A Single Acetylation of 18 S rRNA Is Essential for Biogenesis of the Small Ribosomal Subunit in Saccharomyces cerevisiae*

Background: Post-transcriptional modifications of rRNAs play important roles in biogenesis and function of ribosome. Results: Identification of an essential RNA acetyltransferase Rra1p responsible for forming N4-acetylcytidine at position 1773 in 18 S rRNA. Conclusion: Rra1p and ac4C1773 are required for pre-18 S rRNA processing. Significance: Rra1p modulates 40 S subunit biogenesis through a single acetylation of 18 S rRNA by sensing nuclear acetyl-CoA concentration. 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 N4-acetylcytidine (ac4C) 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 ac4C1773 formation. Using recombinant Rra1p, we could successfully reconstitute ac4C1773 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 ac4C1773 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.

tionally (9). On the other hand, the 18 S and 25 S rRNAs are also subjected to several snoRNA-independent modifications, which are introduced by specific RNA-modifying enzymes. In S. cerevisiae 18 S rRNA, three species of base-specific modifications have been found at four positions (10); 1-methyl-3-(3amino-3-carboxypropyl)pseudouridine (m 1 acp 3 ⌿) at position 1191, 7-methylguanosine at position 1575, and N 6 ,N 6 -dimethyladenosine at positions 1781 and 1782. During biogenesis of m 1 acp 3 ⌿1191, the box H/ACA snoRNA snR35 is responsible for ⌿1191 formation. Next, Emg1p, an essential rRNA methyltransferase, methylates ⌿1191 to form m 1 ⌿1191 in nucleolus (11,12). EMG1 is an essential gene in S. cerevisiae and is well conserved in archaea and eukaryote. Inactivation of Emg1p showed impaired formation of 18 S rRNA with a decreased 40 S subunit. In the last step the 3-amino-3-carboxypropyl group is formed on m 1 acp 3 ⌿1191; this reaction is known to occur in cytoplasm (13), but the responsible modifying enzyme remains unknown. Missense mutations found in human EMG1 are associated with a genetic disorder Bowen-Conradi syndrome characterized by severe developmental growth delays (14). It implies the importance of m 1 acp 3 ⌿1191 modification in 40 S subunit formation. The methyltransferase responsible for 7-methylguanosine (m 7 G) 1575 formation is Bud23p (15). Deletion of BUD23 leads to severe growth defects and reduction of 40 S biogenesis with impaired processing of the 20 S precursor to 18 S rRNA. Dim1p, another essential methyltransferase, is responsible for dimethylation of adenine bases at positions 1781 and 1782 in the 3Ј-terminal region of 18 S rRNA (16). These dimethylations mediated by Dim1p are required for processing at the 5Ј terminus (A1 site) of 18 S rRNA.
In addition to these modifications, N 4 -acetylcytidine (ac 4 C) is present in 18 S rRNA from rat liver, chicken liver, and S. cerevisiae, although the exact locations of ac 4 C are yet to be determined (17). In Dictyostelium discoideum, ac 4 C has been detected specifically at position 1844 in the 3Ј-terminal region of 18 S rRNA (18). Together these previous reports indicate that ac 4 C is highly conserved among eukaryotic 18 S rRNAs. However, the biogenesis and function of this modification remain to be elucidated.
In this study we used mass spectrometric analysis to show that ac 4 C is present at position 1773 in 18 S rRNA from S. cerevisiae (see Fig. 1A). In addition, we found that the essential gene RRA1 (ribosomal RNA cytidine acetyltransferase 1) encodes an RNA acetyltransferase responsible for ac 4 C1773 formation using ATP and acetyl-CoA as substrates. We demonstrate that ac 4 C1773 plays a critical role in processing of 23 S precursor to 18 S rRNA during biogenesis of the 40 S subunit. Moreover, we provide evidence that 18 S rRNA processing is modulated in response to the nuclear concentration of acetyl-CoA.
