RNA modifications in cellular metabolism: implications for metabolism-targeted therapy and immunotherapy.

Generally speaking, the immunometabolism concerns distinctions between activated and resting immune cells. The former metabolizes in a manner similar to malignant cells, Warburg effect without obvious OXPHOS, while the latter obtains energy from FAO and the Krebs cycle. Corresponding metabolic patterns of different immune cell subgroups have been described. 364 Herein, we focus on the contributions of RNA modifications on immunometabolism in diverse immune responses (Fig. 6 ). Fig. 6 Effects of RNA modification on immunometabolism. This figure shows current findings about how RNA modifications regulate immunometabolism. On the one hand, RNA modifications are involved in the intrinsic metabolic adaptation of immune cells, further affecting function and state of immunocytes. On the other hand, m6A modification could mediate several phenotype alterations of immunocytes induced by glucose deficiency and high lactate in TME. The figure is generated with BioRender ( https://biorender.com ) Effects of RNA modification on immunometabolism. This figure shows current findings about how RNA modifications regulate immunometabolism. On the one hand, RNA modifications are involved in the intrinsic metabolic adaptation of immune cells, further affecting function and state of immunocytes. On the other hand, m6A modification could mediate several phenotype alterations of immunocytes induced by glucose deficiency and high lactate in TME. The figure is generated with BioRender ( https://biorender.com ) The concept of tumor immune microenvironment (TIME) emphasizes the interplay between immune cells, tumor cells, and other components in immunity system, which profoundly influences the immune responses, via nutrients depletion and metabolites release. To sustain the rapid proliferation, tumor cells consume large amounts of glucose, glutamine and other amino acids like arginine, generally constituting an adverse residential environment for immune cells. The universal recognition is that glucose exhaustion induced by tumor cells contributes to immunosuppressive TME. Nevertheless, the pioneering work of Reinfeld et al. showed that glucose is not a limiting factor in the TME, and resident immunocytes were capable to enhance glucose uptake in compensation for depleted glutamine. Indeed, multiple pathways are conducive to the impaired glucose metabolism of T cells from TME, but the effects are likely to be context-dependent. Mechanism study about epigenetic regulation on glycolytic reprogramming of T cells remains deficient, but the glycolytic-epigenetic interplay has been found in development of Tfh cells. VHL deficiency induced expression of glycolytic enzyme GAPDH, which acted as an epigenetic regulator to enhance METTL3/METTL14-catalyzed m6A modification on ICOS mRNA, thereby suppressed ICOS expression led to attenuated Tfh cell differentiation. 365 Glucose depletion in TME predisposes the differentiation of macrophages to M2-like TAM, which preferentially employs OXPHOS for ATP synthesis rather than consuming glucose. 366 Recently, METTL3 was considered as the top candidates for regulating M1 macrophages activation via targeting mTOR/NF-κB-mediated metabolic adaptation. 367 However, Ning et al. proposed that m6A modification was responsible for inhibited glycolysis and M1 macrophage polarization, through YTHDF2-mediated degradation of m6A-modified STAT1 mRNA, further attenuating glycolysis-related genes expression. 368 Distinct glutamine acquisition is another essential aspect of nutrient partitioning. Tumor cells tend to have advantage in glutamine consumption over those immune cells, as tumor cells have over-expressed methionine transporter Slc43a2, thereby restricts methionine metabolism and the antitumor function of T cells. 369 However, whether RNA modifications participate in glutamine metabolism of immune cells remains to be explored. Except for nutrient competition, metabolites secreted by tumor cells also exert immunosuppressive effects on anti-tumor immunity. Tumor cells produce large amounts of lactic acid to generate highly acidic regions, as a hallmark of immunosuppressive TME. High concentration of extracellular lactic acid significantly inhibits the survival and activation of T and NK Cells, further blocks the immunosurveillance. 370 m6A-mediated stabilization of circQSOX1 enhance lactic acid accumulation in CRC, thus supports Treg cells and facilitate immune escape, which further impacts efficacy of anti-CTLA-4 therapy in vivo. 229 As mentioned above, the intratumoral myeloid cells have vigorous glycolytic activity, but whether myeloid-derived lactate limits T cell effector functions, remains to be explored. 371 Lactate also negatively influences functions of macrophage and skews the differentiation of macrophages toward the M2 phenotype. M2 macrophage infiltration in endometriosis was positively correlated with lactate accumulation. Mechanism study showed that lactate promoted M2 macrophage polarization via METTL3-mediated m6A modification on Trib1 mRNA, which enhanced its stability. 372 Tumor cell is a significant source of intratumoral lipids, including cholesterol, fatty acid or oxidized lipids, which have a deleterious effect on T cells, DCs, and macrophages. Several studies have confirmed that m6A methylation was involved in maintaining the functional homeostasis of macrophages via targeting the balance between lipid uptake and cholesterol efflux. Elevated METTL3 in oxidized low-density lipoprotein (oxLDL)-treated macrophages facilitates lipid uptake via interacting with DDX5 to target MSR1 mRNA and stabilize it in m6A-dependent manner. 373 FTO is upregulated in macrophages loaded with ox-LDL, which enhances cholesterol efflux via motivating AMPK/ACC phosphorylation to promote ABCA1/G1-mediated efflux, and attenuates cholesterol ester accumulation through restricting PPARγ to reduce CD36 expression. 316 More direct and convincing evidences are expected to elucidate the epigenetic regulation on TAMs. Besides, cholesterol uptake activates PD-1 expression in tumor-infiltrating CD8 + T cells, which in turn facilitates FAO and lipolysis. 374 Extracellular fatty acids are more effectively consumed by Treg cells than effector T cells, which eventually supports Treg accumulation. 375 Oxidized lipids restrain cross-representation in DCs 376 and enhanced uptake of fatty acids and peroxidation lead to dysfunctional state of tumor-derived DCs. 377 But the role of RNA modifications in these cellular processes has not been identified yet. The pathogenesis of infectious diseases is consisted of two parts, deficiency of immune system itself and immune escape of pathogens. On one hand, specific RNA modifications on viral RNAs have been described, including m6A, m5C ac4C, Ψ, and RNA editing, thus affecting viral RNA sensing and signaling. 378 On the other hand, RNA modifications influence host responses to viral infection via regulating immune cell functions. The interferon pathway is the major target of m6A modification to modulate antiviral innate immunity. METTL3/14 enhances the turnover rate of IFNB mRNAs via YTHDF2-mediated manner and accelerates viral propagation. 379 , 380 The coordination between m6A methylation and other RBPs also mediate the negative effects on immunity. DEAD-box helicase 5 (DDX5), hijacked by viruses to promote replication, could interact with METTL3 to facilitate formation of the METTL3/14 complex during vesicular stomatitis virus infection. 381 In addition to interferon pathway, ALKBH5 could promote viral propagation relying on metabolic rewiring. It was showed that viral infection impaired the enzymatic activity of ALKBH5 in posttranslational level and thus downregulated expression of α-ketoglutarate dehydrogenase (OGDH), leading to reduced itaconate production, a metabolite that inhibits viral replication. 382 Beyond that, the association between RNA modification and metabolic processes remains largely unknown. Inflammatory response is achieved through a coordinately regulated gene expression program, including acute and chronic type. 383 In response to microorganisms, autoimmunity, allergies, dysregulated metabolism, and physical damage, different types of inflammation are produced. 384 Until recently, regulatory roles of RNA modification in inflammation and anti-inflammation gene expression have been verified. Previous studies have shown that m6A modification is involved in pathogenesis of autoimmune diseases. For instance, METTL3 is significantly upregulated in RA patients and positively associated with CRP and ESR, the two common markers of RA disease activity. 385 In systemic lupus erythematosus (SLE), decreased m5C level and low NSUN2 expression are found in CD4 + T cells, and hypermethylated m5C-modified upregulated genes in SLE are enriched in inflammatory pathways. 386 Significantly, in DC-dependent inflammatory response, m6A-mediated glycolytic reprogramming is critical for feedback-control of DC migration. Mechanistically, in response to microbial products or inflammatory signals, upregulated CC-chemokine receptor 7 (CCR7) stimulated lnc-Dpf3 via removing its m6A modification to prevent degradation, and lnc-Dpf3 could negatively modulate HIF-1α pathway via binding to HIF-1α and suppressing HIF-1α-dependent transcription of the glycolytic gene Ldha. 387 Also, m6A modification modulates macrophage phenotype in inflammatory responses. Previous study has reported that METTL3 was notably elevated in M1 macrophages and modulated polarization via metabolism reprogramming. In mechanism, m6A methylation contributes to enhanced expression of HDGF, which increases glycolysis and lipids accumulation in M1, therefore aggravating the progression of atherosclerosis. 388 And METTL3-meidated m6A of PGC-1α mRNA is involved in mitochondrial dysfunction and oxLDL-induced inflammation in monocytes. 340 Although there are few studies on RNA modifications regulating inflammatory and autoimmune diseases in the aspect of immunometabolism.

