A sequence-specific RNA acetylation catalyst

N4-acetylcytidine (ac4C) is a ubiquitous RNA modification incorporated by cytidine acetyltransferase enzymes. Here, we report the biochemical characterization of Thermococcus kodakarensis Nat10 (TkNat10), an RNA acetyltransferase involved in archaeal thermotolerance. We demonstrate that TkNat10's catalytic activity is critical for T. kodakarensis fitness at elevated temperatures. Unlike eukaryotic homologs, TkNat10 exhibits robust stand-alone activity, modifying diverse RNA substrates in a temperature, ATP, and acetyl-CoA-dependent manner. Transcriptome-wide analysis reveals TkNat10 preferentially modifies unstructured RNAs containing a 5'-CCG-3' consensus sequence. Using a high-throughput mutagenesis approach, we define sequence and structural determinants of TkNat10 substrate recognition. We find TkNat10 can be engineered to facilitate use of propionyl-CoA, providing insight into its cofactor specificity. Finally, we demonstrate TkNat10's utility for site-specific acetylation of RNA oligonucleotides, enabling analysis of ac4C-dependent RNA-protein interactions. Our findings establish a framework for understanding archaeal RNA acetylation and a new tool for studying the functional consequences of ac4C in diverse RNA contexts.

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Publication Details

Journal
Nucleic Acids Research
Published
2025-03-07
DOI
https://doi.org/10.1093/nar/gkaf217
Citations
1
Primary Topic
RNA modifications and cancer
Type
article
Field-Weighted Citation Impact
0.81

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A sequence-specific RNA acetylation catalyst

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A sequence-specific RNA acetylation catalyst

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article en
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Abstract

N4-acetylcytidine (ac4C) is a ubiquitous RNA modification incorporated by cytidine acetyltransferase enzymes. Here, we report the biochemical characterization of Thermococcus kodakarensis Nat10 (TkNat10), an RNA acetyltransferase involved in archaeal thermotolerance. We demonstrate that TkNat10's catalytic activity is critical for T. kodakarensis fitness at elevated temperatures. Unlike eukaryotic homologs, TkNat10 exhibits robust stand-alone activity, modifying diverse RNA substrates in a temperature, ATP, and acetyl-CoA-dependent manner. Transcriptome-wide analysis reveals TkNat10 preferentially modifies unstructured RNAs containing a 5'-CCG-3' consensus sequence. Using a high-throughput mutagenesis approach, we define sequence and structural determinants of TkNat10 substrate recognition. We find TkNat10 can be engineered to facilitate use of propionyl-CoA, providing insight into its cofactor specificity. Finally, we demonstrate TkNat10's utility for site-specific acetylation of RNA oligonucleotides, enabling analysis of ac4C-dependent RNA-protein interactions. Our findings establish a framework for understanding archaeal RNA acetylation and a new tool for studying the functional consequences of ac4C in diverse RNA contexts.

Nucleic Acids ResearchVol. 53(6)
Leidos (United States) (US), National Institutes of Health (US), Tel Aviv University (IL), Protein Express (United States) (US), National Center for Biotechnology Information (US), Frederick National Laboratory for Cancer Research (US), National Cancer Institute (US), Leidos Biomedical Research Inc. (United States), Weizmann Institute of Science (IL), Colorado State University (US)
National Science Foundation, Israel Science Foundation, Azrieli Foundation, National Institutes of Health, National Cancer Institute, U.S. National Library of Medicine, H2020 European Research Council
Openalex Percentile: Top 30%
RNA modifications and cancer
0.81
DOIOpen Access PDFOpenAlex
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