A single acetylation of 18 S rRNA is essential for biogenesis of the small ribosomal subunit in Saccharomyces cerevisiae

Abstract

Biogenesis of eukaryotic ribosome is a complex event involving a number of non-ribosomal factors. During assembly of the ribosome, rRNAs are post-transcriptionally modified by 2'-O-methylation, pseudouridylation, and several base-specific modifications, which are collectively involved in fine-tuning translational fidelity and/or modulating ribosome assembly. By mass-spectrometric analysis, we demonstrated that N(4)-acetylcytidine (ac(4)C) is present at position 1773 in the 18 S rRNA of Saccharomyces cerevisiae. In addition, we found an essential gene, KRE33 (human homolog, NAT10), that we renamed RRA1 (ribosomal RNA cytidine acetyltransferase 1) encoding an RNA acetyltransferase responsible for ac(4)C1773 formation. Using recombinant Rra1p, we could successfully reconstitute ac(4)C1773 in a model rRNA fragment in the presence of both acetyl-CoA and ATP as substrates. Upon depletion of Rra1p, the 23 S precursor of 18 S rRNA was accumulated significantly, which resulted in complete loss of 18 S rRNA and small ribosomal subunit (40 S), suggesting that ac(4)C1773 formation catalyzed by Rra1p plays a critical role in processing of the 23 S precursor to yield 18 S rRNA. When nuclear acetyl-CoA was depleted by inactivation of acetyl-CoA synthetase 2 (ACS2), we observed temporal accumulation of the 23 S precursor, indicating that Rra1p modulates biogenesis of 40 S subunit by sensing nuclear acetyl-CoA concentration.

Keywords: Acetyl Coenzyme A (Acetyl-CoA); Acetyltransferase; RNA Modification; Ribosomal RNA Processing (rRNA Processing); Ribosome Assembly.

FIGURE 1.

N⁴-acetylcytidine is present at position 1773 in S. cerevisiae 18 S rRNA. A, the secondary structure of S. cerevisiae 18 S rRNA (left), with a detailed description of helix 45 and the surrounding region (right box), including modified nucleosides: N⁴-acetylcytidine (ac⁴C) at position 1773, N⁶,N⁶-dimethyladenosine (m⁶₂A) at positions 1782 and 1783, and 2′-O-methylcytidine (Cm) at position 1639. The RNA segment G1745-A1800 transcribed in vitro, used for ac⁴C formation, is shown in blue. The ac⁴C-containing hexamer fragment generated by RNase T₁ digestion is colored green. Watson-Crick base pairs and wobble pairs are shown as bars and dots, respectively. B, capillary LC/ESI-MS analysis of RNA fragments of S. cerevisiae rRNAs digested with RNase T₁. The upper panel shows a base-peak chromatogram (BPC), and the lower panel represents mass chromatogram for detecting the double-charged ion of the ac⁴C-containing hexamer fragment (UUUCac⁴CGp, m/z 965.6). C, collision-induced dissociation spectrum of the ac⁴C-containing hexamer fragment. The double-charged ion (m/z 965.6) was used as the parent for collision-induced dissociation. The sequence was confirmed by assignment of the product ions. Nomenclature for the product ions is as suggested in a previous report (54).

FIGURE 2.

In vitro reconstitution of ac⁴C1773 and substrate specificity of Rra1p. A, recombinant Rra1p (Kre33p) with C-terminal hexahistidine tag, analyzed by SDS-PAGE. B, in vitro reconstitution of ac⁴C1773 in the 56-mer RNA transcript, including helix 45 (Fig. 1A) in the presence or absence of recombinant Rra1p, ATP, and acetyl-CoA. The left and right panels show mass chromatograms detecting the hexamer fragment carrying ac⁴C1773 (UUUCac⁴CGp, m/z 965.6) or C1773 (UUUCCGp, m/z 944.6), respectively. C, ac⁴C1773 formation in the 26-mer RNA transcript in the presence (upper panels) or absence (lower panels) of recombinant Rra1p. The left and right panels show mass chromatograms detecting the hexamer fragment containing ac⁴C1773 (UUUCac⁴CGp, m/z 965.6) or C1773 (UUUCCGp, m/z 944.6), respectively. D, variants of the 41-mer transcript (G1760-A1800) used in this study. Variant names are the same as those in E and F. E, in vitro acetylation of the 41-mer variants by Rra1p. After the reaction, each variant was resolved by 10% denaturing PAGE and stained by SYBR Safe (upper panels). The radioactivity of the acetylated RNA fragment was visualized and quantified by FLA-7000 imaging analyzer (lower panels). M stands for a size marker. RI, radio isotope. F, relative acetylation activities of Rra1p on the 41-mer transcript variants, normalized by the activity on the wild-type transcript. Averaged values of three independent experiments with S.D. values are shown. Numerical values for activities are indicated at the bottom.