For the currently approved metabolic drugs, an impending challenge of clinic application is development of chemo-resistance owing to rewiring or compensatory metabolic pathways. Thus, the multiple pathways blockade or combined therapy may have superiority over the single-agent therapy. Notably, multiple studies have supported that combined utilization of targeting RNA modifications could improve chemo-resistance to some metabolism-targeted drugs. The influences of m6A modification on CRC resistance to 5-FU is a representative example. Mechanism studies have demonstrated that METTL3 could induce 5-FU resistance of CRC cells via m6A/DGCR8/miR181d/NCALD axis, 389 m6A/IGF2BP1/SEC62/β-catenin axis. 390 Also, m6A methylation facilitates preferential splicing of p53 pre-mRNA to produce p53 R273H mutant protein, leading to multidrug resistance in CRC cells. 391 Moreover, suppressing c-Myc-driven YTHDF1 transactivation was revealed to re-sensitize CRC cells to some anticancer drugs, including 5-FU. 392 Consistently, Jiang et al. found that miR-136-5p could downregulated YTHDF1 to suppress tumor progression and chemoresistance to 5-FU, while miR-136-5p was declined in CRC cell lines and tissues. 393 Moreover, METTL3 was identified to positively modulate gemcitabine (GEM) sensitivity of PC via DBH-AS1/miR-3163/USP44, and low expression level of METTL3 was closely related with GEM resistance. 394 Upregulated METTL14 was observed in GEM-resistant PC cells, which was induced by p65 and downstream facilitated cytidine deaminase (CDA) expression to inactivate GEM in PC. Inhibition of METTL14 effectively re-sensitized GEM in vitro and in vivo, indicating a promising approach for circumvent chemo-resistance. 395 Intriguingly, ALKBH5-mediated demethylation also exerts a positive role in GEM sensitivity of pancreatic ductal adenocarcinoma (PDAC) through suppressing Wnt pathway. 396 Growing researches revealed that m6A regulators markedly affected therapeutic responses against checkpoint blockade. Wang et al. reported that depletion of METTL3/14 enhanced infiltration and cytokines secretion of CTL, augmenting anti-PD-1 therapy efficacy of CRC in vivo, through m6A/YTHDF2/STAT1/IRF1 axis. 397 However, METTL14 could sensitize cholangiocarcinoma to ICB via YTHDF1-mediated degradation of SIAH2 mRNA. 398 Knockdown of YTHDF1 enhances cross-presentation of DCs to CD8 + T cells by suppressing cathepsins expression, further increased IFN-γ secretion of T cells upregulates PD-L1 level in tumor cells. 399 FTO was identified to negatively regulate ICB therapeutic efficacy in melanoma. Ablating FTO decreases expression of several significant melanoma-promoting genes and sensitized anti-PD-1 treatment in vivo. 400 However, FTO was revealed to promote PD-L1 expression in an IFN-γ-independent manner of CRC cells, thus improving ICB treatment. 401 Su et al. demonstrated that inhibition of FTO obviously downregulated immune check point gene LILRB4 in AML cells, with superiority over PD-L1/2, further repressing leukemia stem cell maintenance and immune evasion. 402 In melanoma, FTO participates in rewiring tumor glycolysis metabolism to suppress T cell effector functions, and FTO inhibition synergizes with anti-PD-L1 therapy. 248 Additionally, the silico analysis identified that high FTO level was associated with poor prognosis and unfavorable immunotherapy effect of GC patients. 403 And ALKBH5 was supposed to be a potential predictor for anti-PD-1 blockade efficacy in melanoma. Deficiency of ALKBH5 induced downregulation of MCT4 expression and intra-tumoral lactate content, which negatively influenced polymorphonuclear myeloid derived cells and Tregs. 404 Several bioinformatic studies have underlined the intimate connection between m6A modification and immunotherapy resistance. 405 Moreover, loss of A-to-I editor ADAR1 significantly augmented anti-PD-1 treatment in melanoma and CRC. In mechanism, the interaction of tumor intrinsic type I and type II IFN signaling contribute to sensitize ADAR1-null cells to ICB. 406 Except for checkpoint blockade therapy, targeting m6A modification has demonstrated promising potential in improving adoptive cell therapy. Marvelous progresses have been made in modulating METTL3 and YTHDF2 to enhance the proliferation and cytotoxicity of NK cells in vitro, which might inspire future protocols for NK cell-based immunotherapy. 407 , 408 No attempt to modulate m6A methylation in CAR T cells has been reported yet, but considering the significant roles of m6A regulators in determining functions and fate of T cell, novel therapeutic strategies are expected. Targeting the dysregulated m6A regulators, which are overexpressed in tumor on most occasions, plentiful specific inhibitors have demonstrated exciting anti-tumor effects in vitro and in vivo (Table 6 ). FTO is considered as the most promising target. Within a decade, a series of selective inhibitors have come out, ranging from natural substance to small-molecule compound. The first natural inhibitor Rhein displayed therapeutic activity in leukemia mice, 409 meclofenamic acid 2 (MA2) was observed to suppress glioblatoma progression. 410 Small-molecule compounds CHTB and N-CDPCB were identified with novel binding sites by crystal structure screening. 411 R-2-hydroxyglutarate (R-2HG) exerts anti-leukemia and anti-glioma effects, synergizing with current first-line chemotherapy agents. 412 FB23-2 also significantly attenuates the progression of AML in vitro and in xeno-transplanted mice. 413 Table 6 Specific inhibitors against RNA modification regulators Target Drug Cancer Effect Ref. FTO Rhein AML Anti-leukemia efficacy in vitro and in vivo 409 MA2 GBM Suppresses cell proliferation and tumor progression 410 R-2HG AML/GBM Antitumor effect, synergizing with first-line chemotherapy agents 412 FB23-2 AML Anti-leukemia efficacy in vitro and in vivo 413 CS1/2 AML Potent anti-leukemia efficacy in mouse models, sensitize leukemia cells to T-cell cytotoxicity, overcomes immune evasion 402 Dac15 Melanoma Promotes activation and effector state of T cell, improving anti-PD1 blockade 248 18097 BRCA Restrain in vivo growth and lung colonization 414 FTO-43 GC/AML/GBM Potent anti-tumor effects in mouse model 415 C6 ESCC Anti-tumor efficacy in vitro and in vivo 416 ALKBH5 ALK-04 Melanoma Improve anti-PD-1 therapy efficiency 404 Compound 1/2 AML Anti-proliferative effects in specific AML cell lines 417 METTL3 UZH1a AML Suppress proliferation and viability of tumor cells 418 UZH2 AML/PC More potent anti-proliferative effects in vitro 419 STM2457 AML Anti-leukemia efficacy in vitro and in vivo 420 TRMT6/TRMT61A Thiram HCC Suppresses HCC growth in preclinical models 421 Specific inhibitors against RNA modification regulators Subsequently, more potent inhibitors with potential to improve anti-tumor immunity was developed. CS1/2-induced FTO inhibition not only attenuates leukemia stem cell self-renewal, but reprograms immune responses by downregulating expression of immune checkpoint gene, which overcomes HMA-induced immune evasion and sensitizes leukemia cells to T cell cytotoxicity. 402 Also, Dac15 restores functions of CD8 + T cells, blocks FTO-mediated immune evasion, and synergizes with anti-PD-1 blockade. 248 Recently, progresses have been made in more tumor types rather than AML and glioma. A small molecule inhibitor 18097 significantly restrained in vivo growth and lung colonization of breast cancer cells. 414 Oxetanyl class demonstrated antiproliferative effects in GC, glioblastoma and AML models, while FTO-43 has potency comparable to 5-FU. 415 Compound C6, a 1,2,3-triazole analogs, was suggested as a potential orally antitumor agent for esophageal cancer. 416 Exploitation of inhibitors against other regulators, including METTL3 and ALKBH5, is also proceeding steadily. The ALKBH5 inhibitor ALK-04 effectively sensitized melanoma cells to anti-PD-1 blockade, as ALKBH5 attenuated immunotherapy responses via regulating lactate content and immunosuppressive cell infiltration in the TME. 404 Selberg et al. discovred two compouds, 2-[1-hydroxy-2-oxo-2-phenylethyl]sulfanyl acetic acid and 4-[furan-2-yl]methyl amino-1,2-diazinane-3,6-dione, demonstrated cancer-cell-type-selective antiproliferative effects in selected leukemia cell lines. 417 Targeting SAM binding sites, adenosine was first identified as METTL3 inhibitors. Non-nucleoside inhibitors with higher selectivity and permeability have been developed, such as UZH1a and UZH2. 418 , 419 A novel METTL3 inhibitor STM2457 effectively blocked AML progression and prolonged survival in AML mouse models, without disturbing normal hematopoiesis. 420 Furthermore, RNA m1A methylation is also a potential therapeutic target. The m1A methyltransferase complex, TRMT6/TRMT61A is highly expressed in HCC and correlated with poor prognosis. Wang et al. screened out three potential drugs targeting the interaction of TRMT6 and TRMT61A, thimerosal, phenylmercuric acetate (PMA), and thiram. Among them, the administration of thiram significantly attenuated HCC growth in preclinical models. 421 Though RNA medicine has been facing challenges like efficacy and immunogenicity since from birth, the most recent hit of mRNA vaccines against COVID-19 provide new momentum to this field and bring RNA modifications back into to spotlight. Chemical modification of RNA could protect RNA from hydrolysis and nucleases, and decrease off-target cytotoxic effects. Once therapeutic RNAs form duplexes with targeted sequences, modifications lowering the melting temperature could destabilize the complex and improve target specificity via reducing base-paring with non-target RNAs. Moreover, RNA modifications are utilized for RNA delivery and strengthen the pharmaceutical activity of RNA. 422 Base modifications have been successfully applied in improving the performance of therapeutic RNA, for example, replacement of uridine with the modified base 1-methylpseudouridine (N1-Me) in COVID-19 vaccines (Pfizer’s Comirnaty and Moderna’s Spikevac) effectively facilitates translation and reduces off-target side effects and immunogenicity of therapeutic mRNA. 423 Additionally, m7G cap linked by a 5′-triphosphate to the 5′ end of the mRNA, which replicates the naturally occurring mRNA caps to prevent degradation of the 5′ end of mRNA, has been introduced into mRNA vaccines BNT162/Comirnaty and mRNA-1273/Spikevax. 