Abstract
The eukaryotic small-subunit (SSU) ribosomal RNA (rRNA) has two evolutionarily conserved acetylcytidines. However, the acetylation sites and the acetyltransferase responsible for the acetylation have not been identified. We performed a comprehensive MS-based analysis covering the entire sequence of the fission yeast, Schizosaccharomyces pombe, SSU rRNA and identified two acetylcytidines at positions 1297 and 1815 in the 3′ half of the rRNA. To identify the enzyme responsible for the cytidine acetylation, we searched for an S. pombe gene homologous to TmcA, a bacterial tRNA N-acetyltransferase, and found one potential candidate, Nat10. A temperature-sensitive strain of Nat10 with a mutation in the Walker A type ATP-binding motif abolished the cytidine acetylation in SSU rRNA, and the wild-type Nat10 supplemented to this strain recovered the acetylation, providing evidence that Nat10 is necessary for acetylation of SSU rRNA. The Nat10 mutant strain showed a slow-growth phenotype and was defective in forming the SSU rRNA from the precursor RNA, suggesting that cytidine acetylation is necessary for ribosome assembly.
Highlights
The eukaryotic ribosome is a large ribonucleoprotein complex consisting of two major components, the small subunit and large ribosomal subunit
In fission yeast SSU ribosomal RNA (rRNA), we found 26 fragments carrying a modified nucleotide resulting from post-transcriptional modifications (PTMs)
We report the sites of acetylation in the fission yeast SSU rRNA and the corresponding N-acetyltransferase responsible for this acetylation
Summary
The eukaryotic ribosome is a large ribonucleoprotein complex consisting of two major components, the small subunit and large ribosomal subunit. For more than three decades, identification of the exact acetylation site has remained elusive [11], and little is known about its physiological significance in ribosome biogenesis and function or the presumptive acetyltransferase responsible for the modification. Determination of RNA PTMs has typically depended on RNase mapping techniques, but the large molecular size of the eukaryotic SSU rRNA often precludes application of such techniques [6]. With the use of Ariadne combined with genetics and molecular biology techniques, we were able to identify the positions of AcCs along the sequence of the SSU rRNA of the fission yeast, Schizosaccharomyces pombe, and we determined the enzyme responsible for this modification. It appears that cytidine acetylation has a regulatory role in ribosome assembly, in the process of SSU formation from a precursor rRNA
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