Emerging roles of RNA N⁴-acetylcytidine modification in reproductive health

Abstract

N⁴-acetylcytidine (ac⁴C), an emerging posttranscriptional RNA modification, plays a pivotal role in epigenetic regulation. Ac⁴C is detected not only in tRNA, rRNA, and mRNA, but also in miRNA, lncRNA, viral RNA, and even DNA. Functionally, ac4C stabilizes mRNA, enhances protein translation fidelity, and impacts various biological processes and diseases such as cancer, inflammation, immune regulation, neural diseases, osteogenic differentiation, cardiovascular diseases, viral infections, and replication. Current research primarily focuses on ac⁴C’s roles in cancer progression and immunity, with emerging findings in gynecological diseases and reproduction. However, a comprehensive understanding of ac4C’s implications in reproductive health is lacking. This review provides a historical perspective on ac⁴C’s discovery and detection methods, elucidates its functions in reproductive development and gynecological disorders, and offers insights for further research in reproductive health. This review aims to pave the way for innovative therapeutic approaches and precise diagnostic tools tailored to this field.

Keywords

epigenetic regulation / RNA modification / ac ⁴C / gynecological disease / reproduction

References

[1] An Y, Duan H. The role of m6A RNA methylation in cancer metabolism. Mol Cancer 2022;21:14.

[2] Arango D, Sturgill D, Alhusaini N et al. Acetylation of Cytidine in mRNA promotes translation efficiency. Cell 2018;175:1872–1886.e24.

[3] Arango D, Sturgill D, Oberdoerffer S. Immunoprecipitation and sequencing of acetylated RNA. Bio Protoc 2019;9:e3278.

[4] Arango D, Sturgill D, Yang R et al. Direct epitranscriptomic regulation of mammalian translation initiation through N⁴-acetylcytidine. Mol Cell 2022;82:2797–2814.e11.

[5] Bartee D, Nance KD, Meier JL. Site-specific synthesis of N4-acetylcytidine in RNA reveals physiological duplex stabilization. J Am Chem Soc 2022;144:3487–3496.

[6] Batista PJ, Molinie B, Wang J et al. m⁶A RNA modification controls cell fate transition in mammalian embryonic stem cells. Cell Stem Cell 2014;15:707–719.

[7] Beiki H, Sturgill D, Arango D et al. Detection of ac4C in human mRNA is preserved upon data reassessment. Mol Cell 2024;84:1611–1625.e3.

[8] Boo SH, Kim YK. The emerging role of RNA modifications in the regulation of mRNA stability. Exp Mol Med 2020;52:400–408.

[9] Bortolin-Cavaillé M-L, Quillien A, Thalalla Gamage S et al. Probing small ribosomal subunit RNA helix 45 acetylation across eukaryotic evolution. Nucleic Acids Res 2022;50:6284–6299.

[10] Bruenger E, Kowalak JA, Kuchino Y et al. 5S rRNA modification in the hyperthermophilic archaea Sulfolobus solfataricus and Pyrodictium occultum. FASEB J 1993;7:196–200.

[11] Chen L, Wang W-J, Liu Q et al. NAT10-mediated N4-acetylcytidine modification is required for meiosis entry and progression in male germ cells. Nucleic Acids Res 2022;50:10896–10913.

[12] Chen X, Hao Y, Liu Y et al. NAT10/ac4C/FOXP1 promotes malignant progression and facilitates immunosuppression by reprogramming glycolytic metabolism in cervical cancer. Adv Sci (Weinh) 2023;10:e2302705.

[13] Cui L, Ma R, Cai J et al. RNA modifications: importance in immune cell biology and related diseases. Signal Transduct Target Ther 2022;7:334.

[14] Dang Y, Li J, Li Y et al. N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA. J Virol 2024;98:e0135023.

[15] Furuse Y. RNA modifications in genomic RNA of influenza A virus and the relationship between RNA modifications and viral infection. Int J Mol Sci 2021;22:9127.

[16] Geng Z, Liu P, Yuan L et al. Electroacupuncture attenuates ac4C modification of P16 mRNA in the ovarian granulosa cells of a mouse model premature ovarian failure. Acupunct Med 2022;41:27–37.

[17] Georgeson J, Schwartz S. No evidence for ac4C within human mRNA upon data reassessment. Mol Cell 2024;84:1601–1610.e2.

