A Chemical Signature for Cytidine Acetylation in RNA

Abstract

N4-acetylcytidine (ac4C) is a highly conserved modified RNA nucleobase whose formation is catalyzed by the disease-associated N-acetyltransferase 10 (NAT10). Here we report a sensitive chemical method to localize ac4C in RNA. Specifically, we characterize the susceptibility of ac4C to borohydride-based reduction and show this reaction can cause introduction of noncognate base pairs during reverse transcription (RT). Combining borohydride-dependent misincorporation with ac4C's known base-sensitivity provides a unique chemical signature for this modified nucleobase. We show this unique reactivity can be used to quantitatively analyze cellular RNA acetylation, study adapters responsible for ac4C targeting, and probe the timing of RNA acetylation during ribosome biogenesis. Overall, our studies provide a chemical foundation for defining an expanding landscape of cytidine acetyltransferase activity and its impact on biology and disease.

Figure 1.

(a) Chemical reduction of N4-acetylcytidine to tetrahydro-N4-acetylcytidine. (b) Heat map of UV analysis testing different hydride donors for ablation of ac4C absorbance at 300 nm, indicative of reduction or deacetylation. (c) Heat map of UV analysis testing different hydride donors for production of C (280 nm) from ac4C, indicative of deacetylation. Reaction conditions: ac4C (0.1 mM), reductant (20 mM), H₂O. Additional UV data is provided in Figure S1.

Figure 2.

Chemical reduction of ac4C in polynucleotide RNA. (a) Schematic of synthetic ac4C/C-containing RNA. (b) Dot blot analysis of ac4C reduction in a synthetic RNA substrate (37 °C). (c) Primer extension analysis of ac4C-containing RNAs following NaBH₄ treatment (100 mM, 37 °C, 1 h). (d) Sanger sequencing analysis of PCR-amplified cDNAs (sense strand) generated from ac4C RNAs following NaBH₄ treatment (100 mM, 37 °C, 1 h) and TGIRT RT.

Figure 3.

(a) Schematic of human rRNA ac4C sites. (b) Sequencing analysis of ac4C-dependent mismatches in cDNAs generated from human rRNA helix 34 and (c) helix 45 following borohydride treatment and RT. Sequence corresponds to the cDNA sense strand. Vehicle = water; NaBH₄ = 100 mM sodium borohydride; (+alkali) NaBH₄ = alkali pretreatment (100 mM NaCO₃, pH 10, 60 °C, 1 h), precipitation, then borohydride. (d) Relationship between misincorporation signal and stoichiometry of ac4C in rRNA and (e) a synthetic RNA harboring ac4C in an “ACA” sequence context. For all data, error bars indicate the standard deviation (n = 3). Primary data in the form of full sequencing traces is provided in the Supporting Information.

Figure 4.

Applying borohydride-dependent misincorporation to study ac4C in rRNA biogenesis. (a) RNA modifications in human 18S helix 45. (b) RT of 21S-C pre-rRNA depends on relative timing of m26A and ac4C modifications. “C” and “E” represent sites at which the pre-rRNA is cleaved. Extension of the 21S-C primer, which forms cDNA from pre-rRNAs in which these cleavage sites are intact, may be impeded by m26A and reduced ac4C. (c) ac4C-dependent mismatch can be detected using an RT primer specific for the 21S-C pre-rRNA and its precursors.