NAT10-mediated ac⁴C modification promotes ectoderm differentiation of human embryonic stem cells via acetylating NR2F1 mRNA

Abstract

Cell fate determination in mammalian development is complex and precisely controlled and accumulating evidence indicates that epigenetic mechanisms are crucially involved. N⁴-acetylcytidine (ac⁴C) is a recently identified modification of messenger RNA (mRNA); however, its functions are still elusive in mammalian. Here, we show that N-acetyltransferase 10 (NAT10)-mediated ac⁴C modification promotes ectoderm differentiation of human embryonic stem cells (hESCs) by acetylating nuclear receptor subfamily 2 group F member 1 (NR2F1) mRNA to enhance translation efficiency (TE). Acetylated RNA immunoprecipitation sequencing (acRIP-seq) revealed that levels of ac⁴C modification were higher in ectodermal neuroepithelial progenitor (NEP) cells than in hESCs or mesoendoderm cells. In addition, integrated analysis of acRIP-seq and ribosome profiling sequencing revealed that NAT10 catalysed ac⁴C modification to improve TE in NEP cells. RIP-qRT-PCR analysis identified an interaction between NAT10 and NR2F1 mRNA in NEP cells and NR2F1 accelerated the nucleus-to-cytoplasm translocation of yes-associated protein 1, which contributed to ectodermal differentiation of hESCs. Collectively, these findings point out the novel regulatory role of ac⁴C modification in the early ectodermal differentiation of hESCs and will provide a new strategy for the treatment of neuroectodermal defects diseases.

References

[1] Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282(5391):1145-1147.

[2] Kamelgarn M, Chen J, Kuang L, Jin H, Kasarskis EJ, Zhu H. ALS mutations of FUS suppress protein translation and disrupt the regulation of nonsense-mediated decay. Proc Natl Acad Sci USA. 2018;115(51):E11904-E11913.

[3] Ghosh A, Mizuno K, Tiwari SS, et al. Alzheimer's disease-related dysregulation of mRNA translation causes key pathological features with ageing. Transl Psychiatry. 2020;10(1):192.

[4] 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(6):707-719.

[5] Yang D, Qiao J, Wang G, et al. N⁶-Methyladenosine modification of lincRNA 1281 is critically required for mESC differentiation potential. Nucleic Acids Res. 2018;46(8):3906-3920.

[6] Xing M, Liu Q, Mao C, et al. The 18S rRNA m⁶A methyltransferase METTL5 promotes mouse embryonic stem cell differentiation. EMBO Rep. 2020;21(10):e49863.

[7] Arango D, Sturgill D, Alhusaini N, et al. Acetylation of cytidine in mRNA promotes translation efficiency. Cell. 2018;175(7):1872-1886.e24.

[8] He PC, He C. mRNA acetylation: a new addition to the epitranscriptome. Cell Res. 2019;29(2):91-92.

[9] Arango D, Sturgill D, Yang R, et al. Direct epitranscriptomic regulation of mammalian translation initiation through N4-acetylcytidine. Mol Cell. 2022;9(8):e2106058.

[10] Wang G, Zhang M, Zhang Y, et al. NAT10-mediated mRNA N4-acetylcytidine modification promotes bladder cancer progression. Clin Transl Med. 2022;12(5):e738.

[11] Zong GZ, Wang X, Guo XY, et al. NAT10-mediated AXL mRNA N4-acetylcytidine modification promotes pancreatic carcinoma progression. Exp Cell Res. 2023;428(2):113620.

[12] Zheng X, Wang Q, Zhou Y, et al. N-acetyltransferase 10 promotes colon cancer progression by inhibiting ferroptosis through N4-acetylation and stabilization of ferroptosis suppressor protein 1 (FSP1) mRNA. Cancer Commun. 2022;42(12):1347-1366.

[13] Tsai K, Jaguva VA, Martinez CC, et al. Acetylation of cytidine residues boosts HIV-1 gene expression by increasing viral RNA stability. Cell Host Microbe. 2020;28(2):306-312.e6.

[14] Chen L, Wang WJ, 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(19):10896-10913.

