ADAR1 downregulation by autophagy drives senescence independently of RNA editing by enhancing p16INK4a levels

Abstract

Cellular senescence plays a causal role in ageing and, in mice, depletion of p16^INK4a-expressing senescent cells delays ageing-associated disorders^1,2. Adenosine deaminases acting on RNA (ADARs) are RNA-editing enzymes that are also implicated as important regulators of human ageing, and ADAR inactivation causes age-associated pathologies such as neurodegeneration in model organisms^3,4. However, the role, if any, of ADARs in cellular senescence is unknown. Here we show that ADAR1 is post-transcriptionally downregulated by autophagic degradation to promote senescence through p16^INK4a upregulation. The ADAR1 downregulation is sufficient to drive senescence in both in vitro and in vivo models. Senescence induced by ADAR1 downregulation is p16^INK4a-dependent and independent of its RNA-editing function. Mechanistically, ADAR1 promotes SIRT1 expression by affecting its RNA stability through HuR, an RNA-binding protein that increases the half-life and steady-state levels of its target mRNAs. SIRT1 in turn antagonizes translation of mRNA encoding p16^INK4a. Hence, downregulation of ADAR1 and SIRT1 mediates p16^INK4a upregulation by enhancing its mRNA translation. Finally, Adar1 is downregulated during ageing of mouse tissues such as brain, ovary and intestine, and Adar1 expression correlates with Sirt1 expression in these tissues in mice. Together, our study reveals an RNA-editing-independent role for ADAR1 in the regulation of senescence by post-transcriptionally controlling p16^INK4a expression.

Extended Data Figure 1:. ADAR1 downregulation during senescence

a, Expression of ADAR1 in the indicated control proliferating and quiescent IMR90 cells induced by contact inhibition and serum starvation was determined by immunoblot analysis. The experiment was repeated three times independently with similar results. b, Expression of ADAR2 in control proliferating (PD30), replicative senescent (PD80), and RAS-induced senescent IMR90 cells was determined by immunoblot. Mouse brain tissue was used as a positive control for ADAR2 expression. The experiment was repeated three times independently with similar results. c, IMR90 cells were induced to senesce by Etoposide (100 μM for 48h) and examined for expression of the indicated proteins by immunoblot at the indicated point points. The experiment was repeated three times independently with similar results. d-h, IMR90 cells expressing the indicated sgRNAs were analyzed for expression of the indicated proteins by immunoblot (d), or stained for SA-β-gal activity (e). Percentages of SA-β-Gal positive cells were quantified (from >200 cells) (f). The indicated cells were also subjected to colony formation assay to determine senescence-associated growth arrest (g), and the intensity of colony formed by the indicated cells was quantified by NIH ImageJ software (h). Scale bars = 100 μm. i-j, Mouse embryonic fibroblasts isolated from wildtype control and Adar1 knockout mouse were stained for SA-β-Gal activity (i). Percentages of SA-β-Gal positive cells were quantified (from >200 cells) (j). Scale bars = 100 μm. k-l, Schematic of the construct used to knock down Adar1 expression in mouse (k) and outline of hydrodynamic tail vein injection to induce senescence by Adar knockdown in mouse liver (l). m, Validation of Adar1 knockdown in mouse cells. Mouse ID8 cells expressing the indicated shControl or shAdar1 were examined for expression of the indicated proteins. n-o, ADAR1 mRNA expression was determined by RT-qPCR analysis RAS-induced senescent IMR90 cells (n) or Etoposide induced senescent IMR90 cells (o). Data represent the mean ± s.d. of three biologically independent experiments. P values were calculated using a two-tailed Student’s t-test. Source numerical data and unprocessed blots are available in source data.

