Jhulki, I. | Search bioRxiv

bioRxiv2024

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 TkNat10s 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 use non-native acyl-CoA donors, providing insight into its cofactor specificity. Finally, we demonstrate TkNat10s 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.

biochemistry↗

bioRxiv2022

Chemoproteomics yields a selective molecular host for acetyl-CoA

Chemoproteomic profiling is a powerful approach to define the selectivity of small molecules and endogenous metabolites with the human proteome. In addition to mechanistic studies, proteome specificity profiling also has the potential to identify new scaffolds for biomolecular sensing. Here we report a chemoproteomics-inspired strategy for selective sensing of acetyl-CoA. First, we use chemoproteomic capture experiments to validate the N-terminal acetyltransferase NAA50 as a protein capable of differentiating acetyl-CoA and CoA. A Nanoluc-NAA50 fusion protein retains this specificity and can be used to generate a bioluminescence resonance energy transfer (BRET) signal in the presence of a CoA-linked fluorophore. This enables the development of a ligand displacement assay in which CoA metabolites are detected via their ability to bind the Nanoluc-NAA50 protein host and compete binding of the CoA-linked fluorophore guest. We demonstrate that the specificity of ligand displacement reflects the molecular recognition of the NAA50 host, while the window of dynamic sensing can be controlled by tuning the binding affinity of the CoA-linked fluorophore guest. Finally, we show the methods specificity for acetyl-CoA can be harnessed for gain-of-signal optical detection of enzyme activity. Overall, our studies demonstrate the potential of harnessing insights from chemoproteomics for molecular sensing and provide a foundation for future applications in target engagement and selective metabolite detection.

biochemistry↗