424 As for ribose modifications, modified hydroxyl group on the C-2′ position of the ribose could protect RNA against nuclease digestion and lower the thermal stability of duplexes. N-acetylgalactosamine (GalNAc) groups or lipophilic moieties attached cleavable linkers, including ester-based, peptide-based cleavable groups, could localize therapeutic RNA to target tissue. 425 Moreover, modifications to the phosphate group in the sugar-phosphate backbone shelter RNAs from nucleases, represented by phosphorothioate. And eliminating the negative charge via replacing the oxygens on the phosphates with neutral groups or cations can assist the delivery into cell. 426 Based on CAS Content Collection, a recent study summarized the modification content in approved RNA medicines, including antisense oligonucleotide (ASO), siRNA, aptamer, and mRNA. 427 Thereinto, N1-Me is prominently abundant in two mRNA vaccines, along with 2′-O-methyl, 3′-methyl, m7G, 5′-5′-triphosphate. And 2′ -oxy-methoxyethylguanosine (2′-MOE) is exclusive in ASOs, which protects ASOs from degradation. The approved siRNAs have 2′-fluoro and 2′-O-methyl modification of the ribose, and three of them are 3′-glycosylated with the GalNAc conjugate, which specifically targets siRNAs to hepatocytes. 428 Some gratifying results have been acquired in combined application of m6A regulators inhibitors with current anticancer therapy. The involvement of m6A modification in underlying mechanisms of resistance has been systematically summarized. 429 Herein, we put emphasis on the combined utility of targeting RNA modification to circumvent resistance and improve individualized cancer treatment. A mass of evidences showed that overexpressed METTL3 widely participated in the acquisition of various therapeutic resistance in many cancer types. Knockdown of METTL3 using short hairpin RNA improved sensitivity to anticancer reagents such as gemcitabine, 5-fluorouracil, cisplatin and irradiation in pancreas cancer (PC). 430 Suppression of METTL3 restored chemosensitivity and attenuated CML cells viability. 431 Nevertheless, few studies have ever investigated the utility of METTL3 inhibitors in overcoming chemoresistance. Targeting FTO also shed new light on improving chemoresistance. Upregulated FTO in oral squamous cell carcinoma played a pivotal part in arecoline-induced stemness and chemoresistance to cisplatin. 432 Depletion of FTO sensitized breast cancer to doxorubicin via suppressing de novo synthesis of fatty acid. 433 Specifically, FTO was revealed to facilitate GBM resistance to temozolomide (TMZ), and the inhibitor R-2HG demonstrated a synergistic effect with TMZ in suppressing proliferation of FTO-high glioma cells. 412 Moreover, the feasibility of administrating m6A regulators inhibitors to improve immunotherapy effectiveness needs further investigation. Depletion of METTL3/14 was found to augment ICB therapeutic responses in mismatch-repair-proficient or microsatellite instability-low (pMMR-MSI-L) CRC and melanoma. 434 In accordance, a recent study confirmed that targeting METTL3 by inhibitor STM2457 potentiate ICB efficacy in various CRC mouse models. 435 Knockdown of FTO reduced PD1 expression in melanoma via m6A/YTHDF2-dependent manner, thus sensitized anti-PD-1 blockade. 400 FTO was supposed to enhanced PD-L1 expression independent on IFN-γ in CRC. 436 In AML, FTO inhibition induced by small-molecule compounds CS1/2 leads to downregulation of checkpoint gene LILRB4, reigniting the interest of introducing ICB to AML. 402 Besides, Li et al. reported that the specific inhibitor to ALKBH5, ALK-04 markedly enhanced the efficacy of anti-PD-1 blockade in CRC model. 404 We surveyed ClinicalTrials.gov as of October 20, 2023, to keep up-to-date with clinical implications of RNA modifications, basically including therapeutic effectiveness of agents targeting modifiers, potential as predictive biomarkers, and combined application with current treatment. However, hardly any above-mentioned specific inhibitor has progressed into clinical stage, in spite of the encouraging antitumor results of FTO-targeted agents in various cancers. Most recently, a phase 1, first-in-human study is designed to systematically evaluate the pharmacokinetics, pharmacodynamics and clinical activity of STC-15 in adult subjects with advanced malignancies ( NCT05584111 ). Several studies have evaluated the association between FTO polymorphisms (rs9939609 and rs1558902) and obesity in different populations, including Turkish population ( NCT04205318 ), Indonesian obesity women ( NCT04740528 ), as well as weight loss in overweight carriers induced by calorie restriction ( NCT02940197 ), and intermittent or moderate continuous high intensity training programs ( NCT03568773 ). Furthermore, there are projects aim at assess the correlation between FTO polymorphisms and risk of developing diabetes in Mexican adolescents with overweight and obesity ( NCT02886013 ), and features of metabolic syndrome in children with T1D ( NCT01279161 ). Considering that variants in FTO showed high correlation with body weight and also interact with dopamine signaling in the brain, a clinical trial was designed to develop a genotype-specific and individualized therapy approach for obesity targeting FTO (rs8050136) variant ( NCT03525002 ). Genotyping for FTO was also incorporated into tailored therapeutic model for azathioprine-induced myelosuppression in inflammatory bowel disease patients ( NCT03719118 ).

Modified nucleosides in RNA, beyond the canonical A, U, C and G, have been recognized for more than half a century. Figure 1 illustrates the historical milestones of RNA modifications research. Pseudouridine (Ψ) is the first RNA modification type to be identified in 1950s. 17 In 1965, sequencing of the first biological RNA, alanine tRNA derived from yeast, confirmed 10 modification types. 18 Due to technological advancement, over 170 RNA modifications have been discovered, ubiquitously existing in various coding and non-coding RNAs. However, it was not until last decade that the functional significance of RNA modifications gradually got recognized, prominently the widespread prevalence and biological functions of N6-methyladenosine (m6A). 19 Following 5′ cap and 3′ poly(A) tail of messenger RNA (mRNA), internal modifications on mRNAs were identified, represented by the most common methylation m6A. 20 , 21 These modifications were observed to exert significant roles in every link of mRNA fate, including pre-mRNA splicing, nuclear export, translation, stabilization and degradation. Transfer RNA (tRNA) modification is renowned for the largest number, with an average of 13 modifications per molecule. 22 Their biological roles could be generalized in two aspects, which are maintaining the tertiary structure and facilitating codon–anticodon recognition. 23 For ribosomal RNA (rRNA), RNA modifications are especially indispensable, as rRNA biogenesis is interrupted without pseudouridines and 2′- O -methyls. Modifications of long noncoding RNA (lncRNA) are mainly methyl nucleotide derivatives, including m6A and m5C. 24 Though far from being elucidated, lncRNAs modifications have been revealed to influence the stability, protein interactions, and subcellular distribution of lncRNAs. 25 Human small nuclear RNA (snRNA) contains 2′- O -methyls, pseudouridines, and base methylations, participating in RNA splicing reaction. At present, mainstream RNA-seq methods are incapable for comprehensive and quantitative mapping of modifications on small non-coding RNAs. Here we summarize the current knowledge of RNA modifications, focusing on the regulatory mechanisms and biological consequences of several well-learned types. Fig. 1 The milestone events in RNA modification field. The first RNA modification type Pseudouridine (Ψ) was discovered in 1951. Since then, other RNA modification, including m6A, m1A, m6Am, etc were discovered. Along with the accumulation of epi-transcriptomic knowledge, comprehensive databases like RNAMDB and MODOMICS were incepted. Since Liquid Chromatograph-Mass Spectrometer (LC-MS) technique was utilized for the quantitative analysis of modified ribonucleosides in 2015, more specific high-throughput mapping methods gradually emerged. Recent years have witnessed the application of single-cell sequencing technologies in mapping RNA modification. The figure is generated with BioRender ( https://biorender.com ) The milestone events in RNA modification field. The first RNA modification type Pseudouridine (Ψ) was discovered in 1951. Since then, other RNA modification, including m6A, m1A, m6Am, etc were discovered. Along with the accumulation of epi-transcriptomic knowledge, comprehensive databases like RNAMDB and MODOMICS were incepted. Since Liquid Chromatograph-Mass Spectrometer (LC-MS) technique was utilized for the quantitative analysis of modified ribonucleosides in 2015, more specific high-throughput mapping methods gradually emerged. Recent years have witnessed the application of single-cell sequencing technologies in mapping RNA modification. The figure is generated with BioRender ( https://biorender.com ) To build a general intuition of RNA modifications, we first sketch the distinctions between reversible and non-reversible modifications. Reversible types are usually smaller-scale modifications on chemical side chains, spanning from simple methylation to some appendages of large-molecular mass. These plastic and reversible RNA modifications extensively exist in gene regulation and cellular states. The extensive catalog of nonreversible RNA modifications includes RNA editing, splicing, and transcript-content modification (such as intron retention). Contrary to the reversible type, these modifications directly alter the sequence information, magnifying plasticity and diversity of transcriptome. The chemical structure and distribution of eight RNA modifications were showed in Fig. 2 and Table 1 . Fig. 2 The chemical structure, distribution, and