Extended Data Figure 2:. Autophagy contributes to ADAR1 downregulation during senescence

a-b, Expression of the indicated proteins was determined by immunoblot in IMR90 cells treated with vehicle control or etoposide to induce senescence with or without simultaneous treatment of Lys05 (a) and relative ADAR1 levels were quantified (b). The experiment was repeated three times independently with similar results. c-d, Expression of the indicated protein was determined by immunoblot in proliferating (PD28) and replicative senescent (PD73) IMR90 cells treated with or without Leupeptin (c) and relative ADAR1 levels were quantified (d). The experiment was repeated three times independently with similar results. e, Expression of the indicated proteins was determined by immunoblot in young and proliferative IMR90 (PD30) treated with or without Lys05 (e). The experiment was repeated three times independently with similar results. f-g, Expression of the indicated proteins was determined by immunoblot in IMR90 cells induced to undergo senescence by oncogenic H-RAS^G12V with or without expressing shATG7 (f) and relative ADAR1 levels were quantified (g). h-k, Expression of the indicated proteins was determined by immunoblot in control early passage proliferating or late passage replicative senescent IMR90 (h) or WI38 cells (j) with or without ATG7 knockdown. And relative ADAR1 levels were quantified in i and k. l-m, Representative confocal images of immunostaining of endogenous ADAR1 and LC3 in control proliferating and RAS-induced senescent IMR90 (l). And the percentage of cells with co-localization of cytoplasmic ADAR1 puncta and LC3 (as exemplified by arrows pointed puncta) were quantified in 4 independent biological repeats (m). n, Co-immunoprecipitation analysis between ADAR1 and LC3 in cytoplasmic extracts from the indicated proliferating (PD21) and replicative senescent (PD68) IMR90 cells. Downregulation of ADAR1 was validated in whole cell lysates. o, IMR90 cells transfected with mCherry-ADAR1 p150 were induced senescence by expressing oncogenic H-RAS^G12V. Localization of mCherry-ADAR1p150 was visualized and DAPI counter staining was used to visualize nuclei. Scale bars = 10 μm. Data represent the mean ± s.e.m. of three biologically independent experiments unless otherwise stated. P values were calculated using a two-tailed Student’s t-test. Source numerical data and unprocessed blots are available in source data.

Extended Data Figure 3:. ADAR1 downregulation does not induce robust SASP

a, Immunostaining for macroH2A1.2 in IMR90 cells with or without ADAR1 knockdown (shADAR1 #1). Scale bar = 10 μm. Asterisks indicated inactivated X chromosome in female IMR90 cells and arrows indicate examples of macroH2A1.2 positive SAHF. The experiment was repeated three times independently with similar results. b-c, Immunostaining for macroH2A1.2 in control proliferating and replicative senescent WI38 cells with or without ATG7 knockdown (b). Cells with macroH2A1.2 positive foci were quantified (> 200 cells) (c). Scale bar = 10 μm. d, Global RNA A/I editing was analyzed based on the RNA-seq A/G reads in the indicated cells. e, Changes in expression of SASP genes in the indicated ADAR1 knockdown vs. control IMR90 cells (KD/Ctr) determined by RNA-seq analysis. SASP genes with at least 10 counts at least 1 FPKM level are reported. f, Secreted cytokines in conditioned media from IMR90 cells with or without ADAR1 knockdown (shADAR1 #1) was detected using an antibody-based cytokine array. Conditioned media collected from RAS-induced senescent IMR90 cells was used as a positive control. g-h, Expression of the indicated proteins was determined by immunoblot in IMR90 cells expressing shControl or shADAR1s (g) or sgGFP control or sgADAR1 (h). The experiment was repeated three times independently with similar results. i-j, Representative images of SA-β-gal staining of IMR90 cells cultured with conditioned media from indicated cells (i). Percentages of SA-β-gal positive cells were quantified (from > 200 cells) (j). k-m, IMR90 cells expressing shControl or shADAR1 (#1) with or without ectopically expression of wildtype ADAR1 p110 or a deaminase inactive mutant E912A ADAR1 p110 were analyzed for SA-β-Gal activity (k) and positive cells were quantified (from >200 cells) (l). In addition, the indicated cells were subjected to colony formation assay (m). Scale bars = 100 μm. Data represent the mean ± s.d. in l and m, or s.e.m. in c and j of three biologically independent experiments. P values were calculated using a two-tailed Student’s t-test. P values were calculated using a two-tailed Student’s t-test. Source numerical data and unprocessed blots are available in source data.

