Fig 2: NAT10 acetylates Che-1 at K228. (A) In vitro acetylation experiments were performed with GST-Che-1 and purified Flag-NAT10 or Flag-NAT10 GE as described in the Materials and Methods. Acetylation of Che-1 was evaluated by Western blot using an anti-acetyl-lysine antibody. (B) HCT116 cells were transfected with the indicated plasmids. Acetylation of Che-1 was evaluated by Western blot using an anti-Che-1 antibody on anti-acetyl-lysine antibody-specific immunoprecipitants. Inputs were evaluated by Western blot using the indicated antibodies. (C) Cell lysates were prepared from HCT116 Ctrl cells or HCT116 NAT10 KO cells (c1 or c2). Acetylation of Che-1 was evaluated by Western blot using an anti-Che-1 antibody on the anti-acetyl-lysine immunoprecipitants. The expression of NAT10 and Che-1 was evaluated by Western blot using the indicated antibodies. (D) The schematic diagram represents the Che-1 deletion mutant constructs. (E) In vitro acetylation experiments were performed with purified GST-Che-1-N, GST-Che-1-M or GST-Che-1-C fusion proteins and purified Flag-NAT10 as described in the Materials and Methods. Acetylation of Che-1 was evaluated by Western blot using an anti-acetyl-lysine antibody. (F) The potential acetylation sites on Che-1-M were predicted using the biocuckoo website. (G) In vitro acetylation experiments were performed with the purified GST-Che-1, GST-Che-1-3KR, GST-Che-1-K138R, GST-Che-1-K145R or GST-Che-1-K228R fusion protein and Flag-NAT10 as described in the Materials and Methods. Acetylation of Che-1 was evaluated by Western blot using an anti-acetyl-lysine antibody. (H) HCT116 cells were transfected with the indicated plasmids. Cells were harvested, and immunoprecipitation was performed to evaluate the level of Che-1 acetylation (upper). The expression levels of Flag-tagged proteins in the cell lysates were evaluated by Western blot using the indicated antibodies (lower). (I) U2OS cells were transfected with the indicated plasmids. Immunoprecipitation was performed using an anti-acetyl-lysine antibody to evaluate the level of Che-1 acetylation (upper). The expression levels of Flag-tagged proteins in the cell lysates were evaluated by Western blot using the indicated antibodies (lower). (J) HCT116 cells were cultured in medium containing different concentrations of glucose for 18 h. The acetylation levels of Che-1 in the anti-acetyl-lysine antibody-specific immunoprecipitants were evaluated using an anti-Che-1 antibody (upper). Total Che-1 levels were evaluated by western blot (lower). (K) HCT116 cells were transfected with the indicated plasmids. Cell lysates were prepared and subjected to Western blot for evaluation of the indicated proteins. See also Supplementary Figure S4.

Fig 3: Nucleolar enrichment of NAT10 requires nascent pre-rRNA. (A) U2OS or HEK293T cells were cultured under normal conditions (ctrl), in glucose-deprived medium (Glu (-)) or in AICAR (2 mM)-containing medium for 36 h. Indirect immunofluorescence analysis was performed to determine the cellular localization of NAT10 (red) and UBF1 (green). Nuclei were stained with DAPI. Scale bar, 10 µM. (B) The cellular localization of NAT10 (red) and UBF1 (green) was visualized by indirect immunofluorescence after the cells were permeabilized and incubated with RNase (1 mg/ml, 10 min). Nuclei were stained with DAPI. Scale bar, 10 µM. (C) HEK293T cells transfected with Flag-NAT10 were cultured in the absence or presence of RNase (20 µg/ml). Immunoprecipitation was performed with an anti-Flag antibody. Flag-NAT10 and UBF1 in the immunoprecipitants were evaluated by immunoblot. (D) RNA immunoprecipitation (RIP) experiments were performed with cell lysates from HEK293T cells expressing Flag-NAT10 or Flag-Sirt1. Cell lysates were prepared after cells were exposed to UV light to crosslink the RNA with proteins and then incubated with anti-Flag beads. After elution with a Flag peptide and decrosslinking, coprecipitated RNA was purified and analyzed by RT-qPCR. The percentage of precipitated RNA relative to the total RNA is shown. Error bars indicate the SEM (n = 3). The positions of precipitated rRNA are shown in the upper panel. (E) Purified Flag-NAT10 or Flag-Sirt1 was incubated with streptavidin-coated Dynabeads (ctrl) or Dynabeads containing 5' ETS RNA (+10/+389). Western blotting was performed to evaluate the RNA-bound proteins using an anti-Flag antibody. (F) HCT116 cells expressing Flag-NAT10 were treated with AICAR (2 mM) for 12 h. Flag-NAT10-RNA complexes were isolated and analyzed as described in D. Error bars indicate the SEM (n = 3). The positions of precipitated rRNA are shown in the upper panel of D. (G) HCT116 cells were cultured in medium containing 1.0 g/l, 0.5 g/l, or 0 g/l glucose for 18 h. Immunoprecipitation was performed with an anti-NAT10 antibody, and Che-1 expression in the precipitants was evaluated by immunoblot. (H) HCT116 cells were cultured in glucose-free medium for the indicated amounts of time. Immunoprecipitation was performed with an anti-Che-1 antibody, and NAT10 expression in the precipitants was evaluated by immunoblot. See also Supplementary Figure S5.