[18] Geula S, Moshitch-Moshkovitz S, Dominissini D et al. m⁶A mRNA methylation facilitates resolution of naïve pluripotency toward differentiation. Science 2015;347:1002–1006.

[19] Guo G, Shi X, Wang H et al. Epitranscriptomic N4-acetylcytidine profiling in CD4⁺ T cells of systemic lupus erythematosus. Front Cell Dev Biol 2020;8:842.

[20] Hao H, Liu W, Miao Y et al. N4-acetylcytidine regulates the replication and pathogenicity of enterovirus 71. Nucleic Acids Res 2022;50:9339–9354.

[21] Hu Z, Lu Y, Cao J et al. N-acetyltransferase NAT10 controls cell fates via connecting mRNA cytidine acetylation to chromatin signaling. Sci Adv 2024;10:eadh9871.

[22] Igo-Kemenes T, Zachau HG. On the specificity of the reduction of transfer ribonucleic acids with sodium borohydride. Eur J Biochem 1969;10:549–556.

[23] Ikeuchi Y, Kitahara K, Suzuki T. The RNA acetyltransferase driven by ATP hydrolysis synthesizes N4-acetylcytidine of tRNA anticodon. EMBO J 2008;27:2194–2203.

[24] Ito S, Akamatsu Y, Noma A et al. A single acetylation of 18 S rRNA is essential for biogenesis of the small ribosomal subunit in Saccharomyces cerevisiae. J Biol Chem 2014a;289:26201–26212.

[25] Ito S, Horikawa S, Suzuki T et al. Human NAT10 is an ATP-dependent RNA acetyltransferase responsible for N4-acetylcytidine formation in 18 S ribosomal RNA (rRNA). J Biol Chem 2014b;289:35724–35730.

[26] Jean M, Power D, Kong W et al. Identification of HIV-1 Tat-associated proteins contributing to HIV-1 transcription and latency. Viruses 2017;9:67.

[27] Jégourel D, Delépée R, Breton F et al. Molecularly imprinted polymer of 5-methyluridine for solid-phase extraction of pyrimidine nucleoside cancer markers in urine. Bioorgan Med Chem 2008;16:8932–8939.

[28] Jia Y, Zhang Z, Yan S et al. Voting-ac4C:Pre-trained large RNA language model enhances RNA N4-acetylcytidine site prediction. Int J Biol Macromol 2024;282:136940.

[29] Jiang X, Cheng Y, Zhu Y et al. Maternal NAT10 orchestrates oocyte meiotic cell-cycle progression and maturation in mice. Nat Commun 2023;14:3729.

[30] Jin G, Xu M, Zou M et al. The processing, gene regulation, biological functions, and clinical relevance of N4-acetylcytidine on RNA: a systematic review. Mol Ther Nucleic Acids 2020;20:13–24.

[31] Johansson MJO, Byström AS. The Saccharomyces cerevisiae TAN1 gene is required for N⁴-acetylcytidine formation in tRNA. RNA 2004;10:712–719.

[32] Kawai G, Hashizume T, Miyazawa T et al. Conformational characteristics of 4-acetylcytidine found in tRNA. Nucleic Acids Symp Ser 1989;21:61–62.

[33] Kotelawala L, Grayhack EJ, Phizicky EM. Identification of yeast tRNA Um44 2′-O-methyltransferase (Trm44) and demonstration of a Trm44 role in sustaining levels of specific tRNASer species. RNA 2008;14:158–169.

[34] Kowalski S, Yamane T, Fresco JR. Nucleotide sequence of the “denaturable” leucine transfer RNA from yeast. Science 1971;172:385–387.

[35] Lai F-L, Gao F. LSA-ac4C: a hybrid neural network incorporating double-layer LSTM and self-attention mechanism for the prediction of N4-acetylcytidine sites in human mRNA. Int J Biol Macromol 2023;253:126837.

[36] Lan MD, Xiong J, You XJ et al. Existence of diverse modifications in small-RNA species composed of 16–28 nucleotides. Chemistry 2018;24:9949–9956.

[37] Li B, Qu L, Yang J. RNA-guided RNA modifications: biogenesis, functions, and applications. Acc Chem Res 2023;56:3198–3210.

[38] Li N, Rana TM. Regulation of antiviral innate immunity by chemical modification of viral RNA. WIREs RNA 2022;13:e1720.

[39] Li Z, Jin B, Fang J. MetaAc4C: a multi-module deep learning framework for accurate prediction of N4-acetylcytidine sites based on pre-trained bidirectional encoder representation and generative adversarial networks. Genomics 2024;116:110749.