Preparation of Total RNA and 18 S rRNA-For small-scale preparation, the harvested cells were frozen in liquid nitrogen and crushed in an SK-Mill (FUNAKOSHI). Total RNA was extracted from the crushed cells using TriPure Isolation Reagent (Roche Applied Science). For large scale preparations, the harvested cells were lysed using EmulsiFlex C-3 (AVESTIN) followed by RNA extraction by the acid guanidinium thiocyanate-phenol-chloroform extraction (AGPC) method (21). For mass spectrometric analysis, total RNA was resolved on a 4% PAGE in a gel containing 7 M urea and then stained with ethidium bromide. The 18 S rRNA was excised from the gel, eluted by shaking in elution buffer (0.3 M NaOAc, 1 mM EDTA, 0.1% SDS) at 37°C for 8 h, and precipitated with ethanol.
Isolation of 23 S pre-rRNA by Reciprocal Circulating Chromatography-Total RNA was extracted from a 1-liter culture of the rra1 ts strain cultured at 37°C for 24 h. 23 S pre-rRNA was isolated by reciprocal circulating chromatography (RCC) using an automated RCC device (22), essentially following a previously described method (23). We designed three 5Ј-terminal ethylcarbamate amino-modified DNA probes (listed in Table  1), each of which was covalently immobilized on N-hydroxysuccinimide (NHS)-activated Sepharose 4 Fast Flow (GE Healthcare). The DNA-immobilized resins were packed into custom-made tips attached to a multichannel head on the reciprocal circulating chromatography device. The operation temperatures for hybridization step, washing step, and elution step were set to 66°C, 50°C, and 72°C, respectively. The 23 S pre-rRNA isolated using each of three probes was combined, resolved by 4% PAGE in a gel containing 7 M urea, and stained with ethidium bromide. The 23 S pre-rRNA was excised from the gel, eluted by shaking in elution buffer (0.3 M NaOAc, 1 mM EDTA, 0.1% SDS) at 37°C for 8 h, and precipitated with ethanol.
RNA Mass Spectrometry-Highly sensitive analysis of RNA fragments by mass spectrometry was carried out essentially as described previously (23,24). The isolated 18 S rRNA or 23 S pre-rRNA was digested with RNase T 1 at 37°C for 30 min in a 10-l reaction mixture containing 10 mM ammonium acetate (pH 5.3) and 5 units/l RNase T 1 (Epicenter) followed by the addition of an equal volume of 0.1 M triethylamine acetate (pH 7.0). The digested samples were then subjected to capillary liquid chromatography (LC) coupled with nanoelectrospray ionization (ESI) mass spectrometry using a linear ion trap-orbitrap hybrid mass spectrometer (LTQ Orbitrap XL, Thermo Fisher Scientific).
Northern blotting was carried out using the DIG Northern Starter kit (Roche Applied Science). RNA probes (ϳ200-mers) complementary to 5Ј-ETS or ITS1 were transcribed by T7 RNA polymerase in the presence of digoxigenin-11-UTP. Primer sequences used to amplify the templates for the RNA probes are listed in Table 1. Hybridization was carried out at 68°C overnight. The hybridized bands were visualized by chemiluminescence of CDP-Star (Roche Applied Science) using alkaline phosphatase-conjugated anti-digoxigenin-alkaline phosphatase and detected by LAS 4000 mini (GE healthcare).
Sucrose Density Gradient Centrifugation-The rra1 ts strain (Y40097) and its parental strain (YKL200) were cultured in YPG medium at 37°C and then harvested after 24 h of growth. Preparation of whole cell lysate was carried out basically as described (25). Cell pellets were ground in a mortar with liquid nitrogen and then resuspended in the lysis buffer (50 mM Hepes-KOH (pH 7.7), 100 mM KCl, 5 mM MgCl 2 , 1 mM DTT, 1 mM PMSF, protease inhibitor mixture (Roche Applied Science), and 0.02 units/l SUPERase-In RNase Inhibitor (Invitrogen)). The suspension was centrifuged at 5000 g for 5 min at 4°C to clear the lysate, which was then centrifuged at 10,000 ϫ g for 10 min at 4°C. Each supernatant (500 l) was loaded onto a gradient of 10 -40% (w/v) sucrose in the lysis buffer, and the gra- dients were ultracentrifuged in a Beckman SW-28 rotor at 20,000 rpm for 14 h at 4°C. The gradients were fractionated into Ͼ70 fractions on a Piston Gradient Fractionator (BIOCOMP), and the absorbance at 254 nm of each fraction was measured using an ultraviolet monitor (ATTO AC-5200).

