Locus-specific quantification reveals dynamics of ribosomal ac4C modifications

Eukaryotic ribosomes carry two evolutionarily conserved N4-acetylcytidine (ac4C) modifications within critical functional regions. However, fundamental questions remain regarding their deposition kinetics, potential dynamics, and functional interplay with other rRNA modifications. Here, using ac4C-Sanger, we quantitatively profiled ribosomal ac4C modifications across various rRNA precursors and in mature rRNA under varied conditions in Saccharomyces cerevisiae. We show that ac4C accumulates progressively during 18S rRNA maturation, with distinct kinetics at each site. Moreover, ribosomal ac4C levels are dynamic and respond to growth phase and thermal stress in a site-specific manner. Changes in modifications under heat stress are accompanied by alterations in translation fidelity and efficiency, although evidence is still lacking to establish a causal relationship. Genetic perturbation of the box C/D snoRNA snR40, which guides 2'-O-methylation Gm1271, revealed a differential effect on both ac4C sites, despite the spatial proximity of Gm1271 to only one of them. Beyond ac4C-Sanger, we also demonstrate that High-Resolution Melting (HRM) and SELECT (a single-base elongation- and ligation-based qPCR amplification method) can serve as orthogonal approaches to effectively detect and quantitatively measure ac4C at known sites. Collectively, our work establishes ribosomal ac4C as a dynamic epitranscriptomic mark, suggests intricate interdependencies governing its biogenesis, and provides accessible tools for its investigation, thereby underscoring the integrated and adaptive nature of the ribosomal modification landscape.