Fig 4: NAT10 inhibits the expression of Redd1 and Deptor in mouse tumor xenografts. (A–C) NAT10 KO HCT116 cells were subcutaneously implanted into SCID mice. Tumors were dissected at the end of the experiment (A). The weights and sizes of the tumor xenografts are shown in B and C, respectively. Error bars indicate the SEM (n = 6). P-values were calculated using two-tailed unpaired Student's t-test. **P < 0.01, *P < 0.05. (D) Western blotting was performed on the tumor xenograft lysates to evaluate the levels of the indicated proteins (left). Densitometry scanning analyses of the Che-1, Deptor and Redd1 bands standardized to ß-actin are summarized and shown (right). Error bars indicate the SEM (n = 6). (E) A working model explaining how NAT10 acetylation regulates rRNA biogenesis and autophagy upon glucose starvation.

Fig 5: NAT10 interacts with Che-1. (A) U2OS cells were transfected with Flag-NAT10. After 48 h, the cells were harvested, and whole cell extracts were immunoprecipitated with an anti-Flag antibody affinity resin. NAT10-binding proteins were resolved by SDS-PAGE, detected by silver staining and analyzed by mass spectrometry. (B) HCT116 cell lysates were immunoprecipitated with a control IgG or anti-NAT10 antibody. Immunoprecipitants were immunoblotted with the indicated antibodies. (C) HCT116 cells were fixed, and immunostaining was performed to determine the localization of Che-1 and NAT10. Immunofluorescence images were acquired with a confocal microscope. Scale bars, 10 µM. (D) GST pull-down assay was performed with purified Flag-NAT10 or Flag-NAT10 GE and GST-Che-1 proteins. Che-1-bound Flag-NAT10 was evaluated by Western blot using an anti-Flag antibody. The amounts of GST fusion proteins used in the GST pull-down assay are shown in the lower panel. (E) GST pull-down assay was performed with purified Flag-NAT10 and GST-Che-1 or its deletion mutants. Che-1-bound Flag-NAT10 was evaluated by Western blot using an anti-Flag antibody (upper). The amounts of GST fusion proteins used in the GST pull-down assays are shown (middle). The schematic diagram represents the GST-Che-1 deletion mutant constructs (lower). (F) HCT116 cells were transfected with the indicated plasmids. Twenty-four hours later, the cells were harvested, and proteins from the cell lysates were subjected to immunoblot analysis for the evaluation of Che-1 and NAT10. Beta-actin was evaluated as a loading control. Error bars indicate the SEM (n = 3) (right panel). (G) HCT116 cells were transfected with the Flag-NAT10 or Flag plasmids. Twenty-four hours later, the cells were treated with cycloheximide (CHX) and harvested at the indicated time points. Proteins from the cell lysates were subjected to immunoblot analysis for the evaluation of NAT10 and Che-1. Beta-actin was evaluated as a loading control (upper panels). Relative Che-1 levels standardized to ß-actin at different time points are shown in the lower panel. Error bars indicate the SEM (n = 3).