Expression and Purification of Recombinant Rra1p-
The open reading frame of YNL132W was amplified by PCR from S. cerevisiae genomic DNA with primers listed in Table 1. The PCR product was cloned into the NheI and XhoI sites of pET-21b(ϩ) (Novagen) to generate pET-21b-RRA1. Escherichia coli strain Rosetta (Merck Millipore) was used as a host strain for the expression of recombinant Rra1p. The C-terminal hexahistidine-tagged Rra1p protein was expressed in soluble form by mild induction with 2% lactose at 25°C and then purified on an AKTA chromatography system using a His-trap column (GE Healthcare). Fractions containing the recombinant protein were pooled and dialyzed against a buffer consisting of 20 mM HEPES-KOH (pH 8.5), 300 mM KCl, 10 mM MgCl 2 , and 1 mM DTT. The protein concentration was determined using a Bradford protein assay kit (Bio-Rad) with bovine serum albumin as the standard. Glycerol was added to the protein solution to a final concentration of 33% (v/v).
In Vitro Reconstitution of ac 4 C1773 Formation-The DNA templates for in vitro transcription of RNA fragments containing helix 45 of 18 S rRNA and its variants were constructed by PCR using synthetic DNAs (Table 1). Substrate RNA was transcribed by T7 RNA polymerase basically as described (26). Transcripts were extracted from the reaction mixture with phenol-chloroform and then passed through NAP-5 column (GE Healthcare) to remove free NTPs followed by ethanol precipitation. Each transcript was further purified by 10% PAGE in a gel containing 7 M urea.
In vitro reconstitution of ac 4 C formation was carried out at 37°C for 2 h in a reaction mixture (10 l) consisting of 50 mM HEPES-KOH (pH 7.6), 150 mM KCl, 5 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 1 mM acetyl-CoA, 1 M substrate RNA, and 1.26 M recombinant Rra1p. After the reaction the RNA fragment was extracted with phenol-chloroform followed by ethanol precipitation. The ac 4 C formation was analyzed by mass spectrometry as described above. For the mutation study of RNA substrates, ac 4  After the reaction the substrate RNAs were extracted by phenol-chloroform, precipitated by ethanol, and analyzed by 10% PAGE in a gel containing 7 M urea. The substrate RNAs were stained by SYBR Safe (Invitrogen), visualized, and quantified by FLA-7000 imaging analyzer (Fujifilm). Then the gel was dried in vacuo, and the radioactivities of the acetylated RNA fragments were exposed on the imaging plate which was then visualized and quantified by FLA-7000 imaging analyzer. Relative activity of each variant was normalized by the band intensity on the gel stained by SYBR safe.

Determination of the Position of ac 4 C in S. cerevisiae 18 S
rRNA-To determine the exact position of ac 4 C modification, we performed mass spectrometric analysis on 18 S rRNA from S. cerevisiae. To this end we digested total RNA from S. cerevisiae (BY4741) with RNase T 1 and subjected the digests to the capillary LC coupled with ESI-MS to detect RNA fragments containing modified nucleotides. Most of the RNase T 1 -digested RNA fragments deduced from the 18 S rRNA sequence with modified nucleotides could be assigned in this analysis. We detected the double-charged negative ion of an ac 4 C-containing hexamer fragment (Fig.  1B) and analyzed it by collision-induced dissociation to sequence the fragment. Based on the assignment of product ions, the hexamer was found to be UUUCac 4 CGp (Fig. 1C), showing that ac 4 C was present at position 1773 in helix 45 in the 18 S rRNA (Fig. 1A). This position is equivalent to ac 4 C1844 in D. discoideum 18 S rRNA (18).