Extended Data Figure 4:. ADAR1 regulates SIRT1 expression

a, Expression of p16^INK4a mRNA expression was analyzed by RT-qPCR in the indicated IMR90 cells. b, Heatmap visualization of detected (at least 10 read counts and 1 FPKM) and differentially expressed genes (> 2-fold) between IMR90 cells expressing shControl or shADAR1 as determined by the RNA-seq analysis. c, Expression of SIRT1 mRNA was analyzed by RT-qPCR in the indicated IMR90 cells. d, Heatmap of HuR-associated mRNAs that are significantly reduced by ADAR1 knockdown in IMR90 cells. List of 5 genes related to autophagy pathway was included in the top and the top 25 of the rest of genes identified were listed. IMR90 cells expressing shHuR were used as a positive control. e-f, Expression of the indicated proteins was determined by immunoblot in the IMR90 cells expressing shControl or the indicated shHuR (e) and relative ADAR1 levels were quantified (f). g, Immunoblot of HuR in IMR90 cells with the indicated treatment immunoprecipitated by an anti-HuR antibody. An isotype matched IgG was used as a negative control. h, Expression of the indicated proteins was determined by immunoblot in the indicated IMR90 cells. Data represent the mean ± s.d. in a and c and ± s.e.m. of three biologically independent experiments. P values were calculated using a two-tailed Student’s t-test. Source numerical data and unprocessed blots are available in source data.

Extended Data Figure 5:. Age-associated downregulation of Adar1

a, Expression of the indicated proteins was determined by immunoblot in the indicated tissues harvested from young (4 months) and aged (24 months) mice. The experiment was repeated two times independently with similar results. b-c, The cortex tissues from brains of young (4 months) and aged (24 months) mice were analyzed for expression of the indicated proteins by immunoblot. The intensity of Adar2 (b) or Adar3 (c) immunoblot was quantified by NIH ImageJ software in young and aged mouse cortex tissues and normalized against a loading control β-actin expression. d-g, Expression of Adar1 and Sirt1 was determined by immunoblot in the intestine tissues harvested from young mice (4 months), and aged mice (20 months) treated with either vehicle PBS control or Lys05 (10 mg/kg daily) for 2 weeks (d). The intensity of Adar1 (e) and Sirt1 (f) immunoblot was quantified using NIH ImageJ software in indicated groups by normalizing against a loading control β-actin expression. Correlation between Adar1 and Sirt1 protein expression as determined by a two-sided Pearson correlation analysis (g). h-i, Expression of Adar1 mRNA was analyzed by RT-qPCR in ovaries (h) and intestines (i) from young (4 months) and aged mice (20 months) treated with either vehicle PBS control or Lys05. j-m, Expression of p62 was determined by immunoblot in ovaries (j) and intestines (l) from young mice (4 months) and aged mice (20 months) treated with either vehicle PBS control or Lys05. The intensity of p62 immunoblot in ovaries (k) and intestines (m) was quantified using NIH ImageJ software by normalizing against a loading control β-actin expression. Data represent the mean ± s.d. in b, c, e and f, or s.e.m. in h, I, k and m of biologically independent experiments. P values were calculated using a two-tailed Student’s t-test. Source numerical data and unprocessed blots are available in source data.

Figure 1:. ADAR1 downregulation drives senescence.

a, IMR90 cells at the indicated passage double (PD) were harvested and analyzed for expression of the indicated proteins by immunoblot. The experiment was repeated three times independently with similar results. b, IMR90 cells were infected with retrovirus encoding oncogenic H-RAS^G12V to induce senescence. Cells were harvested at the indicated timepoints to analyze for the expression of the indicated proteins by immunoblot. The experiment was repeated three times independently with similar results. c-e, IMR90 cells infected with the indicated shRNAs were analyzed for expression of the indicated proteins by immunoblot (c) or stained for SA-β-gal activity and quantified for percentage of positive cells (d). In addition, the indicated cells were subjected to colony formation assay and the intensity of colonies formed by the indicated cells were quantified (e). Scale bars = 100 μm. f-g, IMR90 cells expressing shControl or shADAR1 (#1) with or without ectopic ADAR1p110 and ADAR1p150 restoration were analyzed for expression of the indicated proteins by immunoblot (f) or stained for SA-β-Gal activity and quantified for percentage of positive cells (g). Scale bars = 100 μm. h-i, Representative images of SA-β-gal staining of mouse liver from the indicated groups at day 6 post injection (h), and the number of SA-β-gal-positive cells in the indicated groups (n=5 biologically independent mice in shControl and shAdar1 expressing group and n=3 mice for NRas^G12V group). Scale bars = 100 μm. j-k, Immunostaining for NRas and Adar1 in mouse liver from NRas^G12V-expressing group in the hydrodynamic tail vein injection model. DAPI counter staining was used to visualize the nuclei. Asterisks indicated examples of decreased level of Adar1 in NRas^G12V-expressing senescent hepatocyte (j) and relative Adar1 intensity was quantified (k). (n=3 biologically independent mice). Scale bars = 10 μm. Data represents the mean ± s.d. in d, e and i, or s.e.m. in k of three biologically independent experiments. Source numerical data and unprocessed blots are available in source data.