[15] Du ZW, Chen H, Liu H, et al. Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells. Nat Commun. 2015;6:6626.

[16] Lam AQ, Freedman BS, Morizane R, Lerou PH, Valerius MT, Bonventre JV. Rapid and efficient differentiation of human pluripotent stem cells into intermediate mesoderm that forms tubules expressing kidney proximal tubular markers. J Am Soc Nephrol. 2014;25(6):1211-1225.

[17] Jin C, Wang T, Zhang D, et al. Acetyltransferase NAT10 regulates the Wnt/β-catenin signaling pathway to promote colorectal cancer progression via ac⁴C acetylation of KIF23 mRNA. J Exp Clin Cancer Res. 2022;41(1):345.

[18] Wang K, Zhou LY, Liu F, et al. PIWI-interacting RNA HAAPIR regulates cardiomyocyte death after myocardial infarction by promoting NAT10-mediated ac⁴C acetylation of Tfec mRNA. Adv Sci. 2022;9:e2106058.

[19] Huang H, Weng H, Sun W, et al. Recognition of RNA N⁶-methyladenosine by IGF2BP proteins enhances mRNA stability and translation. Nat Cell Biol. 2018;20(3):285-295.

[20] Kamata T, Yang CS, Melhuish TA, Frierson HF, Wotton D, Paschal BM. Post-transcriptional regulation of PARP7 protein stability is controlled by androgen signaling. Cells. 2021;10(2):363.

[21] Shih YY, Lin HY, Jan HM, et al. S-glutathionylation of Hsp90 enhances its degradation and correlates with favorable prognosis of breast cancer. Redox Biol. 2022;57:102501.

[22] Che H, He W, Feng J, et al. WWP2 ameliorates acute kidney injury by mediating p53 ubiquitylation and degradation. Cell Biochem Funct. 2020;38(6):695-701.

[23] Mao Y, Dong L, Liu XM, et al. m⁶A in mRNA coding regions promotes translation via the RNA helicase-containing YTHDC2. Nat Commun. 2019;10(1):5332.

[24] Schmidt EK, Clavarino G, Ceppi M, Pierre P. SUnSET, a nonradioactive method to monitor protein synthesis. Nat Methods. 2009;6(4):275-277.

[25] Wei R, Cui X, Min J, et al. NAT10 promotes cell proliferation by acetylating CEP170 mRNA to enhance translation efficiency in multiple myeloma. Acta Pharm Sin B. 2022;12(8):3313-3325.

[26] Yoo H, Greene GH, Yuan M, et al. Translational regulation of metabolic dynamics during effector-triggered immunity. Mol Plant. 2020;13(1):88-98.

[27] Ito S, Horikawa S, Suzuki T, et al. Human NAT10 is an ATP-dependent RNA acetyltransferase responsible for N⁴-acetylcytidine formation in 18S ribosomal RNA (rRNA). J Biol Chem. 2014;289(52):35724-35730.

[28] Zheng J, Tan Y, Liu X, et al. NAT10 regulates mitotic cell fate by acetylating Eg5 to control bipolar spindle assembly and chromosome segregation. Cell Death Differ. 2022;29(4):846-860.

[29] Liu X, Tan Y, Zhang C, et al. NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2. EMBO Rep. 2016;17(3):349-366.

[30] Russell JO, Camargo FD. Hippo signalling in the liver: role in development, regeneration and disease. Nat Rev Gastroenterol Hepatol. 2022;19(5):297-312.

[31] Martín-Hernández E, Rodríguez-García ME, Chen C, et al. Mitochondrial involvement in a Bosch-Boonstra-Schaaf optic atrophy syndrome patient with a novel de novo NR2F1 gene mutation. J Hum Genet. 2018;63(4):525-528.

[32] 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.

[33] Zhu Z, Xing X, Huang S, Tu Y. NAT10 promotes osteogenic differentiation of mesenchymal stem cells by mediating N⁴-acetylcytidine modification of gremlin 1. Stem Cells Int. 2021;2021:8833527.

