress of nucleic acid research.[1] [2] [3] [4] [5] RNA plays an important role in protein synthesis, and RNA-based drug discovery has attracted great interest, as it contains only four types of nucleotides compared with the 20 different amino acid residues present in proteins.[6] To achieve diversity in RNA structure and function, nature uses a variety of chemical groups for its modification.[7] Since the discovery of the foremost RNA modification in yeast about 60 years ago, over 150 distinct modifications have been identified in all RNA species, including transfer RNA (tRNA), messenger RNA (mRNA), and ribosomal RNA (rRNA).[8] [9] [10] [11] These modifications play crucial roles in genetics, growth, and disease development, including embryonic stem cell differentiation, oocyte meiosis, and cancer occurrence.[12] [13] [14] [15] [16] [17]

RNA modifications enrich the diversity of RNA functions and of genetic information processing. An extensive range of RNA modifications has been identified ([Fig. 1]), and the content and related functions of each type vary greatly. Among them, the methylation of RNA nucleotides is the main form of RNA modification, accounting for approximately two-thirds of the total number of RNA modifications. Currently, the methylation modifications found on mRNA include mainly 5-methylcytosine (m⁵C),[18] 1-methyladenosine (m¹A),[19] N ⁶-methyladenosine (m⁶A),[20] and pseudouridine (Ψ).[21] Compared with other types of RNA modifications, the m⁶A internal modification occurs on the N ⁶ of the adenosine base and is most commonly encountered in eukaryotic mRNA, representing more than 80% of RNA base methylation and having been identified throughout numerous species.[10] [12] [22] [23] [24] [25] [26] [27] It has been reported that m⁶A can occur in the 5′-untranslated region (UTR), coding sequence (CDS), 3′-UTR, and introns of precursor mRNA, as well as in several noncoding RNAs and miRNAs.[28] [29] In addition to the m⁶A modification, other hydrophobic modifications of A also occur, such as 2-methylthio-N ⁶-isopentenyladenosine (ms²i⁶A),[30] N ⁶-isopentenyladenosine (i⁶A),[30] N ⁶-methyl-threonylcarbamoyl adenosine,[31] and N ⁶-threonylcarbamoyladenosine (t⁶A).[32] However, the molecular mechanism underlying these prenyl modifications remains poorly understood ([Fig. 1]; i⁶A, S-geranylated 2-thiouridines (ges²U)). The biosynthesis mechanism, abundance, and distribution regularity of these prenyl modifications, and the molecular mechanism underlying their regulation network in cells, also remain elusive.

Fig. 1 Chemical structures of the typical modifications of RNA.

Sequencing Technology Used for m⁶A Modification

Determining the location of chemical modification groups in transcripts is key information for understanding their basic functions, which is also true for the m⁶A modification. An analysis of m⁶A modification over the entire transcriptome indicated that m⁶A modifications occur across an enormous spectrum of RNA transcripts in unique patterns.[28] [29] In 2012, 30 years after the discovery of the m⁶A modification, m⁶A-seq (also known as MeRIP-seq) afforded the m⁶A landscape in humans and mice. Two independent transcriptome studies have shown that the accurate proportion of m⁶A-modified RNAs among the total mRNAs is 1 per 2,000 nucleotides, on average.[28] [29] Specifically, total RNA was divided into approximately 100 NTs and subjected to immunoprecipitation using anti-m⁶A affinity-purified antibodies ([Fig. 2A]). The library prepared from the immunoprecipitated NTs was sequenced, and an algorithm designed to detect peaks was employed to identify the m⁶A sites.[28] A search of motif databases based on the identified m⁶A peak sites revealed trends in m⁶A occurrence in the RRACH consensus sequence (R = G or A; and H = A, C, or U).[28] Although RRACH sequences are ubiquitous in specific transcriptome species, instances of their methylation constitute only 1 to 5% of transcripts in vivo. Intriguingly, the RRACH motif that undergoes m⁶A methylation is not randomly distributed on transcripts; rather, it occurs in specific CDSs and 3′-untranslated regions (3′-UTRs), and is most commonly observed close to the termination codon. These findings imply that the RRACH motif might not be solely responsible for modulating m⁶A accumulation.[28] The results obtained in eukaryotic organisms, such as yeast,[33] plants,[34] fruit flies,[35] zebrafish,[36] and mammals,[37] have demonstrated that the cis-regulatory RRACH motif together with CDS/3′-UTR enrichment forms signature traits of the m⁶A epigenetic transcriptome, thus illustrating the added value of m⁶A in terms of its function. Given above, the accumulation of m⁶A in eukaryotic organisms is strictly controlled.

Fig. 2 Schematic illustrations of the molecular mechanism underlying the m⁶A modification of RNA. (A) The molecular mechanism underlying the RNA m⁶A modification involves regulatory enzymes, such as writers, readers, and erasers. (B) Pipeline of the m⁶A sequencing technique.