Identification of RRA1 and in Vitro Reconstitution of ac 4 C1773-ac 4 C is a modified nucleoside frequently detected in both tRNAs and rRNA from all domains of life (27). For instance, in E. coli, ac 4 C is present at the wobble position of elongator tRNA Met . We previously identified a bacterial tRNA acetyltransferase, TmcA (tRNA Met cytidine acetyltransferase), that catalyzes ac 4 C formation in the presence of acetyl-CoA and ATP (28). In bacteria, homologs of tmcA (COG1444) are limited to the ␥-proteobacterial subphylum (28). On the other hand, tmcA homologs are widely distributed among archaea and eukaryotes; ac 4 C is found at the wobble positions of archaeal tRNAs and at position 12 in a subset of eukaryotic tRNAs (27). In addition, as above mentioned, ac 4 C can be found in the 3Ј terminal region of 18 S rRNAs in eukaryotes (17,18). Based on these observations, the eukaryotic homolog of TmcA was hypothesized to be an RNA acetyltransferase responsible for ac 4 C formation in tRNA and/or 18 S rRNA (28). The S. cerevisiae ortholog of bacterial TmcA, encoded by YNL132W/KRE33, is an essential protein initially identified as a killer toxin-resistance protein (29). Kre33p, a putative ATPase, is a component of the 90 S pre-ribosomal particle (30), which localizes to the nucleolus (31). A large scale genetic approach revealed that transcription inhibition of KRE33 results in defects in formation and nuclear export of the 40 S subunit (30,32), suggesting that Kre33p acts as an assembly factor in ribosome biogenesis (33). These data prompted us to speculate that Kre33p is an acetyltransferase responsible for ac 4 C1773 formation in 18 S rRNA. To determine whether Kre33p has the appropriate enzymatic activity to catalyze formation of ac 4 C, Kre33p was recombinantly expressed in E. coli and purified homogenously ( Fig. 2A). A 56-mer RNA segment, including helix 45 (G1745-A1800) (Fig. 1A), transcribed in vitro, was used as a substrate for ac 4 C formation mediated by recombinant Kre33p. After the reaction, the RNA segment was digested with RNaseT 1 and then subjected to LC/ESI-MS to detect ac 4 C-modified fragment. As shown in Fig. 2B, an ac 4 Ccontaining hexamer was clearly detected only in the presence of both ATP and acetyl-CoA. Next, we used collision-induced dissociation analysis to determine that ac 4 C is present specifically at position 1773 (data not shown). In addition, we examined ac 4 C formation in a shorter substrate, a 26-mer derived from helix 45 (G1768-G1793) (Figs. 1A). Once again, we clearly detected an ac 4 C-containing hexamer (Fig. 2C), although the modification efficiency was only ϳ4% in this condition.
These data demonstrated that Kre33p is a bona fide acetyltransferase responsible for ac 4 C1773 in 18 S rRNA. Based on its enzymatic activity, we renamed YNL132W/KRE33 as RRA1 (ribosomal RNA cytidine acetyltransferase 1).
Substrate Specificity of Rra1p-To study the substrate specificity of Rra1p, we constructed a series of 41-mer transcripts (G1760-A1800) with various mutations (Fig. 2D). The relative activity of the enzyme on each of these variants was normalized to the activity on the wild-type transcript (WT) (Fig. 2, E and F). When each base pair of helix 45 was individually flipped (mutants F1-F9), ac 4 C formation was significantly reduced. In particular, no activity was detected on the mutants F3, F4, and F5, suggesting that the CCG sequence at positions 1772-1774 is essential for the ac 4 C formation. When the loop sequence of helix 45 was mutated (4L, 3L, and 5L), the activity persisted but at lower levels than on the WT transcript, indicating the bases in the loop sequence are involved in the activity. To investigate the functional significance of base-pairing at the target site (C1773-G1788), we replaced G1788 with A (A4), C (C4), and U (U4). Intriguingly, all of these mutants conferred higher activity than the WT transcript. In addition, when both sides of the target site were mutated, the two variants G1789A (A3) and C1787G (A5) also exhibited elevated activity. Taken together these data indicated that the CCG (1772-1774) sequence is critical for ac 4 C formation, but the Watson-Crick type base pairs of this sequence in helix 45 are not necessary for Rra1p activity. Indeed, to the contrary, disruption of these base pairs increased the activity.
