Probing small ribosomal subunit RNA helix 45 acetylation across eukaryotic evolution

Abstract

NAT10 is an essential enzyme that catalyzes N4-acetylcytidine (ac4C) in eukaryotic transfer RNA and 18S ribosomal RNA. Recent studies suggested that rRNA acetylation is dependent on SNORD13, a box C/D small nucleolar RNA predicted to base-pair with 18S rRNA via two antisense elements. However, the selectivity of SNORD13-dependent cytidine acetylation and its relationship to NAT10’s essential function remain to be defined. Here, we demonstrate that SNORD13 is required for acetylation of a single cytidine of human and zebrafish 18S rRNA. In-depth characterization revealed that SNORD13-dependent ac4C is dispensable for human cell growth, ribosome biogenesis, translation and development. This loss of function analysis inspired a cross-evolutionary survey of the eukaryotic rRNA acetylation ‘machinery’ that led to the characterization of many novel metazoan SNORD13 genes. This includes an atypical SNORD13-like RNA in Drosophila melanogaster which guides ac4C to 18S rRNA helix 45 despite lacking one of the two rRNA antisense elements. Finally, we discover that Caenorhabditis elegans 18S rRNA is not acetylated despite the presence of an essential NAT10 homolog. Our findings shed light on the molecular mechanisms underlying SNORD13-mediated rRNA acetylation across eukaryotic evolution and raise new questions regarding the biological and evolutionary relevance of this highly conserved rRNA modification.

Antisense pairing and SNORD13 structure guide RNA cytidine acetylation

“…Since 18S-B's antisense element is part of stem IV, whose disruption destabilizes SNORD13 (Figure 3c), we used a second-generation mutant of 18S-B (termed 18S-B*) which restores formation of stem IV (Figure Sequence of human SNORD13s analyzed. In addition to wild-type ("WT") sequence, disruptive mutations (18S-A, 18S-B*, 18S-C mutants), and a mutant with increased complementarity ("18S-A/18S-B perfect comp") were explored for rescue of ac 4 C in SNORD13 KO cells. Sequences and verification of expression are provided in Figure S1.…”

“…One of the most highly conserved of these is N4-acetylcytidine (ac 4 C). 1 In humans, ac 4 C has been mapped to two dominant sites in serine and leucine tRNA, as well as two dominant sites in small subunit (SSU) 18S rRNA occurring at helix 34 and 45. 2,3 Acetylation of all of these RNA targets is catalyzed by NAT10, the only RNA acetyltransferase known in the human genome.…”

“…3 Recently, we used genetic ablation to demonstrate that SNORD13 is required for acetylation of a single cytidine residue in helix 45 (h45) of small subunit 18S ribosomal RNA (SSU-ac 4 C1842; Figure 1a). 4 In addition to its biological function, another question is how SNORD13 helps NAT10 recognize its targets in human cells. SNORD13 is a box C/D snoRNA, so termed for the presence of C (5′-RUGAUGA-3′) and D (5′-CUGA-3′) motifs in its primary sequence.…”

“…Our first strategy hinges on the use of a SNORD13 knockout cell line in which SSU-ac 4 C1842 is lost. 4 Ectopic expression of SNORD13 rescues h45 acetylation and can be quantitatively assessed using nucleotide resolution ac 4 C sequencing. Using this assay we evaluated several SNORD13 mutants, allowing us to identify structural and antisense elements critical for activity.…”

“…This assay uses a RNA Polymerase I (Pol I) transcribed minigene to express a nucleolar pre-rRNA fragment which is efficiently acetylated by endogenous NAT10/SNORD13. Systematic mutation of this substrate is used to validate a consensus sequence necessary for ac 4 C deposition and highlights the potential reprogrammability of this system. Our studies demonstrate the power of combining mutational analysis with nucleotide resolution ac 4 C sequencing, and provide the basis for understanding and exploiting RNA-guided RNA modification catalysts in biology, biotechnology, and disease.…”