It should be noted that, although m⁶A-seq can detect m⁶A modifications at a resolution of 100–200 nt, it lacks information at the single-nucleotide resolution. Site-specific cleavage and radioactive labeling followed by ligation-assisted extraction and thin-layer chromatography (SCARLET) can locate m⁶A-modification sites down to the individual nucleotide level in RNA samples. Although it is effective for limited applications, it cannot be easily scaled up for the analysis of entire transcriptomes.[38] To boost the granularity of the m⁶A sequencing information, a technique called photo-crosslinking-assisted m⁶A sequencing (PA-m⁶A-seq) has been developed recently. This method uses UV irradiation at 365 nm to crosslink 4-thiouracil with m⁶A antibodies against mRNA, thereby increasing its resolution to ∼23 nt.[33] miCLIP-seq m⁶A single-nucleotide resolution crosslinking and immunoprecipitation (CLIP) is another method that can be used in this context, which incorporates the implementation of cutting-edge sequencing strategies for precise genomic characterization. By tracing the distinct alterations at the m⁶A site that are induced by the UV irradiation of anti-m⁶A antibodies crosslinked to m⁶A labeling in RNA, it has been shown that m⁶A residues often occur in clusters and are frequently distributed in the DRACH (D = A/G/U) motif of CDSs and 3′-UTRs.[39] However, miCLIP requires an input of 20 μg of poly(A)⁺ mRNA[39] [40]; therefore, it is necessary to optimize its protocols toward a lower input of the initial sample.

The RNA endonuclease MazF from Escherichia coli only cleaves the ACA motif specifically from the 5′ side in the absence of methylation. Based on this discovery, researchers developed a new method termed MAZTER-seq (short for m⁶A-REF-seq) as an antibody-independent method for analyzing m⁶A.[41] [42] Although this new method detects m⁶A methylation only at the ACA site, it identified the enrichment of a distribution pattern near the termination codon, which is consistent with the conclusions drawn from the antibody-based experiments. Furthermore, a sequencing method termed deamination adjacent to RNA modification targets (DART-seq) was developed as an antibody-free m⁶A-seq method[43] in which the cytosine deaminase apolipoprotein B mRNA editing enzyme catalytic subunit 1 (APOBEC1) is fused to the YT521-B homology (YTH) domain, which binds to m⁶A to induce C-U deamination at the m⁶AC sequence, followed by its detection using standard sequencing methods targeting RNA molecules. DART-seq can pinpoint numerous m⁶A sites, even among small quantities of total RNA from cells (down to 10 ng), and can measure temporal fluctuations in m⁶A concentrations. However, because effective transfection remains crucial for the performance of the DART-seq method, its usage in living organisms is currently curtailed.[43]

The antibody-free m⁶A-seq techniques (MAZTER-seq, m⁶A-REF-seq, and DART-seq) rely on m⁶A sequencing or cell transfection.[41] [43] A recent study proposed a revolutionary m⁶A-detection method, i.e., m⁶A-SEAL, which offers antibody-free fat-mass- and obesity-associated (FTO) protein–assisted chemical labeling for specific detection.[44] In m⁶A-SEAL, the FTO enzyme promotes the conversion of m⁶A into a transient hm⁶A intermediary molecule through thiol addition reactions mediated by DL-dithiothreitol (DTT), thus generating the more stable sulfide-containing dm⁶A using a simplified installation of biotinylation and other functional tags. The profiling of human and plant transcriptomes using m⁶A-SEAL confirmed the expected m⁶A distributions. Moreover, based on comparative analyses against the existing m⁶A sequencing techniques and the confirmation of specific m⁶A sites using single-base elongation- and ligation-based qPCR amplification (SELECT), it has been demonstrated that whole-transcriptome scanning via m⁶A-SEAL yields excellent sensitivity, specificity, and reliability. The versatility of m⁶A-SEAL, with its FTO-based oxidation and labeling abilities, renders it suitable for diverse applications, ranging from sequencing to enrichment and imaging, thereby advancing m⁶A research.[44] The combination of this labeling and enrichment strategy with highly specific chemical reactions has attracted great research interest because of its high discrimination ability at the single-base level; furthermore, it has been widely used in studies of the 5-hydroxymethylcytosine, 5-formylcytosine, and pseudouridine (Ψ) RNA modifications.