Rra1p Plays a Critical Role in Pre-18 S rRNA Processing-Because Rra1p is involved in 40 S biogenesis (30,32), we examined whether 18 S rRNA processing is affected by inactivation of Rra1p. To this end we used a degron construct of Rra1p (rra1 ts ) to rapidly deplete the protein upon heat induction (19) because RRA1 is an essential gene in yeast. When the rra1 ts strain was cultured at 37°C for 24 h, the steady-state level of 18 S rRNA was severely reduced relative to the parental strain (YKL200) (Fig. 3B), whereas the 25 S rRNA was unaffected (Fig.  3B). In fact, the 18 S/25 S ratio of the rra1 ts strain dropped to only 16% that of the initial level (Fig. 3C), indicating that Rra1p is required for maturation of 18 S rRNA. In addition, in the rra1 ts strain cultured at 37°C for 24 h, the level of 80 S ribosome was markedly reduced, whereas free 60 S subunit accumulated to high levels (Fig. 3D), indicating that the steady-state level of the 40 S subunit was significantly lower than in the WT (YKL200) strain under the same conditions.
To determine which step in rRNA processing is affected by Rra1p depletion, we detected precursors of 18 S rRNAs by northern blotting after heat induction of both rra1 ts and WT strains (Fig. 3B). In the rra1 ts strain, we observed rapid accumulation of 23 S pre-rRNA (Fig. 3, A and B) immediately after raising the culture temperature to 37°C. Concomitantly, the level of 20 S pre-rRNA (Fig. 3, A and B), the final precursor for 18 S rRNA, was reduced. After 24 h, we observed high levels of accumulation of 23 S pre-rRNA, which produced no 20 S pre-rRNA, eventually resulting in huge reduction of 18 S rRNA (Fig.  3B). This observation neatly explains why Rra1p is an essential protein. By contrast, in the WT strain, little accumulation of 23 S pre-rRNA was observed until 4 h after heat treatment (Fig.  3B). However, 24 h after heat treatment of the WT strain, the level of 23 S pre-rRNA increased (discussed below) and the level of 20 S pre-rRNA decreased, although the steady-state level of 18 S rRNA was not changed, unlike the case of rra1 ts strain (Fig. 3B).
Under normal growth conditions, processing of 35 S pre-rRNA is initiated by endonucleolytic cleavage at A0 site (Fig.  3A). The 5Ј-ETS is removed by cleavages at A0 and A1, whereas cleavage at A2 in ITS1 generates 20 S and 27 S A2 pre-rRNAs, splitting pathways for small 40 S-and large 60 S-subunit maturation. In an alternative pathway, a separating cleavage at A3 site in ITS1, mediated by RNase MRP, produces the 23 S and 27 S A3 pre-rRNAs (Fig. 3A). As observed in the WT strain, 23 S pre-rRNA accumulated at high levels during stationary phase in both S. cerevisiae (34) and Candida albicans (35). Hence, 23 S pre-rRNA is a physiological precursor for 18 S rRNA formation in the WT strain. Therefore, rapid accumulation of 23 S pre-rRNA upon Rra1p depletion can be interpreted as the result of inhibition of cleavage at the A0, A1, and A2 sites. In other words either ac 4 C1773 formation or Rra1p binding to 90 S pre-ribosome is required for 23 S to be processed into 20 S pre-rRNA.
