Profiling Cytidine Acetylation with Specific Affinity and Reactivity

Abstract

The human acetyltransferase NAT10 has recently been shown to catalyze formation of N4-acetylcytidine (ac4C), a minor nucleobase known to alter RNA structure and function. In order to better understand the role of RNA acetyltransferases in biology and disease, here we report the development and application of chemical methods to study ac4C. First, we demonstrate that ac4C can be conjugated to carrier proteins using optimized protocols. Next, we describe methods to access ac4C-containing RNAs, enabling the screening of anti-ac4C antibodies. Finally, we validate the specificity of an optimized ac4C affinity reagent in the context of cellular RNA by demonstrating its ability to accurately report on chemical deacetylation of ac4C. Overall, these studies provide a powerful new tool for studying ac4C in biological contexts, as well as new insights into the stability and half-life of this highly conserved RNA modification. More broadly, they demonstrate how chemical reactivity may be exploited to aid the development and validation of nucleobase-targeting affinity reagents designed to target the emerging epitranscriptome.

Figure 1.

Enzyme-catalyzed RNA acetylation. (a) Domain architecture of NAT10. (b) Catalytic activity of NAT10. (c) Known targets of RNA acetylation in human cells.

Figure 2.

Development of an ac4C antibody. (a) Scheme for ac4C carrier protein conjugation and antibody development. (b) SDS-PAGE gels indicating conjugation of ac4C to ovalbumin (OVA).

Figure 3.

Preparation of ac4C-containing RNA probes. (a) ac4CTP is efficiently used as a substrate in T7 Polymerase-catalyzed in vitro transcription and can be incorporated into multiple templates. Full gel image is provided in the Supporting Information. (b) HPLC analysis verifies incorporation of ac4C into an in vitro transcribed RNA probe. (c) Dot blot analysis demonstrates concentration-dependent detection of in vitro transcribed ac4C-containing RNAs using a monoclonal anti-ac4C antibody.

Figure 4.

Profiling ac4C using its specific reactivity. (a) Scheme for chemical deacetylation of ac4C. (b) Effect of hydroxylamine on ac4C free nucleoside monitored by UV spectroscopy. Reaction conditions: 0.25 mM ac4C [free nucleoside], 50 mM hydroxylamine, Tris at pH 7.0. (c) Effect of hydroxylamine on ac4C in RNA monitored by anti-ac4C dot blot. Reaction conditions: 50 mM hydroxylamine, Tris at pH 8.0, 65 °C, 1 h. (d) Effect of hydroxylamine (50 mM, pH 7.0) on overall cytidine and uridine base content in total RNA analyzed by HPLC. (e) Effect of hydroxylamine (50 mM, pH 7.0, 65 °C, 1 h) on ac4C in total cellular RNA analyzed by LC-MS/MS. (f) Anti-ac4C antibody can detect chemical ablation of ac4C in total cellular RNA.