Biological Function of the M⁶A Modification: Physiological Regulation and Disease Occurrence

RNA methylation plays an essential role in the regulation of many biological functions, with N ⁶-methyladenosine being a prominent example for controlling gene expression, translation, and physiology in many organisms, including humans.[45] [46] Three factors govern the reversible m⁶A modifications ([Fig. 2B]): (1) methyltransferases (Writers), such as methyltransferase-3 (METTL3), methyltransferase-14 (METTL14), and their cofactors, Wilms tumor 1-associated protein (WTAP), KIAA1429 (VIRMA), HAKAI, ZC3H13, RBM15, and RBM15B[47]; (2) m⁶A-binding proteins (Readers), such as YTH-domain-family proteins and YTH domain-containing protein 1 (YTHDC1)[48]; and (3) demethylases (Erasers), such as the FTO protein and alkylation repair homolog protein 5 (ALKBH5) ([Fig. 3]).[49] [50] Being abundant within eukaryotic cells, m⁶A controls crucial processes, such as embryonic growth, stem cell specialization, brain-cell generation, and malignancy.

Fig. 3 Functional roles of ALKBH5 and mRNA m⁶A methylation in trophoblasts. (Reproduced with permission from Li XC, Jin F, Wang BY, et al. The m⁶A demethylase ALKBH5 controls trophoblast invasion at the maternal–fetal interface by regulating the stability of the CYR61 mRNA. Theranostics 2019;9(13):3853–3865[50].)

mRNA m⁶A modulation imparts singular control over transcripts, and its correct deposition in mRNA is crucial for embryonic development.[51] Research has shown that methyltransferase affects the meiosis process in yeast. The fundamental structure of yeast RNA methyltransferase contains three building blocks that form the Mum2–Ime4–Slz1 (MIS) complex: Mum2 (homologous to mammalian WTAP), Ime4 (homologous to mammalian METTL3), and Slz1 (MIS). Mutating any slz1 in yeast does not cause death, but it can lead to impaired meiosis.[52] [53] Arabidopsis thaliana mRNA contains m⁶A, which is similar to that in animal cells. This modification is necessary for embryogenesis.[54] Inactivation of METLL3 in A. thaliana leads to the failure of continuous transformation throughout the early developmental stages of embryos.[34] [54] During the development of mouse follicles, the ability of RNA metabolism mediated by the m⁶A methyltransferase KIAA1429 to maintain oocytes is retained.[55] Moreover, m⁶A determines the transition of endothelial cells to hematopoietic cells during zebrafish embryogenesis, and m⁶A modification in endothelial cells can specifically regulate the inhibition of the endothelial Notch signals that trigger the emergence of blood-forming progenitors/stem cells. Furthermore, maternal mRNAs that are dependent on m⁶A are recognized and cleared by YTHDF2, thus promoting the process from zebrafish-fertilized eggs through the maternal-to-zygotic transition.[56] In turn, YTHDC1 guides splicing and polyadenylation decisions during mouse oocyte maturation.[57] Mice lacking YTHDC2 are infertile because their germ cells do not progress past the zygote stage and are essential for sperm production.[58] Absence of ALKBH5 results in higher concentrations of m⁶A-modified RNAs in male mice, abnormal apoptosis, and reduced fertility.[59] Recently, a transcriptomics investigation identified links between m⁶A marks in 5′ UTRs and fetal development/pre-eclampsia in the human placenta.[60]

The presence of m⁶A affects embryonic stem cell maintenance and fate determination.[13] [61] The primordial pluripotent genes of embryonic stem cells, as well as many lineage-specific regulatory genes, carry m⁶A modifications in their mRNAs.[12] [61] Moreover, the maintenance of stem cell characteristics and destiny relies on proper mRNA m⁶A modification.[62] The inactivation or depletion of METTL3 in mice and humans leads to prolonged expression of NANOG (which is a transcription factor that is involved in the self-renewal of embryonic stem cells) and delays the turnover of embryonic stem cells caused by self-renewal, thereby preventing their differentiation into downstream lineages in the absence of m⁶A. This study underscored the significance of m⁶A for stem cell signaling and homeostasis.[12] Knockout of METTL3 in mice downregulates the m⁶A levels, causing embryo demise during early gestation.[62] In turn, loss of METTL14 impedes the proliferation and hastens the differentiation of mouse embryonic neural stem cells (NSCs), suggesting that the m⁶A modification can enhance NSC self-renewal.[63] Recent research indicates that m⁶A RNA methylation may serve as a promising therapeutic target for various diseases with reduced serum testosterone levels, including azoospermia and oligospermia. A negative correlation exists between the m⁶A modification and autophagy processes in Leydig cells during testosterone synthesis ([Fig. 4]).[64] These studies demonstrate the significance of m⁶A during embryonic growth and starting cell difference projects.