molecular functions of eight RNA modifications. Chemical modification occurs on many types of RNA and modulate every links of RNA metabolism. m6A N6-methyladenosine, m6Am N6,2′-O-dimethyladenosine, m5C 5-methylcytosine, m1A N1-methyladenosine, m7G 7-methylguanosine, ac4C N4-acetylcytidine, ψ pseudouridine, A-to-I editing adenosine-to-inosine RNA editing, CDS coding sequence, UTR untranslated regions, pri-miRNA primary microRNA, pre-miRNA precursor microRNA. The figure is generated with BioRender ( https://biorender.com ) Table 1 The general characteristics of main RNA modification types Type Contribution Class Regulator Function Ref. m6A mRNA, rRNA, snRNA, snoRNA, miRNA, lncRNA, circRNA, eRNA writer METTL3 Catalyzes most of the m6A modifications via forming methyltransferase complex (MTC) with METTL14 34 METTL14 Provides structural support in MTC 35 METTL16 Catalyzes m6A in U6 snRNA 40 eraser FTO Removes m6A/m6Am/m1A modifications 44 , 45 ALKBH5 Demethylates m6A modification exclusively 50 reader YTHDF1 Stabilizes transcripts and initiates translation 56 , 57 YTHDF2 Promotes degradation 56 , 57 YTHDF3 Facilitates translation and degradation 56 , 57 YTHDC1 Mediates RNA splicing, nuclear export and degradation 54 YTHDC2 Promotes translation efficacy and decay 55 IGF2BP1/2/3 Stabilizes transcripts and facilitates translation 58 , 59 HNRNPs Mediate splicing of pre-mRNAs and/or pri-miRNAs 60 m6Am mRNA, snRNA writer PCIF1 Catalyzes m6Am next to the 5′ cap of mRNAs and in snRNAs 77 METTL4 Catalyzes m6Am at position 30 in human U2 snRNA 80 eraser FTO Removes m6A/m6Am/m1A modifications 49 m1A tRNA, rRNA, lncRNA, and mRNA writer TRMT61B Catalyzes mA at positions 58 (m1A58) 92 TRMT10C Catalyzes mA at positions 9 (m1A9) 92 , 93 TRMT6/61A Catalyzes m1A in tRNA at A58 and mRNA 94 TRMT61B Mediates m1A in mitochondrial 16S rRNA 95 eraser ALKBH1 Catalyzes demethylation of most m1A in cyto-tRNAs 97 ALKBH3 Demethylates m1A in both tRNAs and mRNAs 98 , 99 ALKBH7 Demethylates m1A within mitochondrial Leu1 pre-tRNA regions 100 FTO Demethylates m1A in tRNA 49 reader YTHDF1/2/3 YTHDC1 Mediates stabilization, degradation, splicing, translation 101 , 102 m5C writer NSUN1 Catalyzes m5C at position 4413 of 28S rRNA 114 NSUN5 Catalyzes m5C at position 3761 of 28S rRNA 115 NSUN2 Methylates C34, C40, C48, C49, and C50 in several tRNAs 116 NSUN6 Methylates C72 in particular tRNAs 117 DNMT2 Methylates C38 in particular tRNAs 118 NSUN3 Catalyzes m5C in mitochondrial tRNA 119 NSUN4 Catalyzes m5C in 12S rRNA 120 eraser ALKBH1 Demethylates m5C at position 34 of cytoplasmic and mitochondrial tRNA 126 , 127 TET1/2/3 Catalyzes first step of m5C demethylation 128 reader ALYREF Promotes the nuclear export of m5C-modified rRNAs 129 YTHDF2 Modulates the maturation of m5C-modified rRNAs 130 ac4C mRNA, tRNA, rRNA writer NAT10 THUMPD1 and snoRNP are necessary assistants for modifying tRNA and 18S rRNA, respectively 141 , 140 , 141 m7G mRNA, tRNA, rRNA, miRNA writer METTL1 Forms complex with WDR4 to catalyze m7G on tRNA, miRNA, and mRNA 147 RNMT Catalyzes m7G on recapped mRNAs, cooperated with RAM 149 WBSCR22 methylate G1639 in human 18S rRNA, cooperated with TRMT112 150 TGS1 Catalyzes hypermethylation of m7G caps into m2,2,7G in snRNAs and snoRNAs 151 reader eIF4E, CBC recognizes m7G cap and further affect RNA maturation, nuclear export, and translation 152 Ψ writer DKC1 Forms a complex with box H/ACA snRNA to pseudouridylates rRNA 160 A-to-I Pre-mRNA, pri-miRNA writer ADAR1-3 ADAR1 and ADAR2 could catalyze all known A-to-I editing events, while ADAR3 has no deaminase activity 186 The chemical structure, distribution, and molecular functions of eight RNA modifications. Chemical modification occurs on many types of RNA and modulate every links of RNA metabolism. m6A N6-methyladenosine, m6Am N6,2′-O-dimethyladenosine, m5C 5-methylcytosine, m1A N1-methyladenosine, m7G 7-methylguanosine, ac4C N4-acetylcytidine, ψ pseudouridine, A-to-I editing adenosine-to-inosine RNA editing, CDS coding sequence, UTR untranslated regions, pri-miRNA primary microRNA, pre-miRNA precursor microRNA. The figure is generated with BioRender ( https://biorender.com ) The general characteristics of main RNA modification types m6A modification refers to the methylation of the adenosine base at the N-6 position. m6A targeted at consensus sequences DRACH (D = G, A, or U; R = G or A; H = A, C, or U), which are mainly enriched in CDS and 3’UTR region of mRNA, 26 as well as most non-coding RNAs, including rRNAs, lncRNAs, circular RNAs (circRNAs), microRNAs (miRNAs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs). 27 Growing studies have confirmed that m6A could exert significant impacts on various biological processes in mammals, including DNA damage response, cell cycle, circadian rhythm, heat shock response, meiotic progression, development of hematopoietic, central nervous and reproductive systems, myogenesis, and fat differentiation. 28 – 33 m6A deposition in mRNA is dependent on methyltransferase complex (MTC), of which the methyltransferase-like 3/14 (METTL3/14) heterodimer is the key component. 34 Therein, METTL3 exerts catalytic role via transferring methyl group of S-adenosyl methionine (SAM) and METTL14 provides structural support. In METTL3, two methyltransferase domains (MTD) bind to methyl donors, CCH-type zinc finger domain (ZFD) recognizes targets, while nuclear localization signal (NLS) domain and leading helix structure (LH) domain coordinately mediate the interaction between METTL3 and METTL4. 35 , 36 There are several auxiliary subunits for localizing and initiating methylation, including Wilms’ tumor 1-associating protein (WTAP), RNA-binding motif protein 15/15B (RBM15/15B), zinc finger CCCH-type containing 13 (ZC3H13) and vir-like m 6 A methyltransferase-associated (VIRMA, also known as KIAA1429). 37 – 39 METTL16 is responsible for m6A formation in U6 snRNA, targeting a conserved UACAGAGAA sequence. 40 METTL16 also participates in maintaining homeostasis of SAM in a m6A-dependent manner. 41 Zinc Finger CCHC-Type Containing 4 (ZCCHC4) and METTL5 mediate m6A modification of 28 S and 18 S rRNA at A4220 and A1832 region, respectively. 42 , 43 Both m6A demethylases, Fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5), belong to AlkB family of the Fe (II)/α-ketoglutarate-dependent dioxygenase superfamily. FTO is the first discovered m6A eraser for mRNA and snRNA, which also mediates demethylation of N6,2′-O-dimethyladenosine (m6Am) and N1-methyladenosine (m1A). 44 , 45 Which one of m6A and m6Am is the principal substrate of FTO remains controversial. It was reported that FTO catalyzed m6A demethylation at a concentration at least twice that of m6Am. 46 , 47 But Zhang et al. proposed that FTO equivalently demethylated m6A and m6Am deposited on the same RNA sequence. 48 Significantly, Wei et al. discovered that nuclear FTO showed an affinity bias towards m6A, which tends to be inconspicuous in cytoplasm, due to altered abundance of m6A. 49 Whereas, ALKBH5 exclusively catalyzes m6A demethylation in mRNA. 50 The most studied readers are the YT521-B homology (YTH) domain family members, including YTHDF1/2/3 and YTHDC1/2, most of which localize to cytoplasm except for YTHDC1 in nucleus. 51 , 52 The prevailing idea is that YTHDFs bind to different m6A-modified RNAs, but Zaccara et al. hold that all m6A-modified RNAs are subjected to YTHDFs and they act redundantly in mediating RNA degradation. 53 YTHDC1 contributes to RNA splicing, nuclear export and degradation, while YTHDC2 promotes translation efficacy and decay. 54 , 55 YTHDF1 could stabilize transcripts and initiate translation via interacting with eIF3, YTHDF3 not only facilitates translation but works in synergism with YTHDF2 in inducing mRNA degradation. 56 , 57 The insulin-like growth factor 2 mRNA-binding protein family, IGF2BP1/2/3, is another group of readers. IGF2BPs possess 4 repetitive KH domains and bind to m6A sites with KH3/4 to stabilize transcripts and facilitate translation. 58 , 59 The heterogeneous nuclear ribonucleoprotein (HNRNP) family includes HNRNPC, HNRNPG, and HNRNPA2B1. HNRNPs can mediate splicing of precursor (pre)-mRNAs and/or primary (pri)-miRNAs through ‘the m6A-switch’ mechanism, in which m6A alters the local structure of mRNA or lncRNA to facilitate the binding of HNRNPs. 60 HNRNPA2B1 directly binds to pri-miRNAs to mediate alternative splicing. Meanwhile, its interaction with the miRNA microprocessor complex protein DGCR8 promoted primary miRNA processing. 61 And HNRNPG could elicit co-transcriptional m6A-dependent alternative splicing regulation via directly binding to RNA polymerase II (RNAPII). 62 Besides, proline rich coiled-coil 2 A (PRRC2A) and Staphylococcal nuclease and tudor domain-containing 1 (SND1) could serve as readers to stabilize m6A-modified RNAs. 63 , 64 To sum up, m6A modification extensively influences fate of different RNA classes, consequently regulates various cellular processes. In mRNAs, m6A methylation can affect splicing, exportation, stabilization, degradation, and translation. 65 In rRNAs, the A1832 methylation in 18 S rRNA and A4220 methylation in 28 S rRNA are essential for translation. 42 , 43 In miRNAs, m6A could facilitate pri-miRNA processing via recruiting DGCR8, 61 or downregulate several miRNAs via some exclusive mechanism. 66 In lncRNAs, m6A modification could serve as a structural switch to regulate RNA-protein interactions, 67 or stabilize lncRNAs to ensure its function. 68 In cirRNAs, m6A could facilitate cytoplasmic export, 69 translation 70 and degradation. 71 Moreover, m6A participates in modulating splicing and biogenesis of snRNA. 72 Although m6A methylation has been widely investigated, the underlying rationales are far from clarified. For example, m6A modification could modulate RNA life via diverse mechanisms, but how these selective effects are determined in different cellular contexts remains unclear. While previous studies notably focus in mRNAs, the interplay between m6A and non-coding RNAs deserves more attention. The same is true for m6A readers, which are unheeded compared to writers and erasers. And the significance of methodology development cannot be stressed enough, as bona fide m6A mapping and elaborate edition on specific m6A sites will provide a wide scope for future researches. m6Am is produced at a 2′-O-methylated adenosine which is methylated co-transcriptionally at the N6 position. It is discovered in the first position adjacent to the 5′ cap structure in many mRNAs and snRNAs in mammals, and also found as internal modification in the snRNA U2. 