RESUMO
Remodelin is a putative small molecule inhibitor of the RNA acetyltransferase NAT10 which has shown preclinical efficacy in models of the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS). Here we evaluate remodelin's assay interference characteristics and effects on NAT10-catalyzed RNA cytidine acetylation. We find the remodelin chemotype constitutes a cryptic assay interference compound, which does not react with small molecule thiols but demonstrates protein reactivity in ALARM NMR and proteome-wide affinity profiling assays. Biophysical analyses find no direct evidence for interaction of remodelin with the NAT10 acetyltransferase active site. Cellular studies verify that N4-acetylcytidine (ac4C) is a nonredundant target of NAT10 activity in human cell lines and find that this RNA modification is not affected by remodelin treatment in several orthogonal assays. These studies display the potential for remodelin's chemotype to interact with multiple protein targets in cells and indicate remodelin should not be applied as a specific chemical inhibitor of NAT10-catalyzed RNA acetylation.
RESUMO
Cytidine acetyltransferases are an emerging class of nucleic-acid-modifying enzymes responsible for the establishment of N4 -acetylcytidine (ac4C) in RNA. In contrast to histone acetyltransferases, whose activity is commonly studied by western blotting, relatively few methods exist for quickly assessing the activity of cytidine acetyltransferases from a biological sample of interest or the distribution of ac4C across different RNA species. In this protocol, we describe a method for analysis of cellular cytidine acetyltransferase activity using dot- and immuno-northern-blotting-based detection. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Detection of N4 -Acetylcytidine in RNA by dot blotting Basic Protocol 2: Visualizing N4 -Acetylcytidine Distribution in RNA by northern blotting.
Assuntos
Acetiltransferases/análise , Northern Blotting , Citidina/análise , RNA/química , Acetiltransferases/metabolismo , Citidina/metabolismo , Humanos , RNA/metabolismoRESUMO
N4-acetylcytidine (ac4C) is an ancient and highly conserved RNA modification that is present on tRNA and rRNA and has recently been investigated in eukaryotic mRNA1-3. However, the distribution, dynamics and functions of cytidine acetylation have yet to be fully elucidated. Here we report ac4C-seq, a chemical genomic method for the transcriptome-wide quantitative mapping of ac4C at single-nucleotide resolution. In human and yeast mRNAs, ac4C sites are not detected but can be induced-at a conserved sequence motif-via the ectopic overexpression of eukaryotic acetyltransferase complexes. By contrast, cross-evolutionary profiling revealed unprecedented levels of ac4C across hundreds of residues in rRNA, tRNA, non-coding RNA and mRNA from hyperthermophilic archaea. Ac4C is markedly induced in response to increases in temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Visualization of wild-type and acetyltransferase-deficient archaeal ribosomes by cryo-electron microscopy provided structural insights into the temperature-dependent distribution of ac4C and its potential thermoadaptive role. Our studies quantitatively define the ac4C landscape, providing a technical and conceptual foundation for elucidating the role of this modification in biology and disease4-6.
Assuntos
Acetilação , Citidina/análogos & derivados , Células Eucarióticas/metabolismo , Evolução Molecular , RNA/química , RNA/metabolismo , Archaea/química , Archaea/citologia , Archaea/genética , Archaea/crescimento & desenvolvimento , Sequência Conservada , Microscopia Crioeletrônica , Citidina/metabolismo , Células Eucarióticas/citologia , Células HeLa , Humanos , Modelos Moleculares , Acetiltransferases N-Terminal/metabolismo , RNA Arqueal/química , RNA Arqueal/genética , Proteínas de Ligação a RNA/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA , TemperaturaRESUMO
Posttranscriptional modifications of RNA represent an emerging class of regulatory elements in human biology. Improved methods for studying how these elements are controlled and where they occur has the potential to transform our understanding of gene expression in development and disease. Here we describe a chemical method for nucleotide resolution sequencing of N4-acetylcytidine (ac4C), a highly conserved modified nucleobase whose formation is catalyzed by the essential cytidine acetyltransferase enzyme NAT10. This approach enables the sensitive, PCR-amplifiable detection of individual ac4C sites from nanograms of unfractionated cellular RNA. The sensitive and quantitative nature of this assay provides a powerful tool to understand how cytidine acetylation is targeted, profile RNA acetyltransferase dynamics, and validate the sites and stoichiometry of ac4C in novel RNA species.
