Cytidine Acetylation Across the Tree of Life

Abstract

Acetylation plays a critical role in regulating eukaryotic transcription via the modification of histones. Beyond this well-documented function, a less explored biological frontier is the potential for acetylation to modify and regulate the function of RNA molecules themselves. N⁴-Acetylcytdine (ac⁴C) is a minor RNA nucleobase conserved across all three domains of life (archaea, bacteria, and eukarya), a conservation that suggests a fundamental role in biological processes. Unlike many RNA modifications that are controlled by large enzyme families, almost all organisms catalyze ac⁴C using a homologue of human Nat10, an essential disease-associated acetyltransferase enzyme.A critical step in defining the fundamental functions of RNA modifications has been the development of methods for their sensitive and specific detection. This Account describes recent progress enabling the use of chemical sequencing reactions to map and quantify ac⁴C with single-nucleotide resolution in RNA. To orient readers, we first provide historical background of the discovery of ac⁴C and the enzymes that catalyze its formation. Next, we describe mechanistic experiments that led to the development of first- and second-generation sequencing reactions able to determine ac⁴C's position in a polynucleotide by exploiting the nucleobase's selective susceptibility to reduction by hydride donors. A notable feature of this chemistry, which may serve as a prototype for nucleotide resolution RNA modification sequencing reactions more broadly, is its ability to drive a penetrant and detectable gain of signal specifically at ac⁴C sites. Emphasizing practical applications, we present how this optimized chemistry can be integrated into experimental workflows capable of sensitive, transcriptome-wide analysis. Such readouts can be applied to quantitatively define the ac⁴C landscape across the tree of life. For example, in human cell lines and yeast, this method has uncovered that ac⁴C is highly selective, predominantly occupying dominant sites within rRNA (rRNA) and tRNA (tRNA). By contrast, when we extend these analyses to thermophilic archaea they identify the potential for much more prevalent patterns of cytidine acetylation, leading to the discovery of a role for this modification in adaptation to environmental stress. Nucleotide resolution analyses of ac⁴C have also allowed for the determination of structure-activity relationships required for short nucleolar RNA (snoRNA)-catalyzed ac⁴C deposition and the discovery of organisms with unexpectedly divergent tRNA and rRNA acetylation signatures. Finally, we share how these studies have shaped our approach to evaluating novel ac⁴C sites reported in the literature and highlight unanswered questions and new directions that set the stage for future research in the field.

Figure 1.

(A) Structure of N⁴-acetylcytidine (ac⁴C). (B) Canonical base pairing between ac⁴C and G. (C) Positions of ac⁴C characterized in eukaryotic RNA. Reproduced with permission from ref . Copyright (2022) American Chemical Society.

Figure 2.

Acetylation enzymes, substrates, and adapter molecules. (A) Cytidine acetylation enzymes. (B) Adapter proteins used by Nat10/Kre33-type cytidine acetyltransferases to modify tRNA (THUMPD1, human; Tan1, yeast). (C) Short nucleolar RNA (snoRNA) adapters by Nat10/Kre33-type cytidine acetyltransferases to modify rRNA (human helix 45, U13; yeast helix 45, snR45; yeast helix 34, snR4). The identity of snoRNA guiding helix 34 acetylation in human cells remains unknown. Adapted with permission from ref . Copyright (2019) Elsevier.

Figure 3.

(A) Scheme for the ac⁴C sequencing reaction. Note that the second-generation (“2.0”) chemistry is drawn. However, a similar sequence is believed to occur for the first-generation (“1.0”) reaction. The configuration of the new stereocenter that forms at C4 has not been determined. (B) Potential base pairing with adenosine that would explain the specific C → T misincorporation profiles of ac⁴C-seq. (C) Misincorporation at SSU-ac⁴C1842 is dependent on the pH of the reduction reaction, consistent with a role for the protonated nucleobase in the reduction reaction. (D) Consumption of ac⁴C (free nucleoside) by ac⁴C-seq 1.0 and ac⁴C-seq 2.0 sequencing chemistries. (E) Comparison of misincorporation at SSU-ac4C1842 using ac⁴C-seq 1.0 and 2.0 chemistries. Adapted with permission from ref (copyright (2018) American Chemical Society), ref (copyright (2020) Springer Nature), and ref (copyright (2021) Springer Nature).

Figure 4.

(A) Schematic for interfacing ac⁴C-seq 2.0 chemistry with next-generation sequencing. (B) Sequence motifs identified for ac⁴C sites identified in human (HEK-293T) and yeast (S. cerevisiae) mRNA upon high-level overexpression of Nat10/Thumpd1 and Kre33/Tan1, respectively. (C) SLC2A6 and WDR26 are two substrate mRNAs that are acetylated only in Nat10/Thumpd1 overexpression cells. Ectopic expression of these transcripts in the Nat10/Thumpd1 overexpression cells followed by amplicon-specific ac⁴C-seq verifies acetylation at the 5′-CCG-3′ sites. Modification is abolished when the consensus is mutated to 5′-CCA-3′. (D) Applying ac⁴C-seq to endogenous human RNA identifies the dominant sites in rRNA and tRNA. Adapted with permission from ref (copyright (2020) Springer Nature).

Figure 5.

Testing the molecular determinants of the SNORD13 function. (A, top) To test how mutations in SNORD13 affect its ability to facilitate 18S rRNA acetylation, mutant variants were expressed in SNORD13 KO cells. The transfected cells were sorted. RNA was then isolated and subjected to ac4C-seq using endogenous 18S rRNA specific primers. (Bottom) To test how mutations in 18S rRNA affect its ability to be modified by wild-type Nat10/SNORD13:18S, minigene variants were expressed in SNORD13 WT cells. RNA was isolated and subjected to ac⁴C-seq using minigene specific primers. (B) Summary of the findings from parallel SNORD/substrate profiling.

Figure 6.

Cytidine acetylation across the eukaryotic tree of life. (A) Unconventional eukaryotic organisms whose ac⁴C sites have been profiled with ac⁴C-seq. P. polycephalum is a slime mold in which ac⁴C-seq induces a misincorporation in 18S rRNA helix 45 in an unusual nonconsensus sequence. S. fetidae is a commercially available nematode that is useful for fighting garden parasites and cross-evolutionary studies of rRNA modifications. Photographs used with permission from J. Cavaille and A. Dussutour (CNRS, France). (B) ac⁴C-seq and antiac⁴C northern blotting reveal that C. elegans lacks rRNA acetylation. Adapted with permission from ref (copyright (2022) Oxford University Press).

Figure 7.

Cytidine acetylation across the archaeal tree of life. (A) Knockout of TkNat10 causes growth defects at high temperatures. (B) T. kodakarensis modifies the central C of a 5′-CCG-3′ consensus sequence at hundreds of sites in rRNA, tRNA, mRNA, and other RNA types. (C) T. kodakarensis dynamically acetylates RNA in response to thermal stress. (D) Cryo-EM analysis illustrates the site-specific environment-dependent modification of the T. kodakarensis ribosome. At high temperatures, ac⁴C replaces ordered water molecules that are observed at low temperatures. Red = ac⁴C sites. Adapted with permission from ref (copyright (2020) Springer Nature).