RNA-seq and acRIP-seq in NAT10-knockdown PDAC cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE185960 | GEO | 2025/03/26
REPOSITORIES: GEO
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Project description:Pancreatic cancer is a lethal diease with high tendency of metastasis. Howerver, the mechanisms of pancreatic cancer are sitill unclear. To explore the roles of N4-acetylation (ac4C) RNA modification and its involved N-Acetyltransferase 10 (NAT10) in pancreatic ductal adenocarcinoma (PDAC), we performed profiling by high throughput sequencing. In this study, we investigate the effects of NAT10 knockdown on N4-acetylcytidine (ac4C) modification in mRNA within PANC-1 cells using ac4C-seq. By employing RNA interference to specifically knock down NAT10 expression in PANC-1 cells, we aim to elucidate its impact on ac4C RNA modifications, which have been implicated in various cellular processes and cancer progression. Total RNA was extracted and mRNA was captured and treated with sodium borohydride (NaBH4) for detection of ac4C sites.Following library preparation, sequencing was performed on an Illumina Novaseq 6000 platform. Bioinformatics analyses identified significant changes in ac4C modification patterns due to NAT10 depletion. This dataset provides a valuable resource for further exploration of ac4C modifications in mRNA and their role in PDAC.
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Project description:Increased differentiation or activity of osteoclasts is the key pathogenic factor of postmenopausal osteoporosis (PMOP). N4‐acetylcytidine (ac4C) modification, catalyzed by Nat10, is a novel post-transcriptional mRNA modification related to many diseases. However, its impact on regulating osteoclast activation in PMOP remains uncertain. Here, we initially observed that Nat10-mediated ac4C positively correlates with osteoclast differentiation of monocytes and low bone mass in PMOP. The specific knockout of Nat10 in monocytes and remodelin, a Nat10 inhibitor, alleviates ovariectomized (OVX)-induced bone loss by downregulating osteoclast differentiation. Mechanistically, epitranscriptomic analyses reveal that the nuclear factor of activated T cells cytoplasmic 1 (Nfatc1) is the key downstream target of ac4C modification during osteoclast differentiation. Subsequently, translatomic results demonstrate that Nat10-mediated ac4C enhances the translation efficiency of Nfatc1, thereby inducing Nfatc1 expression and consequent osteoclast maturation. Cumulatively, these findings reveal the promotive role of Nat10 in osteoclast differentiation and PMOP from a novel field of RNA modifications and suggest that Nat10 can be a target of epigenetic therapy for preventing bone loss in PMOP.
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Project description:N4-acetylcytidine (ac4C), a conserved chemical modification in eukaryotic prokaryotes that is catalyzed by the N-acetyltransferase 10 (NAT10) enzyme, plays a crucial role in promoting mRNA stability and translation. However, the biological function and mechanisms of NAT10-mediated ac4C in human cancer were poorly defined. In order to investigate the regulatory mechanism of NAT10 in gastric cancer, we performed ac4C RIP-seq(acRIP-seq) analysis in AGS cells with NAT10 knockout compared with control in two repeats.
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Project description:N4-acetylcytidine (ac4C), a newly identified epigenetic modification within mRNAs, has been characterized as a crucial regulator of mRNA stability and translation efficiency. And NAT10 is the only known RNA acetyltransferase. In our study, we documented the down-regulated expression of both ac4C and NAT10 during meiotic maturation of mouse oocytes. NAT10 knockdown resulted in ac4C reduction and impaired mouse oocyte maturation in vitro. These results indicated that NAT10-mediated ac4C modification plays a critical regulatory role in oocyte meiotic maturation. We further performed high-throughput sequencing with NAT10-overexpressed HEK293T cells and NAT10-binding transcripts to investigate the genes modulated by NAT10-mediated ac4C modification.
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Project description:N4-acetylcytidine (ac4C) is an evolutionarily conserved RNA modification that is deposited on diverse RNAs by N-acetyltransferase 10 (NAT10), a protein with high biological significance for aging and cancer. Here, we performed a comprehensive survey of ac4C using metabolic labeling, sodium cyanoborohydride chemical treatment coupled to next generation sequencing (NGS) and ac4C antibody-based cell and molecular biology techniques. Our NGS analysis confirms robust ac4C modification of rRNA and specific tRNA species in a NAT10-dependent manner, but suggests low or spurious ac4C acetylation in mRNA. Analysis of RNA-seq data also revealed an induction of inflammatory responses as well as mutagenesis at transcriptionally active sites in NAT10-KO cells. This finding led us to further explore the role of NAT10 in R-loops, which have recently been shown to induce APOBEC3B-mediated mutagenesis. Our analysis revealed that R-loops are modified with ac4C in a NAT10-dependent manner. Furthermore, NAT10 restrains the levels of R-loops at a subset of differentially expressed genes in a manner dependent on its catalytic activity. Together with cellular biology data showing ac4C containing RNA in endosomal structures, we propose that increased levels of ac4C-unmodified RNAs, likely derived from R-loops, in endosomal structures induce inflammatory responses.