FIGURE 3.

Rra1p plays a critical role in pre-18 S rRNA processing. A, schematic depiction of the rRNA processing pathways in S. cerevisiae. In the canonical pathway 35 S pre-rRNA is processed by endonucleolytic cleavages at the A0, A1, and A2 sites to yield 20 S pre-rRNA. In an alternative pathway A3 site cleavage in ITS1 takes place before cleavages at the A0, A1, and A2 sites to yield the 23 S and 27SA3 pre-rRNAs (27SA3 is not shown is this scheme). Upon depletion of Rra1p, endonucleolytic cleavages at the A0, A1, and A2 sites are inhibited. The position of ac⁴C1773 is indicated by circle flag. B, high level accumulation of 23 S pre-rRNA with severely reduced 18 S rRNA upon Rra1p depletion. Precursors of 18 S rRNA were detected by northern blotting at the indicated times after heat treatment of the rra1^ts (right panels) and its parental (WT/YKL200) (left panels) strains. Steady-state levels of 25 S (top panels) and 18 S (second panels) rRNAs were visualized by ethidium bromide (EtBr) staining. The 23 S (third panels) and 20 S (bottom panels) pre-rRNAs are detected by northern blotting with the ITS1 probe. C, relative 18 S/25 S ratio of WT (open circles) and rra1^ts (closed squares) strains after culture temperature was raised to 37 °C. The data were calculated from the band intensities on the gel stained by EtBr as shown in B. D, sucrose density gradient profiling of ribosomal subunits in cell lysates of the WT (YKL200) (left) and the rra1^ts strain (right). E, capillary LC/ESI-MS analyses of RNase T₁-digested RNA fragments of 18 S rRNAs from the WT (YKL200) (upper panels) and rra1^ts strain (middle panels) and of 23 S pre-rRNA from the rra1^ts strain (lower panels). Mass chromatograms for detecting the double-charged ions of the ac⁴C-containing hexamers (UUUCac⁴CGp, m/z 965.6) and the control fragments A973-G976 (AAmCGp, m/z 669.1) are shown in the left and right panels, respectively. The intensities of the ac⁴C-containing hexamers in the mass chromatograms were normalized to those of the control fragments. F, 23 S pre-rRNA accumulated in the rra1^ts strain resides in the nucleus. Shown are mass chromatograms detecting the m¹acp³Ψ1191-containing fragment (ACm¹acp³ΨCAACACGp, m/z 1105.8) (left panels) and the m¹Ψ1191-containing fragment (ACm¹ΨCAACACGp, m/z 1072.2)(right panels) from 18 S rRNA (WT, YKL200 strain) (upper panels) and 23 S pre-rRNA (rra1^ts strain) (lower panels), respectively. Each peak is indicated by an arrowhead.

FIGURE 4.

The 23 S pre-rRNA accumulates upon Acs2p inactivation. A, schematic depiction of acetyl-CoA metabolism in S. cerevisiae. Acetyl-CoA produced in mitochondria is used in TCA cycle for energy production. Because no ATP-citrate lyase is present in S. cerevisiae, cytoplasmic acetyl-CoA is not produced from mitochondrial citrate. Nuclear acetyl-CoA, which is synthesized only by Acs2p, is used for histone acetylation by histone acetyltransferases (HAT) in nucleus and for formation of ac⁴C1773 in pre-rRNA in the nucleolus. B, accumulation of 23 S pre-rRNA after depletion of Acs2p. Precursors of 18 S rRNA were detected by northern blotting (NB) with the 5′-ETS probe at the indicated times after heat treatment of the acs2^ts strain (YHT652) (right panels) and the control strain (YHT651) (left panels). Total RNA from rra1^ts strain cultured at non-permissive condition was used as a marker for 23 S pre-rRNA (right-most panel). Steady-state levels of 25 S and 18 S rRNAs were visualized by ethidium bromide (EtBr) staining (lower panels).