73 According to quantification studies, m6Am content ranges from 10% to almost 50% in mRNAs of different organisms and cell types. 74 Previous studies have shown that m6Am installed by host PCIF1 on viral RNA mediated immune evasion, while host m6Am exhibited both anti-viral and pro-viral roles. 75 , 76 The enzyme catalyzes m6Am next to the 5′ cap of mRNAs and in snRNAs is “phosphorylated CTD-interacting factor 1” (PCIF1), also known as “cap-specific adenosine methyltransferase” (CAPAM). 77 The core region of PCIF1 contains the methyltransferase domain and helical domain that functions as the RNA-binding surface, 78 and a specific site (m7Gsite) located between the two domains mediated the specific recognition of the m7G cap. 79 It was revealed that knockout of PCIF1 altered cell proliferation under oxidative stress conditions in human HEK293T cell line. 79 Another m6Am writer, METTL4 methylated the internal 2′-O methylated adenine, at position 30 in human U2 snRNA. 80 METTL4 contains a C-terminal domain that is similar to METTL3, a middle domain (MID) and a N-terminal domain (NTD), which enables METTL4 works as a monomer with no need for METTL14. 81 It was indicated that METTL4 was highly conserved and exclusive for U2 snRNA. 82 However, overexpressed METTL4 tends to modify A instead of Am in mRNAs with consensus HMAGKD (H = A/C/U, M = A/C, K = G/U, D = A/G/U). 83 Also, METTL4 was found to catalyze mt-DNA m6A in human cell line. 84 Ablation of METTL4 did not influence viability of HEK293T cell line, but altered adipocyte differentiation of mouse 3T3-L1 cells. 83 , 85 To date, FTO is the only known demethylase for m6Am, which, as mentioned above, show a substrate preference between m6A and m6Am depending on its cellular localization. 49 In cytoplasm, FTO preferentially demethylates cap-adjacent m6Am and internal m6A on mRNAs, while nuclear FTO acts on m6Am in RNA Pol II-transcribed snRNAs, and internal m6Am and m6A in the snRNAs U2 and U6. 49 Studies have identified that FTO distribution was correlated with cell cycle phase and regulated by casein kinase II-mediated phosphorylation. 86 To be mentioned, structural analysis demonstrated that the catalytic activity of FTO was mediated by recognizing N6-methyl of adenine rather than the 2′- O methyl group of the ribose. 48 There are discrepancies exist in present studies on influences of m6Am modification on gene expression, as an inherited issue from the past immature m6Am mapping methodologies. For instance, m6Am methylation was initially suggested to play a positive role in mRNA stability in a cell-type-specific manner. 45 However, a recent study, developing the specific sequencing method m6Am-seq, has clarified that PCIF1 was not required for stabilization of m6Am-modified mRNAs. 77 There are other studies implicated that m6Am did not have direct effects on mRNA stability. 77 , 79 As for translation, the current cognition is that m6Am modifications in mRNA cap exert a cell-specific influence on translation. 79 , 87 And such effects are dependent on 2′-O-methylation modification in the second nucleotide of the cap-structure. 88 Moreover, the effect of m6Am modification in splicing need more verification. It was suggested that METTL4 had no direct influence on U2 snRNA expression levels but rather altered splicing regulation. 80 , 82 To sum up, the cap-adjacent location endows m6Am modification with potential to regulate stability and translation. The significant discovery of PCIF1, specifically catalyzes m6Am in the cap structure, drives relevant exploration. However, methodological deficiency is the major problems in m6Am researches. Most of previous studies adopted m6A mapping protocols instead of specific m6Am mapping methods, which led to poor reproducibility and controversial results. Thus, more specific and efficient methods are in urgent need to clarify the regulatory roles of m6Am modification in gene expression. m1A, the methylation of adenosine at position N1 identified in 1960s, has been found in tRNA, rRNA, lncRNA, and mRNA, among which tRNA is the most heavy-modified class. 89 , 90 Particularly, m1A can transfer to m6A after “Dimroth rearrangement” under alkaline conditions and they also share some regulators. 49 m1A has been identified and enriched in specific regions of viral RNA, but its influences in innate immunity is not yet clear. 91 In mitochondrial tRNA, m1A methylation is catalyzed by tRNA methyltransferase (TRMT61B) and TRMT10C at positions 58 (m1A58) and 9 (m1A9), respectively. 92 , 93 TRMT61A and TRMT6 form a heterotetrameric complex to methylate both cytoplasmic tRNA at A58 and mRNAs with GUUCRA tRNA-like motifs, as TRMT61A functions as the catalytic subunit. 94 TRMT61B mediates m1A in mitochondrial 16 S rRNA, 95 and nucleomethylin (NML, also known as RRP8) methylates 28 S rRNA in nuclei. 96 And no specific m1A writer for mRNA has been reported yet. ALKBH1 catalyzes demethylation of most m1A in cyto-tRNAs, while m1A58 is the major substrate. 97 ALKBH3 demethylates m1A in both tRNAs and mRNAs. 98 , 99 ALKBH7 can demethylate m1A within mitochondrial Leu1 pre-tRNA regions in the nascent polycistronic mitochondrial RNAs. 100 And FTO was also proved to demethylated m1A in tRNA. 49 YTHDF1/2/3 and YTHDC1 have been confirmed to directly bind to m1A marks, with weaker affinity than that of m6A. 101 , 102 The evolutionarily conversed YTH domain was suggested to be the key to methyl recognition, but the mechanistic research remains deficient. 102 The methyl group of m1A carries a positive electrostatic charge, which affects RNA base pairing, and subsequently influences molecule structure and function of modified RNAs. Notably, the electro-chemical interaction of m1A is supposed to play roles in maintaining or stabilizing the T -loop-like structure, and further strengthening the structure. 103 As for translation, m1A modification has effects on initiation or elongation process via regulating tRNA, mRNA and rRNA. Several studies have indicated that m1A on either tRNA or mt-tRNA could facilitate translation. 97 , 104 Whereas, m1A modification on mRNA plays diverse roles in protein synthesis, as m1A in 5’UTR correlates with enhanced translation initiation and efficiency, 105 but m1A in the CDS exerts inhibitory effects. 92 , 106 In rRNA, m1A is likely associated with translation initiation, as loss of yeast RRP8-catalyzed m1A led to incompetent formation of the 80 S initiation complex. 107 Moreover, m1A modification participates in the structural thermostability of tRNAs 108 and the nascent polycistronic mt-RNA processing. 100 As one of the most abundant internal RNA modifications, the machinery and biological functions of m1A remain largely unknown. The roles of YTH domain-containing proteins as m1A readers may provide novel scientific prospects. And whether its impact on RNA base pairing influences RNA interaction, such as miRNA with mRNA, lncRNA, and circRNA, requires more exploration. For decades, methylation of cytosine residues at the position 5 in DNA have been quite familiar. Ever since it was identified in RNA in 1958, m5C has been revealed to distribute widely in RNAs, including tRNA, rRNA, mRNA, enhancer RNA (eRNA), and miRNA. 109 , 110 Studies figured out that m5C modification extensively occurred on maternal mRNA in zygotes of different eukaryotic species, regulating embryogenesis in mouse, zebrafish and Drosophila. 111 – 113 In eukaryotes, m5C modification is catalyzed by members of the NOL1/NOP2/SUN domain (NSUN) family of proteins, NSUN1-7 and DNA methyltransferase (DNMT) homolog DNMT2. For rRNA, NSUN1 and NSUN5 introduce m5C at position 4413 and 3761 of human 28 S rRNA, while their homologs in yeast methylate 25S-C2870/25S-C2278. 114 , 115 For tRNA, NSUN2 could modify several sites in various tRNAs, including C34, C40, C48, C49, and C50. 116 NSUN6 and DNMT2 methylate C72 and C38 in particular tRNAs, respectively. 117 , 118 NSUN3 and NSUN4 are responsible for methylation of mitochondrial tRNA and 12 S rRNA. 119 , 120 And NSUN4 forms a complex with the mitochondrial transcription factor MTERF4 for lack of RNA recognition motif. 121 The m5C methyltransferase specific for mRNAs has not been confirmed yet, but NSUN2 was described to target mRNAs in several studies. 122 , 123 Besides, m5C modifications of ncRNA and eRNA are modified by NSUN2 and NSUN7, respectively. 124 , 125 The identified m5C erasers include ten-eleven translocation (TET) proteins (TET1–3) and ALKBH1. ALKBH1 can successively catalyze m5C into 5-hydroxymethylcytidine (hm5C), 5-formylcytosine (f5C), and 5-carboxylcytosine, at position 34 of cytoplasmic and mitochondrial tRNA, 126 , 127 whereas TETs has been only reported to complete the first step for RNA m5C. 128 Aly/REF Export Factor (ALYREF) is the first identified m5C reader in mRNA, a well-known complex that promotes the nuclear export. 129 Y-box-binding protein 1 (YBX1) is located in cytoplasm and could recruit stability maintainer ELAV like RNA binding protein 1 (ELAVL1) to stabilize m5C-modified mRNAs. 112 Also, YTHDF2 has been reported to modulate the maturation of m5C-modified rRNAs. 130 Collectively, m5C modification plays a crucial role in RNA stabilization, exportation, and translation. m5C at C2278 of 25 S rRNA stabilizes the structural conformation of the ribosome. 115 Hypermethylated mRNAs with m5C are stabilized via YBX1-dependent manner. 131 NSUN2-mediated m5C modifications in vault RNA are significant for its processing into derived small RNAs and protect eRNAs from degradation. 132 Also, NSUN2 modified cyclin-dependent kinase inhibitor 1 A (CDKN1A) mRNA and promoted its nuclear export and translation. 