Fig. 4 The proposed model outlines the connection between METTL14/ALKBH5 and autophagy in human chorionic gonadotropin-treated Leydig cells. (Reproduced with permission from Chen Y, Wang J, Xu D, et al. m⁶A mRNA methylation regulates testosterone synthesis by modulating autophagy in Leydig cells. Autophagy 2020:1–19[64].)

m⁶A and Acute Myeloid Leukemia

The presence of the m⁶A modification on mRNA is vital for the advancement and upkeep of acute myeloid leukemia (AML), similar to that observed for the self-restoration of leukemia stem or initiating cells (LSCs/LICs) ([Fig. 5]).[65] Compared with sound hematopoietic undeveloped/progenitor cells (HSPCs) or different types of malignancy cells, the METTL3 quality in AML cells shows an abundance of mRNA and protein expression.[65] The absence of the shaping compound METTL3 in HSPCs advances cell separation and diminishes cell multiplication. METTL3 controls myeloid separation in AML cells through conditional exhaustion, thus prompting cell separation and apoptosis in living beings and postponing the improvement of leukemia in recipient mice.[65] An examination performed at the single-nucleotide level revealed that m⁶A drives the translation of the cellular myelocytomatosis (c-MYC), B-cell lymphoma 2 (BCL2), and lipid and protein phosphatase and tension homolog (PTEN) mRNAs in a human AML cell line (MOLM-13 cells).[65] Moreover, in AML cells, METTL3 is associated with the CAATT-box binding protein C/EBPZ (CCAAT/enhancer-binding protein zeta) at the transcription initiation site, thus triggering m⁶A to enhance the translation of related mRNAs, which is crucial for the maintenance of the leukemic state.[11] These investigations provide a hypothetical basis for focusing on METTL3 in AML therapeutically.

Fig. 5 Proposed model describing the role of METTL3 in the regulation of myeloid differentiation. (Reproduced with permission from Vu LP, Pickering B, Cheng Y, et al. The N ⁶-methyladenosine (m⁶A)-forming enzyme METTL3 controls myeloid differentiation of normal hematopoietic and leukemia cells. Nat Med 2017;23(11):1369–1376[65].)

m⁶A and Neurological Diseases

Previous research has confirmed that the m⁶A modification of mRNA can control neurological outcomes and is important for human brain development and neurological diseases.[66] [67] [68] Studies have found that METTL14 knockout or METTL3 depletion in the cerebrums of developing mouse hatchlings results in the depletion of m⁶A, thus lengthening the cell cycle and keeping up with the presence of radial nerve cells ([Fig. 6]).[66] The sequencing of m⁶A in the cerebral mantle of mice revealed an overflow of mRNA particles connected with transcription factors, neurogenesis, the cell cycle, and neuronal separation, whereas m⁶A marking advanced its disintegration. The presence of m⁶A also manages human cortical neurogenesis in the organoids of the prefrontal cortex. The examination of the results of m⁶A-mRNA sequencing during mouse and human cortical neurogenesis revealed a wealth of human-explicit m⁶A-marked transcripts connected with qualities that favor illnesses of the mind.[66]

Fig. 6 Illustrations of the downregulation of m⁶A caused by METTL14 nullification during the development of mouse brains, which prompted an expanded cell cycle in radial nerve cells and postponed cortical neurogenesis into postnatal stages. (Reproduced with permission from Yoon KJ, Ringeling FR, Vissers C, et al. Temporal control of mammalian cortical neurogenesis by m⁶A methylation. Cell 2017;171(04):877–889[66].)

Conditional knockout of METTL14 in animals prompted a decline in the number of oligodendrocytes and diminished the myelin layers in the focal sensory system. In vitro, METTL14 depletion can disrupt the maturation of oligodendrocytes after mitosis and has a significant effect on the transcriptomes of precursor cells and oligodendrocytes. In turn, abnormalities of oligodendrocytes can trigger not only demyelinating infections of the focal sensory system, but also neuronal harm or mental issues, and, cerebrum tumors. Moreover, the deletion of METTL14 in oligodendrocyte cell lines induces a strange joining of various RNA molecules.[67] [69] Via behavioral and functional magnetic resonance imaging studies, genetic variations in the m⁶A demethylase FTO can affect the response of the dopamine-dependent midbrain to reward learning, as well as behavioral responses related to learning from negative outcomes.[70]

Based on whole-genome m⁶A analysis and the observation of dynamic m⁶A modifications during postnatal neurodevelopment, it was determined that FTO deficiency causes changes in the articulation of specific fundamental parts of the brain-prompted neurotrophic factor pathway, denoted by m⁶A ([Fig. 7]).[71] These examinations suggest that FTO plays a significant role in neurogenesis, learning, and memory. Concomitantly, previous research has found that the m⁶A demethylase genes FTO and ALKBH5 are associated with major depressive disorder.[72] Furthermore, it has been reported that the insufficiency of FTO can lessen uneasiness and melancholy-like behavior in rodents by changing the gut flora.[73] [74] Moreover, FTO is firmly associated with insulin-inadequacy-related Alzheimer's disease, and conditional deletion of FTO in neurons can decrease intellectual disability in diseased mice.