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Project description:N4-acetylcytidine (ac4C) is an evolutionarily conserved RNA modification that is deposited on diverse RNAs by N-acetyltransferase 10 (NAT10), a protein with high biological significance for aging and cancer. Here, we performed a comprehensive survey of ac4C using metabolic labeling, sodium cyanoborohydride chemical treatment coupled to next generation sequencing (NGS) and ac4C antibody-based cell and molecular biology techniques. Our NGS analysis confirms robust ac4C modification of rRNA and specific tRNA species in a NAT10-dependent manner, but suggests low or spurious ac4C acetylation in mRNA. Analysis of RNA-seq data also revealed an induction of inflammatory responses as well as mutagenesis at transcriptionally active sites in NAT10-KO cells. This finding led us to further explore the role of NAT10 in R-loops, which have recently been shown to induce APOBEC3B-mediated mutagenesis. Our analysis revealed that R-loops are modified with ac4C in a NAT10-dependent manner. Furthermore, NAT10 restrains the levels of R-loops at a subset of differentially expressed genes in a manner dependent on its catalytic activity. Together with cellular biology data showing ac4C containing RNA in endosomal structures, we propose that increased levels of ac4C-unmodified RNAs, likely derived from R-loops, in endosomal structures induce inflammatory responses.
2025-03-26 | GSE274993 | GEO
Project description:N4-acetylcytidine (ac4C) is an evolutionarily conserved RNA modification that is deposited on diverse RNAs by N-acetyltransferase 10 (NAT10), a protein with high biological significance for aging and cancer. Here, we performed a comprehensive survey of ac4C using metabolic labeling, sodium cyanoborohydride chemical treatment coupled to next generation sequencing (NGS) and ac4C antibody-based cell and molecular biology techniques. Our NGS analysis confirms robust ac4C modification of rRNA and specific tRNA species in a NAT10-dependent manner, but suggests low or spurious ac4C acetylation in mRNA. Analysis of RNA-seq data also revealed an induction of inflammatory responses as well as mutagenesis at transcriptionally active sites in NAT10-KO cells. This finding led us to further explore the role of NAT10 in R-loops, which have recently been shown to induce APOBEC3B-mediated mutagenesis. Our analysis revealed that R-loops are modified with ac4C in a NAT10-dependent manner. Furthermore, NAT10 restrains the levels of R-loops at a subset of differentially expressed genes in a manner dependent on its catalytic activity. Together with cellular biology data showing ac4C containing RNA in endosomal structures, we propose that increased levels of ac4C-unmodified RNAs, likely derived from R-loops, in endosomal structures induce inflammatory responses.
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Project description:N4-acetylcytidine (ac4C), a conserved but recently rediscovered RNA modification on tRNAs, rRNAs and mRNAs, is catalyzed by N-acetyltransferase 10 (NAT10). Lysine acylation is a ubiquitous protein modification that controls protein functions. Our latest study demonstrates a NAT10-dependent ac4C modification, which occurs on the polyadenylated nuclear RNA (PAN) encoded by oncogenic DNA virus Kaposi's sarcoma-associated herpesvirus (KSHV), can induce KSHV reactivation from latency and activate inflammasome. However, it remains unclear whether a novel lysine acylation occurs in NAT10 during KSHV reactivation and how this acylation of NAT10 regulates tRNAs ac4C modification. Here, we showed that NAT10 was lactylated by α-tubulin acetyltransferase 1 (ATAT1), as a writer at the critical domain, to exert RNA acetyltransferase function and thus increase the ac4C level of tRNASer-CGA-1-1. Mutagenesis at the ac4C site in tRNASer-CGA-1-1 inhibited its ac4C modifications, translation efficiency of viral lytic genes, and virion production. Mechanistically, KSHV PAN orchestrated NAT10 and ATAT1 to enhance NAT10 lactylation, resulting in tRNASer-CGA-1-1 ac4C modification, eventually boosting KSHV reactivation. Our findings reveal a novel post-translational modification in NAT10, as well as expand the understanding about tRNA-related ac4C modification during KSHV replication, which may be exploited to design therapeutic strategies for KSHV-related diseases.
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Project description:Mammalian oocyte maturation is driven by strictly translational regulation of maternal mRNAs stored in the cytoplasm. However, the function and mechanism of post-transcriptional chemical modifications especially the newly identified N4-acetylcytidine (ac4C) catalyzed by N-acetyltransferase 10 (NAT10) in this process are previously unknown. In this study, we developed a low-input ac4C sequencing technology—ac4C LACE-seq and mapped 8241 ac4C peaks at the whole transcriptome level using 50 mouse oocytes at the germinal vesicle (GV) stage. We profiled the mRNA landscapes of NAT10-interactions and ac4C modifications. The NAT10-interacted and ac4C modified transcripts displayed association with high translation efficiency in oocytes. Oocyte-specific Nat10 knockout wiped out ac4C signals in oocytes and caused severe defects in meiotic maturation and female infertility. ac4C LACE-seq results indicated that Nat10 deletion led to a failure of ac4C deposition on mRNAs encoding key maternal factors such as MAY2, ZAR1, BTG4 and cyclin B1 that regulate transcriptome stability and maternal-to-zygotic transition. Nat10-deleted oocytes had decreased mRNA translation efficiencies during meiotic maturation, partially due to the direct inhibition ac4C sites on specific transcripts. In sum, we developed low-input, high-sensitivity mRNA ac4C profiling approach and highlighted the important physiological function of ac4C in precise regulation of the oocyte meiotic maturation by enhancing translation efficiency.
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