Conspectus
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. N
4-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.