Fig. 7 Proposed mechanism of FTO protein regulation of adult neurogenesis. FTO, fat-mass- and obesity-associated. (Reproduced with permission from Li L, Zang L, Zhang F, et al. Fat mass and obesity-associated (FTO) protein regulates adult neurogenesis. Hum Mol Genet 2017;26(13):2398–2411[71].)

m⁶A and Cancers

The m⁶A modification of mRNA is deeply implicated in the origins and progression of malignancies.[75] [76] The downregulation of the m⁶A methyltransferase METTL3 or METTL14 significantly promotes the growth, self-renewal, and tumorigenesis of human glioblastoma stem cells (GSCs).[77] [78] METTL3 coordinates the successful execution of carcinogenic pathways in GSCs.[79] An increasing number of studies have confirmed that METTL3 promotes the progression of liver cancer,[80] bladder cancer,[81] gastric cancer ([Fig. 8]),[82] [83] breast cancer ([Fig. 9]),[84] colorectal cancer ([Figs. 10] and [11]),[85] [86] prostate cancer,[87] and other tumors in an m⁶A-dependent manner.

Fig. 8 Graphical representation of the manner in which METTL3 affects tumor glycolysis and angiogenesis, leading to enhanced growth and liver metastasis of gastric cancer tumors. (Reproduced with permission from Wang Q, Chen C, Ding Q, et al. METTL3-mediated m⁶A modification of HDGF mRNA promotes gastric cancer progression and has prognostic significance. Gut 2020;69(07):1193–1205[82].)

Fig. 9 Schematic representation of the manner in which the oncoprotein HBXIP upregulates METTL3 by suppressing the levels of the tumor suppressor let-7g, which contributes to breast cancer progression. (Reproduced with permission from Cai X, Wang X, Cao C, et al. HBXIP-elevated methyltransferase METTL3 promotes the progression of breast cancer via inhibiting tumor suppressor let-7g. Cancer Lett 2018;415:11–19[84].)

Fig. 10 Proposed model outlining the role of METTL3 as an oncogene in the maintenance of SOX2 expression via an m⁶A-IGF2BP2-dependent process in colorectal cancer cells. The model also suggests a potential biomarker panel for predicting outcomes in patients with CRC. (Reproduced with permission from Li T, Hu PS, Zuo Z, et al. METTL3 facilitates tumor progression via an m⁶A-IGF2BP2-dependent mechanism in colorectal carcinoma. Mol Cancer 2019;18(01):112[85].)

Fig. 11 Proposed model of the manner in which METTL3 enhances CRC cell proliferation via the stabilization of CCNE1 mRNAs in an m⁶A-dependent manner. CRC, colorectal cancer; METTL3, methyltransferase-like 3. (Reproduced with permission from Zhu et al. Methyltransferase 3 promotes colorectal cancer proliferation by stabilizing CCNE1 mRNA in an m⁶A-dependent manner. J Cell Mol Med 2020;24(06):3521–3533[86].)

A recent study confirmed that the miR-186–METTL3 axis promotes the growth of colorectal cancer through the Wnt–β-catenin signaling pathway. The β-catenin signaling pathway promotes the development of hepatoblastoma ([Fig. 12]).[88] In addition, METTL3 can promote chemotherapy and radiation resistance in various tumors, such as pancreatic cancer.[89]

Fig. 12 Suggested model outlining the functional role of the miR-186–METTL3 axis in hepatoblastoma. (Reproduced with permission from Cui X, Wang Z, Li J, et al. Cross-talk between RNA N ⁶-methyladenosine methyltransferase-like 3 and miR-186 regulates hepatoblastoma progression through Wnt/beta-catenin signaling pathway. Cell Prolif 2020;53(03):e12768[88].)

Recently, another study found that the silencing of METTL3 downregulates the mesenchymal–epithelial transition factor (c-Met), phosphorylated Akt (p-Akt), and cell-cycle-related proteins in uveal melanoma (UM) cells, leading to G1 arrest of the cell cycle and restricting UM cell multiplication, aggregation, and mobility.[90] The Epstein–Barr virus (EBV) is a ubiquitous cancer-causing virus that can induce various types of tumors. The reprogramming of EBV antigenic determinants through METTL14 is crucial for the development of virus-associated tumors ([Fig. 13]).[91] The aforementioned studies revealed that the m⁶A methyltransferase METTL3 or METTL1 may be a potential target for cancer treatment.

