Biogenesis of eukaryotic ribosome is a complex event involving a number of non-ribosomal factors. During assembly of the ribosome, rRNAs are post-transcriptionally modified by 2´-O-methylation, pseudouridylation, and several base-specific modifications, which are collectively involved in fine-tuning translational fidelity and/or modulating ribosome assembly. By mass-spectrometric analysis, we demonstrated that N⁴-acetylcytidine (ac⁴C) is present in the 3’ terminal helix of 18S rRNA from Saccharomyces cerevisiae and human. In addition, we found that an essential gene in yeast, KRE33, which we renamed RRA1 (Ribosomal RNA cytidine Acetyltransferase 1) encoding an RNA acetyltransferase responsible for ac⁴C1773 formation in S. cerevisiae. Using recombinant Rra1p, we could successfully reconstitute ac⁴C1773 in a model rRNA fragment in the presence of both acetyl-CoA and ATP as substrates. Upon depletion of Rra1p, the 23S precursor of 18S rRNA was accumulated significantly, which resulted in complete loss of 18S rRNA and small ribosomal subunit (40S), suggesting that ac⁴C formation catalyzed by Rra1p plays a critical role in processing of the 23S precursor to yield 18S rRNA. When nuclear acetyl-CoA was depleted by inactivation of Acetyl CoA Synthetase 2 (ACS2), we observed temporal accumulation of the 23S precursor, indicating that Rra1p modulates biogenesis of 40S subunit by sensing nuclear acetyl-CoA concentration.

We also identified N-acetyltransferase 10 (NAT10) as a human homolog of RRA1. NAT10 has been known to be a lysine acetyltransferase that targets microtubules and histones, and plays an important role in cell division. NAT10 is highly expressed in malignant tumors, and is also a promising target for therapies against laminopathies and premature aging. We showed that NAT10 catalyzes ac⁴C at position 1842 in the terminal helix of mammalian 18S rRNA. RNAi-mediated knockdown of NAT10 resulted in growth retardation of human cells, and this was accompanied by high-level accumulation of the 30S precursor of 18S rRNA, suggesting that ac⁴C1842 formation catalyzed by NAT10 is involved in rRNA processing and ribosome biogenesis in mammals.