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NAT10-mediated N4-acetylcytidine mRNA modification regulates self-renewal in human embryonic stem cells.

ABSTRACT: NAT10-catalyzed N4-acetylcytidine (ac4C) has emerged as a vital post-transcriptional modulator on the coding transcriptome by promoting mRNA stability. However, its role in mammalian development remains unclear. Here, we found that NAT10 expression positively correlates with pluripotency in vivo and in vitro. High throughput ac4C-targeted RNA immunoprecipitation sequencing (ac4C-RIP-seq), NaCNBH3-based chemical ac4C sequencing (ac4C-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays revealed noticeable ac4C modifications in transcriptome of hESCs, among which transcripts encoding core pluripotency transcription factors are favorable targets of ac4C modification. Further validation assays demonstrate that genetic inactivation of NAT10, the ac4C writer enzyme, led to ac4C level decrease on target genes, promoted the core pluripotency regulator OCT4 (POU5F1) transcript decay, and finally impaired self-renewal and promoted early differentiation in hESCs. Together, our work presented here elucidates a previously unrecognized interconnectivity between the core pluripotent transcriptional network for the maintenance of human ESC self-renewal and NAT10-catalyzed ac4C RNA epigenetic modification.

SUBMITTER: Liu R

PROVIDER: S-EPMC10484679 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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NAT10-mediated N4-acetylcytidine mRNA modification regulates self-renewal in human embryonic stem cells.

Liu Rucong R Wubulikasimu Zibaguli Z Cai Runze R Meng Fanyi F Cui Qinghua Q Zhou Yuan Y Li Yang Y

Nucleic acids research 20230901 16

NAT10-catalyzed N4-acetylcytidine (ac4C) has emerged as a vital post-transcriptional modulator on the coding transcriptome by promoting mRNA stability. However, its role in mammalian development remains unclear. Here, we found that NAT10 expression positively correlates with pluripotency in vivo and in vitro. High throughput ac4C-targeted RNA immunoprecipitation sequencing (ac4C-RIP-seq), NaCNBH3-based chemical ac4C sequencing (ac4C-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/ ...[more]

PMID: 37497776

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Project description:BackgroundDysregulation of the epitranscriptome causes abnormal expression of oncogenes in the tumorigenic process. Previous studies have shown that NAT10 can regulate mRNA translation efficiency through RNA acetylation. However, the role of NAT10-mediated acetylation modification in bladder cancer remains elusive.MethodsThe clinical value of NAT10 was estimated according to NAT10 expression pattern based on TCGA data set and the tumor tissue array. Acetylated RNA immunoprecipitation sequencing was utilized to explore the role of NAT10 in mRNA ac4C modification. Translation efficiency and mRNA stability assay were applied to study the effect of NAT10-deletion on target genes. The nude mouse model and genetically engineered mice were conducted to further verify the characteristics of NAT10 in promoting BLCA progression and regulating downstream targets.ResultsNAT10 was essential for the proliferation, migration, invasion, survival and the stem-cell-like properties of bladder cancer cell lines. NAT10 was responsible for mRNA ac4C modification in BLCA cells, including BCL9L, SOX4 and AKT1. Deficient NAT10 in both xenograft and transgenic mouse models of bladder cancer reduced the tumor burden. Furthermore, acetylated RNA immunoprecipitation sequencing data and RNA immunoprecipitation qPCR results revealed that NAT10 is responsible for a set of ac4C mRNA modifications in bladder cancer cells. Inhibition of NAT10 led to a loss of ac4C peaks in these transcripts and represses the mRNA's stability and protein expression. Mechanistically, the ac4C reduction modification in specific regions of mRNAs resulting from NAT10 downregulation impaired the translation efficiency of BCL9L, SOX4 and AKT1 as well as the stability of BCL9L, SOX4.ConclusionsIn summary, these findings provide new insights into the dynamic characteristics of mRNA's post-transcriptional modification via NAT10-dependent acetylation and predict a role for NAT10 as a therapeutic target in bladder cancer.HighlightsNAT10 is highly expressed in BLCA patients and its abnormal level predicts bladder cancer progression and low overall survival rate. NAT10 is necessary and sufficient for BLCA tumourigenic properties. NAT10 is responsible for ac4C modification of target transcripts, including BCL9L, SOX4 and AKT1. NAT10 may serve as an effective and novel therapeutic target for BLCA.

Project description:BackgroundMetabolic dysfunction associated steatotic liver disease (MASLD), closely linked to excessive lipogenesis, induces chronic liver disease. MASLD often cause other metabolic diseases, such as cardiovascular disease, diabetes and obesity. However, the mechanism of N-acetyltransferase 10 (NAT10)-mediated N4-acetylcytidine (ac4C) mRNA modification in lipogenesis of MASLD has not been fully elucidated. This study investigated the role of NAT10 in lipogenesis targeting mRNA ac4C modification.MethodsThe expression of NAT10 in mouse liver was assessed after a 12-week high-fat diet. In addition, the expression of NAT10 also was detected after AML12 hepatocytes cells were treated with 150 µmol/L palmitic acid (PA). The ac4C mRNA modification was performed by dot blotting. Oil red O staining and the mRNA expression of Srebf1, Acaca and Fasn were used to assess lipogenesis in AML12 cells with NAT10 overexpression or knockdown. acRIP-PCR and NAT10 RIP-PCR were used to verify the Srebf1 and Scap mRNA ac4C modification by NAT10. Furthermore, the liver lipogenesis was evaluated by AAV-mediated target knockdown of NAT10 in mouse liver and treating a specific inhibitor, Remodelin.ResultsThis study revealed that NAT10 is significantly upregulated in liver lipogenesis after a 12-week high-fat diet. NAT10 and ac4C mRNA modification were also drastically increased in AML12 cells after treated with 150 µmol/L PA. Silencing of NAT10 notably inhibited the lipogenesis in AML12 cells and AAV-mediated target knockdown of NAT10 in mouse liver. The acRIP-PCR and NAT10-RIP-PCR revealed that NAT10 ac4C modified Srebf1 and Scap mRNA, the critical modulator of liver lipogenesis, to regulate liver lipogenesis. Besides, Remodelin strongly inhibited liver lipogenesis, including liver TG, serum ALT, AST, TG and TC level and glucose metabolism.ConclusionsNAT10 mediates ac4C modification of Srebf1 and Scap mRNA, thereby affecting lipogenesis in the liver. This study provided a new target for the treatment of MASLD.

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NAT10-mediated N4-acetylcytidine mRNA modification regulates self-renewal in human embryonic stem cells.

Publications

NAT10-mediated N4-acetylcytidine mRNA modification regulates self-renewal in human embryonic stem cells.

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