No ac 4 C1773 Is Formed in 23 S pre-rRNA Accumulated in the rra1 ts Strain-We next compared levels of ac 4 C1773 in 18 S rRNAs between the WT and rra1 ts strains. To this end we cultured both strains at 37°C overnight and analyzed their 18 S rRNAs by capillary LC/ESI-MS (Fig. 3E). We detected the ac 4 C1773-containing fragment (UUUCac 4 CGp) in both strains. Unexpectedly, after normalization to the levels of the control fragment A973-G976 (AAmCGp), there was no reduction in ac 4 C1773 level in the rra1 ts strain, showing that 18 S rRNA from the rra1 ts strain was fully modified with ac 4 C1773. Next, we analyzed the 23 S pre-rRNA accumulated in the rra1 ts strain. For this experiment, we isolated 23 S pre-rRNA by the reciprocal circulating chromatography (22) from the rra1 ts strain cultured at 37°C. The isolated 23 S pre-rRNA was further purified by PAGE and then subjected to RNaseT 1 digestion followed by capillary LC/ESI-MS analysis. Several fragments derived from 5Ј-ETS and ITS1 were detected (data not shown), confirming the successful isolation of 23 S pre-rRNA. To confirm whether the 23 S pre-rRNA accumulated in the rra1 ts strain resides in the nucleus, we searched for a fragment containing m 1 acp 3 ⌿1191, because 3-amino-3-carboxypropyl formation of m 1 acp 3 ⌿1191 takes place in the cytoplasm (13). When we analyzed 18 S rRNA as a control, we clearly detected the m 1 acp 3 ⌿1191-containing fragment (ACm 1 acp 3 ⌿CAACACGp) but no hypomodified species, such as an m 1 ⌿1191-containing fragment (ACm 1 ⌿CAACACGp) (Fig. 3F). By contrast, in our analysis of the accumulated 23 S pre-rRNA, we did not detect an m 1 acp 3 ⌿1191-containing fragment but clearly detected an m 1 ⌿1191-containing fragment (Fig. 3F). These results strongly suggested that 23 S pre-rRNA resides in the nucleus of the rra1 ts strain. Regarding ac 4 C1773 formation, we detected no ac 4 C1773containing fragment (UUUCac 4 CGp) in the isolated 23 S pre-rRNA (Fig. 3E), strongly suggesting that 23 S pre-rRNA without ac 4 C1773 is not further processed into 20 S pre-rRNA. Therefore, it is likely that in the rra1 ts strain, the 18 S rRNA fully modified with ac 4 C1773 originated from a residual pool of 40 S subunit that accumulated in the cytoplasm before heat treatment.
Accumulation of 23 S Pre-rRNA upon Depletion of Nuclear Acetyl-CoA-The findings described above clearly demonstrate that ac 4 C1773 formation mediated by Rra1p is required for processing 23 S pre-rRNA during the biogenesis of the 40 S subunit. On the basis of these observations, we hypothesized that rRNA processing and ribosome biogenesis could be controlled by nuclear acetyl-CoA concentration.
Acetyl-CoA is produced in the mitochondrial matrix by the pyruvate dehydrogenase complex and then used for acetylation of oxaloacetate to generate citrate in the tricarboxylic acid (TCA) cycle for energy production (Fig. 4A). In mammals, mitochondrial citrate can be exported to cytoplasm, where acetyl-CoA is produced from citrate and ATP in a reaction catalyzed by ATP-citrate lyase (36). However, no enzyme corresponding to ATP-citrate lyase is present in S. cerevisiae (37). Hence, nuclear and cytoplasmic acetyl-CoA cannot be supplied from the mitochondria in S. cerevisiae. Instead, nuclear acetyl-CoA is synthesized by acetyl-CoA synthetase 2 (Acs2p), which catalyzes the ligation of acetate and CoA (Fig. 4A) (38). Acs1p, another paralog of acetyl-CoA synthetase, is transcriptionally repressed at high concentrations of glucose (39). Thus, nuclear acetyl-CoA can be depleted in the temperature-sensitive strain, acs2 ts (20), cultured at non-permissive temperature in the presence of glucose. Judging from the deacetylation kinetics of  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 ac 4 C1773 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 T 1 -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 ac 4 C-containing hexamers (UUUCac 4 CGp, 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 ac 4 C-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 m 1 acp 3 ⌿1191-containing fragment (ACm 1 acp 3 ⌿CAACACGp, m/z 1105.8) (left panels) and the m 1 ⌿1191-containing fragment (ACm 1 ⌿CAACACGp, 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. acetylated histones, rapid depletion of nuclear acetyl-CoA is achieved within 1 h after heat treatment (20). We cultured the acs2 ts strain at 37°C and monitored rRNA precursors by northern blotting at early times (Fig. 4B); longer term cultivation of this strain at non-permissive temperature resulted in global repression of transcription and extensive lethality (20). As shown in Fig. 4B, 23 S pre-rRNA clearly appeared 1-2 h after heat treatment in the acs2 ts strain, whereas no accumulation of 23 S pre-rRNA was observed in the control strain YHT651. Transcription of rRNA precursors was not repressed over the time scale of this experiment, judging from the intensity of the 35 S pre-rRNA band 2 h after heat treatment (Fig. 4B). These data indicate that Rra1p modulates biogenesis of 40 S subunit by sensing the nuclear concentration of acetyl-CoA, thereby helping to coordinate ribosome synthesis with the cell's energy budget.