[40] Liebich HM, Lehmann R, Xu G et al. Application of capillary electrophoresis in clinical chemistry: the clinical value of urinary modified nucleosides. J Chromatogr B Biomed Sci Appl 2000;745:189–196.

[41] Lin J, Xiang Y, Huang J et al. NAT10 maintains OGA mRNA stability through ac4C modification in regulating oocyte maturation. Front Endocrinol 2022;13:907286.

[42] Liu R, Wubulikasimu Z, Cai R et al. NAT10-mediated N4-acetylcytidine mRNA modification regulates self-renewal in human embryonic stem cells. Nucleic Acids Res 2023;51:8514–8531.

[43] Liu R, Zhang Y, Wang Q et al. TransAC4C—a novel interpretable architecture for multi-species identification of N4-acetylcytidine sites in RNA with single-base resolution. Brief Bioinform 2024a;25:bbae200.

[44] Liu W-W, Zheng S-Q, Li T et al. RNA modifications in cellular metabolism: implications for metabolism-targeted therapy and immunotherapy. Signal Transduct Target Ther 2024b;9:70.

[45] Liu Y, Li J, Xu J et al. m⁶A-driven NAT10 translation facilitates fatty acid metabolic rewiring to suppress ferroptosis and promote ovarian tumorigenesis through enhancing ACOT7 mRNA acetylation. Oncogene 2024c;43:3498–3516.

[46] Long Y, Ren Y, Wei Q et al. NAT10-mediated RNA acetylation enhances HNRNPUL1 mRNA stability to contribute cervical cancer progression. Int J Med Sci 2023;20:1079–1090.

[47] Lou L-L, Qiu W-R, Liu Z et al. Stacking-ac4C: an ensemble model using mixed features for identifying n4-acetylcytidine in mRNA. Front Immunol 2023;14:1267755.

[48] Mezzar S, de Schryver E, Van Veldhoven PP. RP-HPLC-fluorescence analysis of aliphatic aldehydes: application to aldehyde-generating enzymes HACL1 and SGPL1. J Lipid Res 2014;55:573–582.

[49] Nance KD, Gamage ST, Alam MM et al. Cytidine acetylation yields a hypoinflammatory synthetic messenger RNA. Cell Chem Biol 2022;29:312–320.e7.e317.

[50] Oashi Z, Murao K, Yahagi T et al. Characterization of C+ located in the first position of the anticodon of Escherichia coli tRNA^Met as N⁴-acetylcytidine. Biochim Biophys Acta 1972;262:209–213.

[51] Orita I, Futatsuishi R, Adachi K et al. Random mutagenesis of a hyperthermophilic archaeon identified tRNA modifications associated with cellular hyperthermotolerance. Nucleic Acids Res 2019;47:1964–1976.

[52] Parthasarathy R, Ginell SL, De NC et al. Conformation of N4-acetylcytidine, a modified nucleoside of tRNA, and stereochemistry of codon-anticodon interaction. Biochem Biophys Res Commun 1978;83:657–663.

[53] Pham NT, Terrance AT, Jeon Y-J et al. ac⁴C-AFL: a high-precision identification of human mRNA N4-acetylcytidine sites based on adaptive feature representation learning. Mol Ther Nucleic Acids 2024;35:102192.

[54] Relier S, Schiffers S, Beiki H et al. Enhanced ac4C detection in RNA via chemical reduction and cDNA synthesis with modified dNTPs. RNA 2024;30:938–953.

[55] Sas-Chen A, Thomas JM, Matzov D et al. Dynamic RNA acetylation revealed by quantitative cross-evolutionary mapping. Nature 2020;583:638–643.

[56] Sharma S, Langhendries J-L, Watzinger P et al. Yeast Kre33 and human NAT10 are conserved 18S rRNA cytosine acetyltransferases that modify tRNAs assisted by the adaptor Tan1/THUMPD1. Nucleic Acids Res 2015;43:2242–2258.

[57] Sharma S, Yang J, van Nues R et al. Specialized box C/D snoRNPs act as antisense guides to target RNA base acetylation. PLoS Genet 2017;13:e1006804.

[58] Shen S, Zhang L-S. The regulation of antiviral innate immunity through non-m⁶A RNA modifications. Front Immunol 2023;14:1286820.

[59] Sinclair WR, Arango D, Shrimp JH et al. Profiling cytidine acetylation with specific affinity and reactivity. ACS Chem Biol 2017;12:2922–2926.

