Antisense pairing and SNORD13 structure guide RNA cytidine acetylation
- Supuni Thalalla Gamage1,5,
- Marie-Line Bortolin-Cavaillé2,5,
- Courtney Link1,
- Keri Bryson1,
- Aldema Sas-Chen3,
- Schraga Schwartz4,
- Jérôme Cavaillé2 and
- Jordan L. Meier1
- 1Chemical Biology Laboratory, National Cancer Institute, Frederick, Maryland 21702, USA
- 2Molecular, Cellular and Developmental Biology unit (MCD), Centre de Biologie Integrative (CBI), University of Toulouse III; UPS; CNRS; 31062 Cedex 9, Toulouse, France
- 3The Shmunis School of Biomedicine and Cancer Research, The George S. Wise Faculty of Life Sciences, Tel Aviv University, 6195001 Tel Aviv, Israel
- 4Department of Molecular Genetics, Weizmann Institute of Science, 7610001 Rehovot, Israel
- Corresponding authors: jordan.meier{at}nih.gov, jerome.cavaille{at}univ-tlse3.fr
-
↵5 These authors contributed equally to this work.
Abstract
N4-acetylcytidine (ac4C) is an RNA nucleobase found in all domains of life. The 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. After applying comparative sequence analysis and site-directed mutagenesis to provide evidence that SNORD13 folds into three main RNA helices, 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 that 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. By combining mutational analysis of these reconstituted systems with nucleotide resolution ac4C sequencing, our studies reveal plasticity in the molecular determinants underlying RNA-guided cytidine acetylation that is distinct from deposition of other well-studied rRNA modifications (e.g., pseudouridine). Overall, our studies provide a new approach to reconstitute RNA-guided cytidine acetylation in human cells as well as nucleotide resolution insights into the mechanisms governing this process.
Keywords
- Received May 13, 2022.
- Accepted September 2, 2022.
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