133 As mentioned above, the dizzying matchup between m5C modifiers and their specific targets brings out challenges as well as opportunities. Targeting certain writers or manipulating specific modification sites reserve great therapeutic potential. ac4C, acetylation of the N4 position of cytosine, is the first acetylation event described. As initially found in tRNA and rRNA, ac4C was also confirmed to be widely present on mRNAs. 134 In tRNA, ac4C is located at the wobble of tRNA Met and the D-arm of tRNA Ser/Leu . 135 In eukaryotic 18 S rRNA, ac4C is deposited in helix 34 and helix 45 near the decoding site. 136 In mRNA, ac4C is detected in the CDS region and 5ʹUTR, enriched in the third codon encoding amino acid. 134 Advances in the study of RNA ac4C modification in cell cycle, inflammatory stress, tumors, premature diseases and viral infection have been reported. 91 , 137 , 138 Currently, N-acetyltransferase 10 (NAT10) is the only identified ac4C writer, with acetyl-CoA providing acetyl and ATP/GTP hydrolysis supplying energy. 139 When modifying tRNA, the assistance of THUMP domain containing 1 (THUMPD1) is necessary, 140 while box C/D snoRNPs act as antisense to guide 18 S rRNA acetylation. 141 For now, no ac4C eraser has been identified and it remains unknown whether ac4C modification is reversible. The presence of ac4C on tRNA helps maintain the thermal stability of tRNA and a high heat tolerance of cells, and improves fidelity and efficiency of translation. 134 , 142 ac4C on mRNA CDS region significantly enhance mRNA stability and facilitate translation, probably by preserving codon-anticodon interaction. 143 However, ac4C on 5ʹUTR mainly regulates translation initiation in a location-specific manner, as ac4C downstream a weak translation initiation site could promote translation, but the one adjacent to a strong AUG start codon disturbs translation. 144 In 18 S rRNA, ac4C modification is crucial for maintaining translation accuracy, pre-rRNA processing and ribosome synthesis. 140 The cognition of ac4C modifiers and molecular functions remains largely unknown. Since cofactors of NAT10 have been identified during ac4C formation in human rRNA or tRNA, whether novel cofactors exist in catalyzing mRNA ac4C is noteworthy. Particularly, no erasers or readers has been found yet, whether a deacetylation mechanism exist require more validation. m7G, referring to the RNA methylation of guanine at position N7, was first found at the 5′ cap (m7GPPPN) of mRNA, stabilizing transcripts and further mediating cap-related biological functions. 145 Until now, m7G has been discovered at internal position within mRNA, tRNA, and rRNA, 146 , 147 and tRNA nucleotide position 46 (m7G46) in the variable loop region is the most prevalent m7G methylation site. 148 The most well-characterized m7G writer is METTL1, which forms a functional complex with WD repeat domain 4 (WDR4) to install m7G on tRNA, miRNA, and mRNA. 147 RNA guanine-7 methyltransferase (RNMT) is responsible for m7G on recapped mRNAs, cooperated with RNMT-activating mini-protein (RAM). 149 Williams–Beuren syndrome chromosome region 22 (WBSCR22) methylate G1639 in human 18 S rRNA, requiring tRNA methyltransferase activator subunit 112 (TRMT112). 150 Trimethylguanosine synthase 1 (TGS1) might also function as a modifier, catalyzing hypermethylation of m7G caps into m2,2,7 G in snRNAs and snoRNAs. 151 The eukaryotic translation initiation factor eIF4E and the cap-binding complex (CBC) can recognize m7G cap and further affect RNA maturation, nuclear export, and translation. 152 Notably, m7G modification is extensively involved in various biological processes. For mRNA, the m7G cap could regulate pre-mRNA slicing, nuclear export, translation, 152 and indirectly enhance translational capacity by driving ribosome biogenesis. 153 And internal m7G also influences translation. 154 For tRNA, METTL1/WDR4-mediated m7G methylome plays pivotal roles in maintaining tRNA structural integrity, thereby facilitating translation and reducing ribosome pausing. 155 For rRNA, m7G modification participates in 18 S rRNA precursor biogenesis and nuclear export of the 40 S rRNA. 150 , 156 Moreover, m7G on G-quadruplex structures in pri-miRNA could promote miRNA processing. 157 At present, our understanding of m7G regulators is apparently limited. No specific demethylase has been identified to regulate the global balance of m7G. And whether m7G modification regulating gene expression via affecting the secondary structure of RNA or recruiting RNA binding proteins remains unclear. Furthermore, the interplay among m7G and other post-transcription attracts growing attention, more explorations are imperative to unravel the underlying mechanism. Ψ, the 5–riboside isomer of uridine, is the first discovered and most abundant RNA modification. 17 , 158 The C5 atom, instead of N1, forms a new carbon-carbon bond (C5–C1′) with pentose at its non-Watson-Crick edge, endowing Ψ with unique chemical properties. Ψ is present in a wide range of RNAs, including tRNA, rRNA, and various snRNAs, which is highly conserved among species. 158 , 159 The widespread distribution determines its importance in regulating gene expression, steering cellular programs both in development and disease. The pseudouridylation is mainly catalyzed by pseudouridine synthases (PUSs), via RNA-dependent or -independent manner. The RNA-dependent mechanism involves Dyskerin pseudouridine synthase 1 (DKC1), which forms a complex with box H/ACA snRNA to pseudouridylates rRNA. 160 The RNA-independent PUSs includes PUS1, PUSL1, PUS3, TRUB1, TRUB2, PUS7, PUS7L, RPUSD1–4, and PUS10. 161 – 163 Regrettably, no Ψ eraser or reader has been documented. And it was speculated that C5–C1′ bond render pseudouridylation irreversible. 164 Ψ on tRNA is critical for stabilizing tRNA structure and tRNA codon–anticodon base pairing, further affecting translation processes. Also, Ψ-modified tRNA-derived fragments could restrain aberrant protein synthesis. 165 Besides, Ψ is also involved in pre-mRNA processing, structure and stability of mRNA, translational fidelity and termination. 166 , 167 The rRNA Ψ plays a functional role in rRNA processing and protein synthesis. 168 It was demonstrated that hypo-pseudouridylated rRNAs decreased affinity for tRNA of ribosomes, impairing translational fidelity. 169 snRNP Ψ participates in its biogenesis and splicing. 170 Ψ35 in the 5′ end of the U2 snRNA was considered as necessary for early spliceosome formation. 171 Although discovered 70 years ago, there are still plenty of vacancies in knowledge on the mechanisms and functions of Ψ. Elucidating whether pseudouridylation is reversible will be one of the key directions in the future. Since efforts to approach inducible pseudouridylation have generated exciting results, which open up new avenues for exploring potential therapeutics. Remarkably, Ψ has already been validated to make critical contribution to COVID-19 mRNA vaccines. 172 RNA editing modifies primary mRNA and miRNA in posttranscriptional level, altering coding information of DNA. It was first discovered in trypanosome mitochondrial mRNA in 1986. 173 So far, RNA editing has been found in tRNA, rRNA and miRNA. 174 – 176 The most prevalent type is conversion of adenosine into inosine (A-to-I editing), 177 and then inosine is recognized as guanine by the translational machinery. It has been implicated that ADAR1-mediated A-to-I editing was involved in stem cell pluripotency and maintenance, neurological development and function, and immune response. 178 , 179 A-to-I editing only occurs in the double-stranded regions of RNAs made from inverted Alu repetitive elements (Alu dsRNAs), and is far less frequent in coding sequences than noncoding sequences such as UTRs and introns. 180 Precursors of certain miRNAs are also common targets. 181 The editing levels dramatically vary in cell and tissue type of different origins and development stages, ranging from 2%-100%. 182 , 183 The conversion is catalyzed by adenosine deaminase acting on RNA (ADAR) protein. 184 In vertebrates, the isoforms of ADAR protein, ADAR1-3 have identified. These ADAR enzymes possess a C-terminal conserved catalytic deaminase domain, and double-stranded RNA binding domain (dsRBD) at the N-terminus, three for ADAR1 and two for ADAR2-3. 185 Functionally, ADAR1 and ADAR2 are responsible for all known A-to-I editing events, while ADAR3 has no documented deaminase activity. 186 The mechanism of ADAR substrate specificity remains unclear, in which length and structure of dsRNA was suggested to play an important role, 187 and editor modulators like snoRNAs also participated in. 188 The consequences of A-to-I editing in coding sequences includes alternative splicing, nonsynonymous amino acid substitutions, nuclear retention and degradation of mRNA. Also, these editing could regulate gene expression via influencing splicing enhancers/silencers recognition sites of ncRNAs in non-coding sequence. 189 For several miRNAs, A-to-I editing negatively affects the expression and function of the mature miRNAs. 181 In opposition, ADAR1 could facilitate miRNA processing and RNA interference (RNAi) efficacy via forming a complex with Dicer. 