DISCUSSION
In this study we detected ac 4 C at position 1773 in 18 S rRNA of S. cerevisiae and showed that an essential acetyltransferase, Rra1p, is responsible for ac 4 C1773 formation. Previous reports have demonstrated that this modified base is present in 18 S rRNAs from rat, chicken, D. discoideum; thus, ac 4 C in the terminal helix of the small 40 S subunit rRNA is likely to be a highly conserved modification among eukaryotes. Homologs of RRA1/tmcA (COG1444) are widely distributed among all domains of life. Bacterial homologs are only found in the ␥-proteobacterial subphylum (28), suggesting that bacterial TmcA is responsible for ac 4 C formation at the wobble position of tRNA Met in these bacteria. Among the archaea, both Euryarchaeota and Crenarchaeota have RRA1 homologs. The RNA substrates for archaeal RRA1 remain to be elucidated, as ac 4 C is found in both tRNAs and rRNAs of some archaeal species (40 -42). Among eukaryotes, RRA1 homologs are widely distributed in organisms from fungi to mammals. RNAi-mediated knockdown of Caenorhabditis elegans homolog nath-10 resulted in embryonic lethality (43), and disruption of the Drosophila melanogaster homolog CG1994 by P element insertion caused increased mortality during the early development (44). These observations support the functional and physiological importance of RRA1 in other eukaryotes.
The mammalian homolog of RRA1 is NAT10. Human NAT10 (also known as hALP) was initially identified as a transcriptional factor that interacts with the promoter region of hTERT (telomerase reverse transcriptase) (45). Recombinant NAT10/hALP (amino acids 164 -834) lacking the N-terminal domains had an ability to acetylate calf thymus histones in vitro in the presence of acetyl-CoA (without ATP), indicating that NAT10/hALP has a histone acetyltransferase activity that might promote hTERT transcription via decondensation of chromatin structure (45). However, based on the crystal structure of TmcA, the N-terminal domain of the recombinant NAT10/hALP that was missing from that construct contains a functionally important region (DUF1726) that constitutes the RNA helicase/ATPase domain required for the tRNA acetyltransferase activity (28,46). Therefore, it remains to be conclusively determined whether full-length NAT10 has an efficient and specific histone acetyltransferase activity. NAT10 is predominantly localized in the nucleolus in interphase, but in the mitotic midbody during telophase (47). Knockdown of NAT10 caused defects in nucleolar assembly and cytokinesis and also reduced the level of acetylated ␣-tubulin, leading to G 2 /M arrest (47). NAT10 can acetylate porcine tubulin in vitro (48), although the precise positions that are acetylated have not yet been determined. These observations suggest that NAT10 plays an important role in cell division by facilitating re-formation of the nucleolus and midbody in the late phase of cell mitosis as well as stabilization of microtubules. Assuming that NAT10 is a functional homolog of RRA1, it may play multiple functions required for both ribosome biogenesis and cell division. Furthermore, this protein is associated with several human diseases; 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 ac 4 C1773 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 5Ј-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).
NAT10 was highly expressed specifically in malignant tumors (47), and it is essential for growth of a subtype of epithelial ovarian cancer with poor prognosis (49). More recent work showed that NAT10 is the molecular target of a chemical compound that treats laminopathies, including premature aging syndromes, by correcting nuclear architecture (48). Functional studies of NAT10 as an rRNA-acetyltransferase should facilitate the development of innovative strategies for treating cancer and premature aging.