Fig. 13 Illustration of the interaction between the EBV antigens EBNA3C and METTL14 and EBV transcripts that are modified by METTL14 and its associated proteins. EBV, Epstein–Barr virus; METTL14, methyltransferase-14. (Reproduced with permission from Lang F, Singh RK, Pei Y, Zhang S, Sun K, Robertson ES. EBV epitranscriptome reprogramming by METTL14 is critical for viral-associated tumorigenesis. PLoS Pathog 2019;15(06):e1007796[91].)

The m⁶A-modified mRNA-binding protein YTHDF1 is overexpressed in various cancers, including non-small-cell lung cancer ([Fig. 14]),[92] colorectal cancer,[93] liver cancer,[94] and ovarian cancer ([Fig. 15])[95]; moreover, the depletion of YTHDF1 can inhibit tumor development. Furthermore, in vitro studies have shown that YTHDF1 knockdown can facilitate the responsiveness to chemotherapy agents, such as fluorouracil and oxaliplatin.[96] Snail is a key transcription factor in the epithelial–mesenchymal transition. Studies of loss and gain of function have confirmed that the YTHDF1-mediated m⁶A modification of the snail mRNA enhances its translation.[97] Research has demonstrated that by promoting the translation of the m⁶A-modified cathepsin mRNA, YTHDF1 enhances antigen degradation in phagosomes and limits the cross-presentation of new antigens in dendritic cells, which may become a target for tumor immunotherapy.[98] Moreover, YTHDC2 upregulates HIF-1α and other factors, thus driving tumor spread through the modulation of gene/protein expression.[99] In turn, the inhibition of YTHDC2 can greatly promote the proliferation of tumors, including esophageal squamous cell carcinoma, by affecting several cancer-related signaling pathways.[100] [101]

Fig. 14 Proposed model of non-small-cell lung cancer development under normal oxygen conditions: increased or high expression of YTHDF1 primarily boosts the translation efficiency of m⁶A-modified target transcripts, such as the cell–cycle regulators CDK2, CDK4, and Cyclin D1, within cancerous cells, resulting in uncontrolled cancerous cell proliferation. (Reproduced with permission from Shi Y, Fan S, Wu M, et al. YTHDF1 links hypoxia adaptation and non-small cell lung cancer progression. Nat Commun 2019;10(01):4892[92].)

Fig. 15 The proposed model explorations of the role of YTHDF1-mediated EIF3C translation in ovarian cancer. (Reproduced with permission from Liu T, Wei Q, Jin J, et al. The m⁶A reader YTHDF1 promotes ovarian cancer progression via augmenting EIF3C translation. Nucleic Acids Res 2020;48(07):3816–3831[95].)

The insulin-like growth factor-2 mRNA-binding proteins 1, 2, and 3 (IGF2BP1/2/3) are unique families of m⁶A “readers” that target thousands of mRNA transcripts by recognizing the common GG (m⁶A) C sequence. These factors may play a carcinogenic role in cancer cells by stabilizing the methylation of mRNAs that target carcinogenic targets, such as c-MYC ([Fig. 16]).[102] [103] [104] In addition, IGF2BP1 can disrupt the stability of the highly upregulated lncRNA in liver cancer through the CCR4–NOT (a highly conserved specific gene silencer) adenylate kinase complex, which is highly linked to liver cancer progression and severity.[105] The IGF2BP3 gene is a downstream target of the carcinogenic effector Lin28b. In a mouse model, it was found that the overexpression of IGF2BP3 drives liver tumorigenesis.[106] Finally, IGF2BP2 and IGF2BP2-2 can promote the occurrence of liver cancer.[107] [108]

Fig. 16 IGF2BP-mediated control of m⁶A mRNA expression. mRNAs undergo methylation in the nucleus and selectively associate with IGF2BPs, thus maintaining the stability of P-bodies and enhancing translation until their sequestration in SGs under heat shock. (Reproduced with permission from Huang H, Weng H, Sun W, et al. Recognition of RNA N ⁶-methyladenosine by IGF2BP proteins enhances mRNA stability and translation. Nat Cell Biol 2018;20(03):285–295[102].)

It has been reported that the m⁶A demethylases FTO and ALKBH1 are associated with various malignant tumors,[109] [110] [111] [112] [113] such as the carcinogenic role of FTO in AML ([Fig. 17]).[114] Through autophagy and NF-κ, nutrient-deprivation-triggered FTO in the B pathway leads to heightened melanoma onset in human and murine models. This study suggests that FTO inhibition combined with anti-programmed death-1 (anti-PD-1) blockade may reduce the resistance of melanoma to immunotherapy ([Fig. 18]).[115] By modulating the m⁶A status of the E2F1 and MYC transcripts, FTO potentiates the proliferation and movement of cervical cancer cells.