190 Recently, RNA editing, represented by A-to-I editing, has emerged as a powerful tool to correct pathogenetic mutations, modulate gene expression and protein function. And its transient pharmacodynamic effects could be applied in treatment of several diseases like viral infection, obesity, inflammation, and acute pain. In addition, the transient modulation of protein functions opens up new avenues for oncology and regenerative drugs. To our knowledge, there have been 15 databases established for RNA modifications, two of which are concentrated on biochemical features of RNA modifications, and the rest aimed at elucidating the biological roles. The latter part includes reversible RNA modification database, which can be further classified as comprehensive and type-specific, and nonreversible RNA modification database, namely RNA editing database (Table 2 ). Table 2 Databases of RNA modifications Name Description URL Biochemical RNA modification database RNAMDB A databse of basic chemical characterizations of 109 RNA modified nucleosides https://mods.rna.albany.edu/ MODOMICS the most comprehensive RNA modification pathway source http://modomics.genesilico.pl Comprehensive reversible RNA modification databases . m6A-Atlas a comprehensive knowledgebase for unraveling the m6A epitranscriptome www.xjtlu.edu.cn/biologicalsciences/atlas m7GHub v2.0 a resource deciphering the location, regulation, and pathogenesis of internal mRNA m7G epitranscriptome www.xjtlu.edu.cn/biologicalsciences/m7ghub m5C-Atlas a database for decoding and annotating the m5C epitranscriptome https://www.xjtlu.edu.cn/biologicalsciences/m5c-atlas MeT-DB v2.0 a database for investigation of m6A and its previous version is the first comprehensive resource for m6A in transcriptome http://compgenomics.utsa.edu/MeTDB/ RMBase v2.0 a database deciphering the map of RNA modification from epitanscriptome sequencing data http://rna.sysu.edu.cn/rmbase/ REPIC an atlas of m6A methylome with cell lines or tissue specificity https://repicmod.uchicago.edu/repic Specialized reversible RNA modification database CVm6A a visualization and exploration database for global m6A patterns across cell lines http://gb.whu.edu.cn:8080/CVm6A RMVar a database of functional variants involved in RNA modifications http://rmvar.renlab.org RMDisease a database unveiling the association between disease-associated variants and their epi-transcriptome disturbance www.xjtlu.edu.cn/biologicalsciences/rmd RNA Editing Database REDIdb a specialized database for RNA editing modifications in plant organelles http://srv00.recas.ba.infn.it/redidb/index.html RADAR a rigorously annotated database of A-to-I RNA editing in humans, mice and flies http://RNAedit.com DARNED a repository for RNA editing in humans, centralized on A-to-I editing https://darned.ucc.ie/ REDIportal the largest and specialized repository for A-to-I editing occurring in a variety of human tissues http://srv00.recas.ba.infn.it/atlas/ Databases of RNA modifications With the advances in next-generation sequencing (NGS) technologies, many experimental methods have been designed to profile RNA modifications. Generally, the principles of sequencing methods could be classified as two types. The first type is based on antibody or chemical label to capture modified RNA fragments, such as MeRIP-seq for m6A profiling. Another strategy is using enzyme-assisted reaction or a specific chemical reaction on the modified bases, such as Pseudo-seq for Ψ. And these reactions bring about base deletions, substitutions, or truncations, either before or after the modified bases. Here we briefly introduce characteristics of current sequencing methods in Table 3 . Table 3 Sequencing methods of RNA modifications Technologies Year Resolution Description Ref. MeRIP-seq (m6A-seq) 2012 100-200nt m6A-specific sequencing method based on antibody-mediated capture and massively parallel sequencing 442 miCLIP 2015 single nucleotide individual nucleotide resolution cross-linking and immunoprecipitation method for m6A and m6Am 443 PA-m6A-seq 2015 23nt m6A sequencing assisted by photo-crosslinking 444 m6A-REF-seq 2019 single nucleotide antibody-independent m6A mapping based on the m6A-sensitive RNA endoribonuclease 445 DART-seq 2019 single nucleotide an antibody-free method for m6A targeting deamination adjacent to modification sites 446 m6ACE-seq 2019 single nucleotide m6A cross-linking exonuclease sequencing method 447 m6A-SEAL-seq 2020 single nucleotide FTO-assisted m6A selective chemical labeling method 448 m6A-label-seq 2020 single nucleotide a metabolic labeling method for m6A 449 m1A-seq 2016 50-200nt a protocol for mapping m1A at single-nucleotide resolution 101 m1A-ID-seq 2016 \ a m1A profiling method based on immunoprecipitation and the inherent ability of m1A to stall reverse transcription 450 m1A-MAP 2017 single nucleotide a misincorporation- assisted profiling method for m1A 451 Aza-IP 2013 \ 5-azacytidine-mediated RNAimmunoprecipitation 452 Bisulfite sequencing 2017 single nucleotide a RNA bisulfite sequencing method of m5C 453 m5C-RIP-seq 2017 \ a m5C profiling using RNA immunoprecipitation followed by a deep sequencing 454 Pseudo-seq 2014 single nucleotide a genome-wide, single-nucleotide-resolution method for pseudouridine 167 Ψ-seq/Psi-seq 2014 single nucleotide a protocol for transcriptome-wide quantitative mapping of Ψ 455 CeU-seq 2015 single nucleotide N3-CMC–enriched Ψ sequencing method 456 m7G-MeRIP-Seq 2019 100-200nt m7G-methylated immunoprecipitation sequencing method 154 m7G-MaP-seq 2019 single nucleotide high-throughput m7G mutational profiling sequencing 457 m7G-miCLIP-Seq 2019 single nucleotide m7G individual-nucleotide-resolution cross-linking and immunoprecipitation with sequencing method 458 ac4C-RIP-seq 2018 \ transcriptome-wide ac4C-targeted RNA immunoprecipitation sequencing 134 ac4C-seq 2021 single nucleotide a protocol for the quantitative single-nucleotide resolution mapping of ac4C 459 ICE-seq 2011 \ inosine chemical erasing method with deep sequencing method 460 Sequencing methods of RNA modifications

With advent of high-throughput methodologies, precision and sensitivity of RNA modification sequencing invented in an unprecedented space. Currently, mainstream MeRIP- and miCLIP-based methods have been widely accepted, yet with several disadvantages to be overcome. The poor sensitivity of antibody-based methods is first limitation, and chemical-assisted labeling is recognized as a promising approach. On account of the strong affinity of biotin-streptavidin binding, the m6A seal (m6A selective chemical labeling) method dramatically enhance enrichment efficiency via introducing a biotin tag to modified bases. 437 In addition, to solve the incapability of quantifying modification ratio, m 6 A-LAIC-seq (m 6 A-level and isoform-characterization sequencing), originated from MeRIP-seq, could quantify m6A levels for all isoforms of transcripts for each gene via isolating m6A-positive and m6A-negative post-RIP fractions and sequencing full-length transcripts. 438 Adding synthetic modification-free RNA molecules as internal reference is another strategy to realize quantitative sequencing. 439 To be noted, single-cell sequencing technologies is an emerging hotspot in tumor immunology, which could effectively profile the intricate immune landscape in tumor TME. For instance, DART-seq (deamination adjacent to RNA modification target sequencing) is designed to monitor m6A at the single-cell level, which successfully reveal the heterogeneity in m6A scenarios across individual cells and identify differentially methylated mRNAs across the cell cycle. 440 However, further application of DART-seq in clinics is limited by its dependency on overexpression of the APOBEC1-YTH fusion protein in cells. Hence, a free of genetic manipulation single-cell method for deciphering RNA modification is warranted. With advanced methodologies of detecting and profiling RNA modifications, rapid-accumulated enormous epi-transcriptome data call for centralized bioinformatics platforms to mine the underestimated treasure. For both experimental and computational studies of RNA modifications, such valuable resources will be of great help. Researchers focus on structural biology could take full advantage of comprehensive databases like RNAMDB and MODOMICS, while computational biologists perform their researches based on relational databases such as MetDBv2.0, m6A-Atlas, RMBase v2.0 etc. Exploration of novel types or modifiers of RNA modification fully relies on current knowledgebases. For clinics, these databases advance understanding of the role epi-transcriptomics plays in disease pathology. Not a few databases have provided information about relationship between disease-related varients with RNA modifications, such as m6A-Atlas, RMVar, and RMDisease. Meanwhile, epitranscriptomics-disease links are highlighted in newest update of MODOMICS. The new section exhibits association between malfunction or misregulation of a given RNA-modifying enzymes with specific disease conditions. To better elucidate the post-transcriptional regulatory networks, multi-omics analysis is highly rated. Moreover, context-specificity of RNA modification should be taken into consideration via distinguishing species, cell type, and tissues. Finally, more user-friendly interface and webserver tools are significant for improving accessibility of these resources. During differentiation and development of immune cells, various clusters of functionally coordinated genes are under sophisticated control of RNA modifications. The highly selectivity and specificity of RNA modifying machinery still remains largely defined. The top priority is to distinguish different targeted transcripts according to a framework of classical immunological systems such as polarization of macrophages and CD4 + T cell differentiation. It was suggested that additional factors such as region-enriched cis-regulatory elements exerted a certain effect on selectivity of RNA marking. Besides, increasing evidences have implicated the crosstalk between RNA modifications on non-coding chromosome-associated regulatory RNA (carRNA) and chromatin modifications, thus RNA modifications may control immune responses to environmental stimuli via shaping the chromatin environment of immune cells. In the aspect of tumor immunology, the discrepancy of epi-transcriptome between tumor and immune cells is acknowledged as an essential influencing factor of antitumor immune responses. However, relevant research is still in its infancy. Thus, rigorous dissection of RNA modification marks and regulators in tumor cells and immune cells is considered as a fundamental and crucial for developing effective interventions. Following marker-informed sorting of cell populations of interest, methods like mass spectrometry are used for profiling dynamic RNA modifications. Furthermore, integration of single-cell scale and transcriptome methods with RNA modification sequencing may provide valuable insights into dysregulated RNA modification in the TME. Aside from highly-specific RNA modification-targeted inhibitors, modification editing in immune cells is another promising direction for treating immune-related diseases. Established