[34] Zhang Y, Jing Y, Wang Y, et al. NAT10 promotes gastric cancer metastasis via N4-acetylated COL5A1. Signal Transduct Target Ther. 2021;6(1):173.

[35] Dalhat MH, Altayb HN, Khan MI, Choudhry H. Structural insights of human N-acetyltransferase 10 and identification of its potential novel inhibitors. Sci Rep. 2021;11(1):6051.

[36] Yang W, Li HY, Wu YF, et al. ac4C acetylation of RUNX2 catalyzed by NAT10 spurs osteogenesis of BMSCs and prevents ovariectomy-induced bone loss. Mol Ther Nucleic Acids. 2021;26:135-147.

[37] Duffy EE, Finander B, Choi G, et al. Developmental dynamics of RNA translation in the human brain. Nat Neurosci. 2022;25(10):1353-1365.

[38] Ding Q, Markesbery WR, Chen Q, Li F, Keller JN. Ribosome dysfunction is an early event in Alzheimer's disease. J Neurosci. 2005;25(40):9171-9175.

[39] Weikum ER, Liu X, Ortlund EA. The nuclear receptor superfamily: a structural perspective. Protein Sci. 2018;27(11):1876-1892.

[40] Bosch DG, Boonstra FN, Gonzaga-Jauregui C, et al. NR2F1 mutations cause optic atrophy with intellectual disability. Am J Hum Genet. 2014;94(2):303-309.

[41] Llorens-Bobadilla E, Zhao S, Baser A, Saiz-Castro G, Zwadlo K, Martin-Villalba A. Single-cell transcriptomics reveals a population of dormant neural stem cells that become activated upon brain injury. Cell Stem Cell. 2015;17(3):329-340.

[42] Zhang K, Yu F, Zhu J, et al. Imbalance of excitatory/inhibitory neuron differentiation in neurodevelopmental disorders with an NR2F1 point mutation. Cell Rep. 2020;31(3):107521.

[43] Foglio B, Rossini L, Garbelli R, et al. Dynamic expression of NR2F1 and SOX2 in developing and adult human cortex: comparison with cortical malformations. Brain Struct Funct. 2021;226(4):1303-1322.

[44] Hoch RV, Lindtner S, Price JD, Rubenstein JLR. OTX2 transcription factor controls regional patterning within the medial ganglionic Eminence and regional identity of the septum. Cell Rep. 2015;12(3):482-494.

[45] Park J, Jia S, Salter D, Bagnaninchi P, Hansen CG. The Hippo pathway drives the cellular response to hydrostatic pressure. EMBO J. 2022;41(13):e108719.

[46] Fu M, Hu Y, Lan T, Guan KL, Luo T, Luo M. The hippo signalling pathway and its implications in human health and diseases. Signal Transduct Target Ther. 2022;7(1):376.

[47] Zhou D, Conrad C, Xia F, et al. Mst1 and Mst2 maintain hepatocyte quiescence and suppress hepatocellular carcinoma development through inactivation of the Yap1 oncogene. Cancer Cell. 2009;16(5):425-438.

[48] Yu FX, Zhao B, Panupinthu N, et al. Regulation of the hippo-YAP pathway by G-protein-coupled receptor signaling. Cell. 2012;150(4):780-791.

[49] Dai X, Liu H, Shen S, et al. YAP activates the Hippo pathway in a negative feedback loop. Cell Res. 2015;25(10):1175-1178.

[50] Wang W, Huang J, Wang X, et al. PTPN14 is required for the density-dependent control of YAP1. Genes Dev. 2012;26(17):1959-1971.

[51] Zaltsman Y, Masuko S, Bensen JJ, Kiessling LL. Angiomotin regulates YAP localization during neural differentiation of human pluripotent stem cells. Stem Cell Rep. 2019;12(5):869-877.

[52] Kadamb R, Anton ML, Chua V, et al. Abstract A009: NR2F1 inhibits Gaq/11-YAP-TAZ signaling and limits reactivation of uveal melanoma disseminated cancer cells via induction of dormancy. Cancer Res. 2023;83:A009.

RIGHTS & PERMISSIONS

2023 2023 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.