[60] Song B, Wang X, Liang Z et al. RMDisease V2.0: an updated database of genetic variants that affect RNA modifications with disease and trait implication. Nucleic Acids Res 2023;51:D1388–D1396.

[61] Stern L, Schulman LH. The role of the minor base N4-acetylcytidine in the function of the Escherichia coli noninitiator methionine transfer RNA. J Biol Chem 1978;253:6132–6139.

[62] Sturgill D, Arango D, Oberdoerffer S. Protocol for base resolution mapping of ac4C using RedaC:T-seq. STAR Protoc 2022;3:101858.

[63] Su D, Chan CTY, Gu C et al. Quantitative analysis of ribonucleoside modifications in tRNA by HPLC-coupled mass spectrometry. Nat Protoc 2014;9:828–841.

[64] Su W, Xie X-Q, Liu X-W et al. iRNA-ac4C: a novel computational method for effectively detecting N4-acetylcytidine sites in human mRNA. Int J Biol Macromol 2023;227:1174–1181.

[65] Sun H, Li K, Liu C et al. Regulation and functions of non-m6A mRNA modifications. Nat Rev Mol Cell Biol 2023;24:714–731.

[66] Szymańska E, Markuszewski MJ, Markuszewski M et al. Altered levels of nucleoside metabolite profiles in urogenital tract cancer measured by capillary electrophoresis. J Pharm Biomed Anal 2010;53:1305–1312.

[67] Tang C, Klukovich R, Peng H et al. ALKBH5-dependent m⁶A demethylation controls splicing and stability of long 3′-UTR mRNAs in male germ cells. Proc Natl Acad Sci USA 2018;115:E325–E333.

[68] Taniguchi T, Miyauchi K, Sakaguchi Y et al. Acetate-dependent tRNA acetylation required for decoding fidelity in protein synthesis. Nat Chem Biol 2018;14:1010–1020.

[69] Taoka M, Ishikawa D, Nobe Y et al. RNA cytidine acetyltransferase of small-subunit ribosomal RNA: identification of acetylation sites and the responsible acetyltransferase in fission yeast, Schizosaccharomyces pombe. PLoS One 2014;9:e112156.

[70] Tardu M, Jones JD, Kennedy RT et al. Identification and quantification of modified nucleosides in Saccharomyces cerevisiae mRNAs. ACS Chem Biol 2019;14:1403–1409.

[71] Thalalla Gamage S, Bortolin-Cavaillé ML, Link C et al. Antisense pairing and SNORD13 structure guide RNA cytidine acetylation. RNA 2022;28:1582–1596.

[72] Thalalla Gamage S, Howpay Manage SA, Chu TT et al. Cytidine acetylation across the tree of life. Acc Chem Res 2024;57:338–348.

[73] Thalalla Gamage S, Sas-Chen A, Schwartz S et al. Quantitative nucleotide resolution profiling of RNA cytidine acetylation by ac4C-seq. Nat Protoc 2021;16:2286–2307.

[74] Thomas G, Gordon J, Rogg H. N⁴-acetylcytidine. A previously unidentified labile component of the small subunit of eukaryotic ribosomes. J Biol Chem 1978;253:1101–1105.

[75] Thomas JM, Briney CA, Nance KD et al. A chemical signature for cytidine acetylation in RNA. J Am Chem Soc 2018;140:12667–12670.

[76] Thomas JM, Bryson KM, Meier JL. Nucleotide resolution sequencing of N⁴-acetylcytidine in RNA. Methods Enzymol. 2019;621:31–51.

[77] Tsai K, Cullen BR. Epigenetic and epitranscriptomic regulation of viral replication. Nat Rev Microbiol 2020;18:559–570.

[78] Tsai K, Jaguva Vasudevan AA, Martinez Campos C et al. Acetylation of cytidine residues boosts HIV-1 gene expression by increasing viral RNA stability. Cell Host Microbe 2020;28:306–312.e6.

[79] Wang C, Hou X, Guan Q et al. RNA modification in cardiovascular disease: implications for therapeutic interventions. Signal Transduct Target Ther 2023a;8:412.

[80] Wang C, Ju Y, Zou Q et al. DeepAc4C: a convolutional neural network model with hybrid features composed of physicochemical patterns and distributed representation information for identification of N4-acetylcytidine in mRNA. Bioinformatics 2022a;38:52–57.