Depletion of Rra1p resulted in high level accumulation of 23 S pre-rRNA, leading to a huge reduction of 18 S rRNA (Fig.  3B) and the 40 S subunit (Fig. 3D). No ac 4 C1773 was present in the 23 S pre-rRNA that accumulated in the rra1 ts strain, whereas residual 18 S rRNA was fully modified with ac 4 C1773 (Fig. 3E). These results demonstrated that ac 4 C1773 formation is essential for processing of 23 S pre-rRNA at the A0, A1, and A2 sites to yield 20 S pre-rRNA (Fig. 3A). Cleavages at these sites in 35 S and 23 S pre-rRNAs are dependent on the presence of U3 snoRNA, which base pairs with pre-rRNA to assemble the small 40 S subunit processome, a complex that contains Ͼ70 proteins (3,34). Given that Rra1p is a component of the 90 S pre-ribosomal particle, which contains U3 snoRNP and other components (50), it is likely that Rra1p itself or ac 4 C1773 in pre-rRNA plays a critical role in processing at the A0, A1, and A2 sites. For example, the RNA helicase-like activity of Rra1p might modulate the small 40 S subunit processome to facilitate the rRNA processing (33). Alternatively, a putative reader protein that specifically recognizes ac 4 C1773 might recruit unidentified endonucleases responsible for cleavage at these sites.
Based on the results of our in vitro ac 4 C1773 formation assay, Rra1p preferentially recognizes the conserved sequence and structure of helix 45 in 18 S rRNA (Fig. 2E). In particular, the CCG sequence at positions 1772-1774 (Fig. 1A) is essential for ac 4 C1773 formation. Intriguingly, we observed higher rates of ac 4 C1773 formation when the base pairs of the CCG sequence were disrupted. Considering that Rra1p has an RNA helicase domain that is essential for ac 4 C1773 formation, Rra1p might unwind the local duplex of helix 45 via its helicase activity to acetylate C1773. A similar mechanism was previously proposed based on a structural study of TmcA, which is responsible for ac 4 C formation in tRNA (46).
To investigate the physiological significance of ac 4 C modification in 18 S rRNA, we asked whether rRNA processing might be controlled by cellular energy status. Immediately after depletion of nuclear acetyl-CoA in the acs2 ts strain, we clearly observed accumulation of the 23 S pre-rRNA over time (Fig. 4B), strongly indicating that low levels of nuclear acetyl-CoA result in hypomodification of ac 4 C1773, leading in turn to accumulation of 23 S rRNA. This finding suggests that 18 S rRNA processing can be attenuated by reduced acetyl-CoA concentration in the nucleus under some stress conditions, including nutrient starvation. Because ribosome biogenesis is a cellular process that consumes a lot of energy (5), it is physiologically reasonable that nutrient-starved cells would regulate ribosome assembly at the stage of rRNA processing. In particular, the 40 S subunit is responsible for translation initiation, a step targeted in numerous strategies for regulation of protein synthesis (51,52). Regulation of biogenesis of the 40 S subunit is a more direct means to control overall protein synthesis than alteration of translational efficiency by regulation of the 60 S subunit. Upon acetyl-CoA depletion under certain stress conditions, shutting off the supply of 18 S rRNA by reducing the ac 4 C level in pre-rRNA would provide an effective means for translational control. The cellular concentration of acetyl-CoA is reduced upon entry into stationary phase from logarithmic phase (53), and rRNA transcription is down-regulated in response to such reduced levels of acetyl-CoA (20). Thus, regulation of 18 S rRNA processing might also occur in stationary phase.
In summary, we showed that ac 4 C1773 is present in S. cerevisiae 18 S rRNA and that Rra1p catalyzes ac 4 C1773 formation using ATP and acetyl-CoA as substrates. We also demonstrated that ac 4 C1773 is an essential modification required for processing of 23 S pre-rRNA to yield 20 S pre-rRNA. Together, our results reveal that biogenesis of the 40 S subunit can be regulated by sensing nuclear acetyl-CoA concentration.