Human NAT10 is an ATP-dependent RNA acetyltransferase responsible for N4-acetylcytidine formation in 18 S ribosomal RNA (rRNA)

Abstract

Human N-acetyltransferase 10 (NAT10) is known to be a lysine acetyltransferase that targets microtubules and histones and plays an important role in cell division. NAT10 is highly expressed in malignant tumors, and is also a promising target for therapies against laminopathies and premature aging. Here we report that NAT10 is an ATP-dependent RNA acetyltransferase responsible for formation of N(4)-acetylcytidine (ac(4)C) at position 1842 in the terminal helix of mammalian 18 S rRNA. RNAi-mediated knockdown of NAT10 resulted in growth retardation of human cells, and this was accompanied by high-level accumulation of the 30 S precursor of 18 S rRNA, suggesting that ac(4)C1842 formation catalyzed by NAT10 is involved in rRNA processing and ribosome biogenesis.

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

FIGURE 1.

N⁴-Acetylcytidine is present at position 1842 in human 18 S rRNA. A, the secondary structure of human 18 S rRNA (left) with a detailed description of helix 45 and the surrounding region (right box), with modified nucleosides: ac⁴C at position 1842, N⁶,N⁶-dimethyladenosine (m⁶₂A) at positions 1850 and 1851, 2′-O-methylcytidine (Cm) at position 1703, and N⁶-methyladenosine (m⁶A) at position 1832. The in vitro transcribed RNA segment (G1820–A1869) used for ac⁴C formation is shown in blue. The ac⁴C-containing hexamer fragment generated by RNase T₁ digestion is shown in green. Watson-Crick base pairs and wobble pairs are shown as bars and dots, respectively. B, capillary LC/ESI-MS analysis of fragments of RNase T₁-digested 18 S rRNA from HEK293 cells. The upper panel shows the base-peak chromatogram (BPC), and the lower panel shows the mass chromatogram of the doubly charged ion of the ac⁴C-containing hexamer fragment (UUUCac⁴CGp, m/z 965.6). C, CID spectrum of the ac⁴C-containing hexamer fragment. The doubly charged ion (m/z 965.6) was used as the parent for CID. The sequence was confirmed by assignment of the product ions. The nomenclature for product ions is according to a previous study (24).

FIGURE 2.

NAT10 is an ATP-dependent RNA acetyltransferase involved in 18 S rRNA processing. A, Western blotting (WB) of endogenous NAT10 to estimate the knockdown efficiency of three siRNAs against NAT10 (siNAT10) and an siRNA against luciferase (siLUC), used as a negative control. Coomassie Brilliant Blue (CBB) staining of each sample is shown as a loading control (LC). Knockdown efficiencies of the three siRNAs, estimated from the levels of NAT10 mRNA (normalized by ACTB mRNA), were 10.6% (siNAT10a), 12.5% (siNAT10b), and 3.2% (siNAT10c), respectively. B, growth curves of HeLa cells treated with siRNAs against NAT10 (green and blue lines for siNAT10b and siNAT10c, respectively) or luciferase (black line for siLUC). Each plot is the average of three independent cultures (bars, ±S.D.). C, flow cytometry of HeLa cells treated with siRNAs against NAT10 (right panel) or luciferase (left panel), stained with annexin V-FITC (x-axis) and propidium iodide (y-axis). D, capillary LC/ESI-MS analyses of fragments of RNase T₁-digested 18 S rRNAs from the HEK293 cells treated with siNAT10a against NAT10 (lower panels) or luciferase (upper panels). Mass chromatograms of the doubly charged ions of the ac⁴C-containing hexamers (UUUCac⁴CGp, m/z 965.6) and control fragments (AUUAmAGp, m/z 987.1) 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. E, schematic depiction of the rRNA processing pathways in humans. In the canonical pathway, 47 S pre-rRNA is processed by endonucleolytic cleavages at sites 01 and 02 to yield 45 S pre-rRNA, which is then processed into 30 S pre-rRNA by cleavage at site 2 in ITS1. Precursors for large subunit formation are not shown. 30 S pre-rRNA is further processed into 21 S pre-rRNA by removal of the 5′-ETS. Finally, 18 S rRNA is produced by removal of ITS1. Complementary regions of probes a, b, and c are indicated. The position of ac⁴C1842 is indicated by the circle flag. Processing sites in pre-rRNA are indicated. F, accumulation of 30 S pre-rRNA and disappearance of 21 S pre-rRNA upon NAT10 depletion by siNAT10b. Precursors of 18 S rRNA were detected by Northern blotting. Steady-state levels of 28 S and 18 S rRNAs were visualized by EtBr staining, as a loading control. The 45 S and 30 S pre-rRNAs were detected by all three probes, whereas 21 S pre-rRNA was detected only by probes b and c. G, sucrose density gradient centrifugation profiling of ribosomal subunits in HeLa cells treated with siRNAs for luciferase (siLUC, black line) and NAT10 (siNAT10c, blue line). Absorbance at 254 nm was monitored from the top to bottom of the centrifugation tubes. H, in vitro reconstitution of ac⁴C1842 in the 50-mer RNA segment, including helix 45 (Fig. 1A) in the presence or absence of recombinant Nat10, ATP, and acetyl-CoA. The left and right panels show mass chromatograms of the hexamer fragments carrying ac⁴C1842 (UUUCac⁴CGp, m/z 965.6) and C1842 (UUUCCGp, m/z 944.6), respectively. I, confirmation of the reconstituted ac⁴C1842 in the 50-mer RNA fragment. A CID spectrum of the ac⁴C-containing hexamer fragment digested with RNase T₁ is shown. The doubly charged ion (m/z 965.6) was used as the parent for CID. The sequence was confirmed by assignment of the product ions. Nomenclature for product ions is as suggested in a previous study (24).