[81] Wang M, Cheng R, He H et al. N⁴-acetylcytidine of Nop2 mRNA is required for the transition of morula-to-blastocyst. Cell Mol Life Sci 2023b;80:307.

[82] Wang S, Li Q, Wang Y et al. Peptidylprolyl isomerase D circular RNA sensitizes breast cancer to trastuzumab through remodeling HER2 N4-acetylcytidine modification. J Appl Genet 2024;65:797–807.

[83] Wang S, Xie H, Mao F et al. N⁴-acetyldeoxycytosine DNA modification marks euchromatin regions in Arabidopsis thaliana. Genome Biol 2022b;23:5.

[84] Wei W, Zhang S, Han H et al. NAT10-mediated ac4C tRNA modification promotes EGFR mRNA translation and gefitinib resistance in cancer. Cell Rep 2023;42:112810.

[85] Wiener D, Schwartz S. The epitranscriptome beyond m⁶A. Nat Rev Genet 2020;22:119–131.

[86] Xiang Y, Zhou C, Zeng Y et al. NAT10-mediated N4-acetylcytidine of RNA contributes to post-transcriptional regulation of mouse oocyte maturation in vitro. Front Cell Dev Biol 2021;9:704341.

[87] Xu K, Yang Y, Feng G-H et al. Mettl3-mediated m⁶A regulates spermatogonial differentiation and meiosis initiation. Cell Res 2017;27:1100–1114.

[88] Yan S, Lu Z, Yang W et al. Antibody-free fluorine-assisted metabolic sequencing of RNA N⁴-acetylcytidine. J Am Chem Soc 2023b;145:22232–22242.

[89] Yan Q, Zhou J, Gu Y et al. Lactylation of NAT10 promotes N⁴-acetylcytidine modification on tRNA^Ser-CGA-1-1 to boost oncogenic DNA virus KSHV reactivation. Cell Death Differ 2024;31:1362–1374.

[90] Yan Q, Zhou J, Wang Z et al. NAT10-dependent N⁴-acetylcytidine modification mediates PAN RNA stability, KSHV reactivation, and IFI16-related inflammasome activation. Nat Commun 2023a;14:6327.

[91] Yang J, Sharma S, Watzinger P et al. Mapping of complete set of ribose and base modifications of yeast rRNA by RP-HPLC and mung bean nuclease assay. PLoS One 2016;11:e0168873.

[92] Yu X-M, Li S-J, Yao Z-T et al. N4-acetylcytidine modification of lncRNA CTC-490G23.2 promotes cancer metastasis through interacting with PTBP1 to increase CD44 alternative splicing. Oncogene 2023;42:1101–1116.

[93] Zaccara S, Ries RJ, Jaffrey SR. Reading, writing and erasing mRNA methylation. Nat Rev Mol Cell Biol 2019;20:608–624.

[94] Zachau HG, Dütting D, Feldmann H. The structures of two serine transfer ribonucleic acids. Hoppe Seylers Z Physiol Chem 1966;347:212–235.

[95] Zhang C, Yuan L, Zou Q et al. CircMAST1 inhibits cervical cancer progression by hindering the N4-acetylcytidine modification of YAP mRNA. Cell Mol Biol Lett 2024a;29:25.

[96] Zhang H, Lu R, Huang J et al. N4-acetylcytidine modifies primary microRNAs for processing in cancer cells. Cell Mol Life Sci 2024b;81:73.

[97] Zhang S, Liu Y, Ma X et al. Recent advances in the potential role of RNA N4-acetylcytidine in cancer progression. Cell Commun Signal 2024c;22:49.

[98] Zhang T, Wu X, Ke C et al. Identification of potential biomarkers for ovarian cancer by urinary metabolomic profiling. J Proteome Res 2012;12:505–512.

[99] Zhao CC, Sun X, Chen J et al. NAT10-mediated mRNA N4-acetylcytidine modification of MDR1 and BCRP promotes breast cancer progression. Thorac Cancer 2024;15:820–829.

[100] Zhao W, Zhou Y, Cui Q et al. PACES: prediction of N4-acetylcytidine (ac4C) modification sites in mRNA. Sci Rep 2019;9:11112.

[101] Zheng P, Li N, Zhan X. Ovarian cancer subtypes based on the regulatory genes of RNA modifications: novel prediction model of prognosis. Front Endocrinol 2022;13:972341.

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The Author(s) 2025. Published by Oxford University Press on behalf of Higher Education Press.