Fig. 17 Illustrations of FTO as a viable drug target. The use of small-molecule inhibitors to target FTO may offer potential therapeutic benefits for AML. AML, acute myeloid leukemia; FTO, fat-mass- and obesity-associated. (Reproduced with permission from Huang Y, Su R, Sheng Y, et al. Small-molecule targeting of oncogenic FTO demethylase in acute myeloid leukemia. Cancer Cell 2019;35(04):677–691[114].)

Fig. 18 Proposed model outlining the regulatory and functional impact of FTO on melanoma development and the response to anti-PD-1 blockade therapy. anti-PD-1, anti-programmed death-1; FTO, fat-mass- and obesity-associated. (Reproduced with permission from Yang S, Wei J, Cui YH, et al. m⁶A mRNA demethylase FTO regulates melanoma tumorigenicity and response to anti-PD-1 blockade. Nat Commun 2019;10(01):2782[115].)

Intrahepatic cholangiocarcinoma (ICC) is second only to primary liver cancer as the deadliest form of primary liver cancer with a high prevalence. A Kaplan–Meier survival analysis showed that low expression of FTO promotes the development of ICC, indicating a poor prognosis of ICC.[116] [117] Head and neck squamous cell carcinoma (HNSCC) is the sixth most-common cancer worldwide, with oral squamous cell carcinoma (OSCC) being an aggressive form of HNSCC. Recent research has found that DDX3 (a highly conserved subfamily of the DEAD-box proteins) regulates ALKBH5 to downregulate the m⁶A methylation level in FOXM1 (Forkhead box, FOX) and NANOG (Nanog Homeobox) new transcripts, thus promoting cisplatin resistance in OSCC.[118] A recent study confirmed that decreased levels of the ALKBH5 and FTO mRNAs are associated with shorter overall survival and cancer-specific survival after nephrectomy.[119] These investigations offer possibilities for novel therapeutic and prognostic avenues for cancer management. The implication of FTO in cancer advancement has prompted interest in drugs such as FB23 and FB23-2, which bind to FTO and inhibit its m⁶A function.[114] In addition, fluorescein-based compounds have shown potential for targeting FTO demethylation.[120] Meclofenamic acid has been verified as a potent inhibitor that is specific to FTO, with a higher selectivity for FTO versus ALKBH5.[121] The physiological regulation and occurrence of human diseases discussed above focus only on m⁶A, which is by far the most important chemical modification. In addition to chemical modifications, such as methylation (for example, m⁶A, m⁵C, etc.), there is a great need for basic research on the molecular mechanisms of other chemical modifications, together with their links to illness onset and progression in humans, especially the geranyl modification and nicotinamide adenine dinucleotide (NAD) cap modification reported recently.

Recent Research on Newly Discovered RNA Modifications and Their Implications for Cellular Biology

Although the mechanism underlying the RNA chemical modifications remains unclear, especially that of the m⁶A modification, its role in biological processes has gained increasing recognition, despite the uncertainty about its impact on humans. This review focuses on the progress of high-throughput sequencing technology for the m⁶A modification; its physiological regulation; and its role in cancer occurrence, cancer development, and tumor suppression. Accumulating evidence indicates that RNA m⁶A modifiers offer promising biomarker prospects for cancer prognosis and therapy planning, such as in the context of rectal cancer,[122] non-small-cell lung cancer,[123] and renal cell carcinoma.[124] Recent research has found that m⁶A-mediated long noncoding RNA00958 (LINC00958) upregulation increases adipogenesis and represents a promising nanomedicine target for hepatocellular carcinoma.[125] Because of its pivotal role in multiple diseases, targeting m⁶A may prove beneficial for the diagnosis and management of illnesses such as AML, glioblastoma, and breast cancer. In addition, scientists recently used the human metapneumovirus (HMPV) as a representative sample, and it was discovered that m⁶A confers evasion of RNA sensors such as RIG-I on viral RNA (RIG-I), thus confirming that viral RNA m⁶A can be used as a target for the development of attenuated HMPV vaccines ([Fig. 19]).[126]

Fig. 19 Schematic representation outlining the RIG-I-triggered interferon response after HMPV entry. Viral particles travel to the cytoplasm, and RdRp transcribes starting at the terminal 3′ genomic section, forming full antigenomes that serve as templates for producing copycat viral RNA. (Reproduced with permission from Lu M, Zhang Z, Xue M, et al. N ⁶-methyladenosine modification enables viral RNA to escape recognition by the RNA sensor RIG-I. Nat Microbiol 2020;5(04):584–598[126].)

In summary, unraveling the significance of and the mechanisms underlying m⁶A in nucleic acids holds promise for designing new antitumor treatments, which can provide insights for the development of novel therapeutic strategies.

