Antisense pairing and SNORD13 structure guide RNA cytidine acetylation

Abstract

N4-acetylcytidine (ac4C) is an RNA nucleobase found in all domains of life. Establishment of ac4C in helix 45 (h45) of human 18S ribosomal RNA (rRNA) requires the combined activity of the acetyltransferase NAT10 and the box C/D snoRNA SNORD13. However, the molecular mechanisms governing RNA-guided nucleobase acetylation in humans remain unexplored. Here we report two assays that enable the study of SNORD13-dependent RNA acetylation in human cells. First, we demonstrate that ectopic expression of SNORD13 rescues h45 in a SNORD13 knockout cell line. Next, we show mutant snoRNAs can be used in combination with nucleotide resolution ac4C sequencing to define structure and sequence elements critical for SNORD13 function. Finally, we develop a second method that reports on the substrate specificity of endogenous NAT10-SNORD13 via mutational analysis of an ectopically-expressed pre-rRNA substrate.