RNA cytidine acetyltransferase of small-subunit ribosomal RNA: identification of acetylation sites and the responsible acetyltransferase in fission yeast, Schizosaccharomyces pombe

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.

Figure 1. The Nat10 null mutant is inviable.

Tetrad dissection experiments were carried out using segregates shown below the panel. The four spores from each given tetrad are grouped horizontally. The dissected spores were grown at 30°C for 3 days on YE medium. Parental strain: h+/h– leu1-32/+ his3-D1/+ ura4-D18/ura4-D18 nat10::ura4/+.

Figure 2. Analysis of the Nat10_G285D temperature-sensitive strain.

A. Effects of Nat10 on the temperature sensitivity of the Nat10_G285D mutant. The Nat10_G285D strain (deficient in leu1 gene) was supplemented with Nat10 cDNA in pREP1 vector containing the nmt1 promoter and an auxotrophic marker, leu2 gene, and incubated for 3 days on an SD plate without leucine at 30°C or 37°C. The cDNAs used were: 1) mock vector; 2) Nat10_G285D cDNA in the vector; 3) wild-type Nat10 cDNA in the vector; and 4) no vector and cDNA. Note that the temperature-sensitive mutant grows at 37°C with the expression of Nat10 cDNA. B. Effects of Nat10 on the growth rate of Nat10_G285D mutant. Doubling time during the logarithmic growth phase (5.0×10⁶ to 2.5×10⁷ cells/ml) at 30°C was measured in SD medium without leucine. The strains used were Nat10_G285D supplemented with pREP1-Nat10_wt or the mock vector. Each value represents the mean ± standard deviation of three independent assays. The arrow indicates a significant difference as determined by the Student's t test (p<0.05). Note that the growth rate of the Nat10_G285D mutant was recovered upon expression of Nat10 cDNA.

Figure 3. MS-based identification of acetylcytidines in S. pombe 18S rRNA.

A. Extracted ion monitoring of RNase T1 fragments of 18S rRNA containing acetylcytidine (AcC) and cytidine (C)-1297 (upper panel) and AcC and C-1815 (lower panel). The 18S rRNAs were purified from strain SP6 or Nat10_G285D grown at 30°C in YE medium, digested by RNase T1 and applied to the LC-MS system (50 fmol each). The sequences and m/z values of AcC and C-containing nucleotides are indicated. A mass window of 3 ppm was used for the extractions. Y axis indicates the peak intensity relative to the most intensive peak in each panel. Note that the MS signals of AcC-containing nucleotides were completely lost in the Nat10_G285D mutant strain (indicated by arrows). B. MS/MS spectra of AcC-containing fragments. The acetylated RNA ions [C(AcC)Gp¹⁻, m/z 1014.14; UUUC(AcC)Gp²⁻, m/z 965.60, in A) were analyzed by collision-induced dissociation. The position of acetylcytidine residues was identified by manual interpretation of the a-, c-, w- and y-type series ions and other specific product ions as indicated in the figure. The series ions assigned are indicated on the RNA sequence in the inset.

Figure 4. Effect of Nat10 on the acetylation of cytidines 1297 and 1815 in 18S rRNA.

Extracted ion monitoring of RNase T1 fragments of 18S rRNA containing AcC-1297 (upper panel) and AcC-1815 (lower panel) is shown. The analysis was performed for 18S rRNAs purified from strain SP6 supplemented with the mock vector, strain Nat10_G285D supplemented with the mock vector, or strain Nat10_G285D supplemented with pREP1-Nat10_wt, respectively, as indicated. Each yeast strain was grown at 30°C in EMMmedium without leucine. The rRNA was digested by RNase T1 and applied to the LC-MS system (50 fmol each). The sequences and m/z values of AcC-containing nucleotides are indicated. A mass window of 3 ppm was used for the extractions. Y axis indicates the peak intensity relative to the most intensive peak in each panel. Note that the MS signals of AcC-containing nucleotides at the positions indicated by arrows appear in the Nat10_G285D mutant strain upon expression of Nat10 cDNA.

Figure 5. Effect of Nat10 on ribosome assembly.

A. Ultracentrifugation analysis of ribosome assembly in the Nat10_G285D mutant. Ribosomal and polysomal fractions prepared from strain SP6 (upper panel) or Nat10_G285D mutant (lower panel), grown at 30°C in YE medium were analyzed with 15–50% sucrose density gradient centrifugation. The UV profiles (A₂₅₄) of the separation are depicted. The scales under each UV profile denote individual fractions, and the electrophoretograms shown below the UV profile are the results of agarose gel electrophoresis of each fraction. B. Effect of Nat10 on ribosome assembly of the Nat10_G285D mutant. Ribosomal and polysomal fractions derived from strain Nat10_G285D supplemented with the mock vector (upper panel) or with pREP1-Nat10_wt (lower panel) grown at 30°C in EMM medium without leucine were analyzed with 15–50% sucrose density gradient centrifugation under the conditions as in A. Note that the apparently aberrant phenotype in ribosome assembly of the Nat10_G285D mutant was rescued upon expression of Nat10, as indicated by the decrease in free large subunit (LSU) and increase in SSU and monosomes.