As a popular research field in recent years, the recognition of new types of RNA modification, the investigation of gene activities involved in RNA modifications, and the elucidation of pathogenic mechanisms are valuable avenues for drug development. A typical example is the NAD variant of the RNA cap that commonly occurs outside of the canonical m⁷G type reported by Liu et al in 2009 ([Fig. 20]). With the advancement of detection technology, NAD cap structures were first discovered in E. coli,[127] followed by yeast, cell lines, plants, and mouse tissues.[128] [129] [130] [131] [132] According to the reported results, NAD cap formation occurs simultaneously with RNA transcription. Concomitantly, the existing RNA sequencing technology has shown that the enzyme NUDT12 (a deNADding enzyme), which removes the NAD cap structure from RNA, has a close regulatory effect on the circadian clock in the liver. Although significant achievements have been reached, e.g., for NAD tagSeq., research on the underlying molecular mechanism remains very limited.

Fig. 20 (A) Schematic illustrations of the RNA 5′-NAD capping process and (B) its capturing sequencing approach (NAD tagSeq).

Another typical example was reported in 2012 by Liu et al in which the strongest hydrophobic geranyl (pentenyl dimer) modification to date exists at position 34 of the bacterial tRNA. Geranylation of RNA in vivo is achieved by the enzyme tRNA 2-selenouridine synthase (SelU) ([Fig. 21]). Preliminary studies revealed that the geranyl-modified group can shape the structure and operational properties of RNAs after binding to a specific RNA, as well as its localization within the cell. However, the molecular mechanism underlying this lipid modification is completely unknown.[133] The development of specific markers and localization and enrichment technologies for aryl-modified RNAs will be of great research significance, by laying a solid foundation for explaining its molecular mechanism and even exploring its association with human diseases.

Fig. 21 Overall illustrations of the possible pathway for geranylated RNA generation. (A) Geranylated RNA (ges²U-RNA) was synthesized from s²U-RNA, which was mediated by the SelU enzyme. (B) Proposed pathway of selenide RNA (se²U-RNA) generation through the geranylated RNA intermediate (ges²U-RNA).

Another noteworthy example is the epoxy-queuosine (oQ) modification reported by Bandarian et al in 2011. The biosynthetic pathway for this modification is shown in [Fig. 22]; however, the molecular mechanism via which the modifying enzymes QueA and QueG regulate the levels of modified oQ and Q remains unknown, especially regarding its possible link to human diseases.[134]

Fig. 22 Proposed biosynthetic pathway for the oQ modification of RNA.

Summary

With the in-depth development of multidisciplinary and interdisciplinary research, an increasing number of new chemically modified RNAs have been reported using comprehensive methods, such as those based on chemistry, life sciences, medicine, and pharmacy. Although these new chemically modified groups perform diverse structural and functional roles in RNA, little is known about their regulatory molecular mechanisms. The development of simple and highly selective chemical and biological methods for their detection, labeling, and localization is of practical significance for elucidating their underlying molecular mechanisms. In particular, the development of highly specific chemical and biological labeling and detection tools for these new chemically modified groups, as well as high-throughput sequencing, will shed light on the molecular underpinnings of RNA modifications. Thus, the development of reliable tools targeting these RNA modifications has great application potential.

In addition, the manner in which these new chemical modification groups are implicated in the pathogenesis of human ailments is particularly noteworthy. The development and application of inhibitors, regulation strategies, and corresponding mRNA-targeted degradation technologies for this new chemically modified enzyme (including encoding and decoding) targets will have practical research significance.

In recent years, in particular, COVID-19 has been rampant worldwide, with very few specific therapeutic drugs or vaccines being available for this condition.[135] Research has shown that there are many unknown chemical modifications in the RNA of this virus that have not been reported. The impact of these modifications on viral characteristics remains elusive, as well as the types, molecular mechanisms, and targets of these modifications.[136] At present, the role played by these new chemical modifications (NAD, prenyl, and oQ modifications) remains unknown,[137] [138] [139] [140] [141] [142] [143] [144] [145] although it has been speculated that they may help the virus avoid host attacks (for example m⁶A, m¹A, and Ψ). Therefore, elucidating the types and characteristics of these chemical modifications is expected to provide new clues for the fight against COVID-19 and many other RNA-relevant viruses. In addition to the application of mature technological tools, the development of new technological toolsets to identify them and the implementation of highly specific small-molecule inhibitors are of great importance to human society.

In summary, significant advancements have been made in qualitative/quantitative m⁶A detection, high-throughput sequencing, and research linking it to disease. Efforts continue toward the development of simplified and more accessible chemical/biological technologies that will contribute to precision medicine, thus benefiting society and patients alike.

Conflict of Interest

None declared.

Acknowledgments

The authors thank the starting fund from Huazhong University of Science and Technology (HUST) and Shenzhen Huazhong University of Science and Technology Research Institute for financial support.

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Publication History

Received: 24 October 2023

Accepted: 24 January 2024

Article published online:
11 March 2024