on the understanding of metabolism, the application prospects of targeting m6A methylation in immunotherapy mainly consisted of two possibilities. One is to circumvent therapeutic resistance mediated by the metabolic antagonism in TME, the other is to potentiate proliferation efficiency and effector functions of immune cells for adoptive cell therapy. Recent advances clued some potential strategies: 1) a programmable m6A editing machinery to fine-tune RNA modifications of specific genes with minimal off-target alterations, 2) effective manners to manipulate m6A system ex vivo for optimal generation of NK cells and T cells, 3) efficient targeted delivery of m6A editors into cells, like nanoparticles, 4) inhibitors against m6A regulators with potential to modulate anti-tumor immunity. The current understanding is that metabolic phenotypes evolve as cancers process from premalignant lesions, localized invasive malignancies to metastatic cancers, even therapy-resistant states. The dynamic RNA modification along with emerging metabolic vulnerabilities in evolutionary process provide attractive clinical opportunities. In some cases, tumors exhibited stereotyped metabolic alterations without detectable mutations or DNA methylation abnormities, 441 implicating the presence of other epigenetic regulation like RNA modifications. Delineating the evolving genetic, epigenetic, immune-metabolic landscape is quite necessary for designing effective strategies to preclude metastasis. Progresses in spatial-omics techniques and system biology research may help to address it. Hyperactive metabolic pathways lead to brisk adaptation to nutrient deprivation, contributing to resistance to antimetabolic chemotherapy agents like antifolates. Metabolic coupling, characterized by catabolites transfer, is common in tumor for overcoming nutrient deficiency. Thus, combination of targeting glycolysis and OXPHOS was proposed as a promising strategy. Considering the underlying toxicity, the alternative is suppressing dysfunctional signals to indirectly target glycolysis, while directly targeting OXPHOS. Increasing studies have indicated the significance of epigenetic regulation in metabolic diseases. Up to now, none of epigenetic drugs have been approved for metabolic diseases, and the efficacy of RNA modification-targeted agents have not been verified in metabolic diseases. Thus, investigation whether inhibition of RNA modifiers can be used for treatment of metabolic diseases is requested. Given that environmental factors shed influences on epi-transcriptome via intracellular metabolic changes, molecular insights of RNA modification in development of metabolic diseases remains largely unknown. Indeed, the universal distribution and broad functionality of RNA modification is a double-edged sword. For anticancer treatment, targeting a single identified driver sometimes turns out an unfavorable result, as a consequence of various reasons including the development of resistance and intra- or intertumoral heterogeneity. From this point, targeting RNA modification is advantageous to cover a network of targets. Especially, these RNA modifiers tend to be overexpressed or more active in cancerous tissue compared to matched normal control tissue. However, the essentiality and specificity of these RNA modifiers remain significant concerns. For currently developed agents targeting RNA modifying enzymes, poor specificity and selectivity remain the main obstacle in their progression into clinical researches. And such deficiency is anticipated to be improved via optimized bioinformatic prediction models and high-throughput enzymatic tests. Here are several other outstanding questions to be further investigated. If pharmacologic inhibition of RNA modification enzyme is capable to reproduce the phenotypic activity induced by genetic deletion? If redundance in modifying enzymes, like METTL3/14 complex and METTL6, potentially induce resistance to pharmacological inhibition. Given that RNA modifiers tend to be overexpressed in tumor tissue but still present in normal tissues, an appropriate therapeutic window in a certain therapeutic context may be necessary. Aside from specific inhibitors or activators to those modifiers, RNA modifications have been applied to improve the stability, efficacy and target specificity of RNA-based therapies. Common strategies include utilizing synthetic chemical or naturally occurring modifications, and modulating sequence context or location of these modifications. For all current therapeutic RNA, RNA modifications are extensively present and poised to further enhance their effectiveness. In summary, epigenetic regulation of RNA modifications exerts a crucial role in cellular metabolism in diverse physiological and pathological situations. Growing evidences suggest that such metabolic-epigenetic interplay significantly affects immune responses, via modulating biological activities of immune cells and remodeling immune context. Thus, delineating the evolving genetic, epigenetic, immune-metabolic landscape is quite necessary for designing effective strategies to preclude pathogenesis, including various metabolic disorders, immune-related diseases, and cancer. Recent years have witnessed remarkable advancements in methods for detecting and profiling RNA modifications, accompanied with a series of serviceable databases and tools springing up. At present, attempts to targeting RNA modification for improving current therapy have obtained some inspiring advances, but relevant researches are still in its infancy. And we can count on further in-depth exploration to accelerate the development of RNA modification-targeted therapy, metabolism-targeted therapy and immunotherapy.

Since the first documentation of RNA modification as early as 1950s, over 170 types have been identified, ubiquitously existing in coding RNAs and non-coding RNAs. 1 The contributions of nucleoside base modifications in developing mRNA vaccine against COVID-19, which is awarded the Nobel Prize for 2023, immensely refresh the biologists studying RNA-based therapeutics. In most situations, the RNA modifications we talked about are reversible type, similar to DNA methylation. These modifications are deposited, removed and recognized by dedicated machineries, composed of writers, erasers and readers. These post-transcriptional modifications alter the canonical ribose and base structure to determine RNA fates, including splicing, trafficking, degradation, translation, and so on. Via regulating gene expression and cellular phenotypes, RNA modifications are extensively involved in various cellular processes. 2 Cellular metabolism, a sophisticated network involving multitudes of biochemical reactions, continuously invigorates scientific researches. “Metabolism reprogramming” was originally proposed in cancer research, and gradually expanded to other non-tumor diseases and normal physiological processes. Used to be defined as “changes of tumor cellular bioenergetics”, the current perception tends to regard it as an inherent adaptive capacity of all cells, which is strengthened in tumor cells via abnormally activated pre-existing processes. 3 Such metabolic adaptability is based on the interaction between cells and environment. During these biochemical processes, epigenetic modifications adjust the cell-environment relationship in a context-dependent manner. According to the alarming statistics of several recent public health researches, metabolic diseases appear as an increasingly severe burden in human society. There have been more than 1.9 billion adults and over 650 million adults qualified as obese and overweight globally in 2016. 4 According to International Diabetes Federation, 537 million adults had diabetes in 2021, which has become the ninth major cause of death worldwide. 5 NAFLD is the most common chronic liver disease worldwide, the global prevalence of which was 25%. 6 On the other hand, recent clinical trials targeting cancer metabolism come out with unsatisfactory efficacy and frustrating adverse reactions. 7 – 9 Leaving dietary interventions alone, metabolic therapy is divided into agents targeting nucleotide metabolism and non-nucleotide metabolism. Not a few metabolic drugs targeting nucleotide metabolism, mostly nucleotide analogs, have been commonly employed in clinical practice. But development of the non-nucleotide metabolism-targeted drug remains in its nascent stages. Therefore, for better insights into pathophysiology and optimized therapeutic strategies, integrated multi-omics analysis of metabolism is imperative. Not a few excellent studies have discussed the regulatory roles of RNA modifications on metabolism in specified pathological situations, with a particular focus on cancers. 10 – 12 However, there is a deficiency of a comprehensive and wide-scale review on the epigenetic-metabolic interaction covering health and disease context. Notably, immunometabolism is emerging field, expected to provide novel therapeutic strategies in cancer, autoimmunity, and metabolic diseases. The concept illustrates metabolic changes occurring in immune cells during their differentiation and activation processes. Studies have confirmed that various immune cells, including T cells, macrophages, NK cells, and DCs, proceeded metabolism remodeling to fulfill their specific functions in discrete contexts. 13 – 16 According to current knowledge, RNA modification could exert influences on immunometabolism through cell-intrinsic and extrinsic mechanisms. The former is intrinsic programs, including mTORC1 signaling and metabolic-related genes expression. The latter refers to tissue microenvironment and nutrient availability. In this review, we first introduce the history and current understanding of RNA modifications, and focus on their regulatory roles in cellular metabolism to construct epigenetic-metabolic landscape in physiological and pathological situations. And the influences of RNA modifications on immunometabolism in different immune responses is discussed separately. Eventually, we highlight the clinical implications of RNA modifications and provide perspectives for further studies.