Figure 2:. Autophagy-dependent downregulation of ADAR1 during senescence.

a, The indicated IMR90 cells were treated with or without Lys05 and analyzed for expression of the indicated proteins. The experiment was repeated three times independently with similar results. b, Co-immunoprecipitation analysis between ADAR1 and LC3 in cytoplasmic extracts from the indicated IMR90 cells. The experiment was repeated three times independently with similar results. c, Scheme of ADAR1 showing the location of four LC3-interacting region (LIR) motifs. d-e, Co-immunoprecipitation analysis was performed between ectopic ADAR1 and LC3 in ER:RAS-expressing IMR90 cells with endogenous ADAR1 knockdown and expression of mCherry-GFP tagged ectopic wildtype or the LIR motif mutant ADAR1 with or without RAS induction (d). In addition, expression of ectopically expressed ADAR1 wildtype or mutants was examined by immunoblot using an anti-ADAR1 antibody (e). The experiment was repeated three times independently with similar results. Note that shADAR1 #1 does not target ectopic ADAR1 because shADAR1 targets 3’ UTR region. f-g, Immunostaining for LAMP1 in IMR90 cells expressing mCherry-GFP-ADAR1p110 with or without RAS induction. The arrows indicate examples of cytoplasmic ADAR1 puncta with strong mCherry and faded GFP signal, indicating the localization of ADAR1 in acidic (auto)lysosomes (f). The relative intensities of the mCherry, GFP and LAMP1 signals of a typical senescent cell as in f were quantified (g). Dash lines indicate nuclei. Scale bars = 10 μm. The experiment was repeated three times independently with similar results. h-i, IMR90 cells transfected with mCherry-ADAR1 p110 were induced to senescence by expressing RAS, and treated with or without p38 inhibitor SB203580. Localization of mCherry-ADAR1p110 was visualized (h), and the indicated distribution pattern was quantified (i). Dash lines indicate nuclei. Scale bars = 10 μm. j-k, Immunoblot of the indicated proteins in IMR90 cells expressing RAS, and treated with or without p38i SB203580 or MG132 (j). And the relative expression of ADAR1p110 was quantified (k). Data represents the mean ± s.e.m. in i and k of three biologically independent experiments. P values were calculated using a two-tailed Student’s t-test. Source numerical data and unprocessed blots are available in source data.

Figure 3:. ADAR1 loss promotes heterochromatin formation independent of its RNA editing function

a, IMR90 cells expressing indicated shRNAs were analyzed for expression of the indicated proteins. The experiment was repeated three times independently with similar results. b, IMR90 cells expressing the indicated sgRNAs were analyzed for expression of the indicated proteins. The experiment was repeated three times independently with similar results. c-d, Immunostaining for H3K9me2 in mouse livers from indicated groups in the hydrodynamic tail vein injection model (c), which was quantified (d) from three biologically independent mice. The arrow indicates an example of clusters of shAdar1-expressing hepatocytes with an increase in H3K9me2 levels. e-f, Immunostaining for HIRA and PML in the indicated IMR90 cells (d), and the percentage of HIRA/PML foci positive cells were quantified (> 200 cells) (e). g-h, Immunostaining for macroH2A1.2 in IMR90 cells expressing the indicated sgRNAs (f), and the percentage of macroH2A1.2 foci positive cells were quantified (> 200 cells) (g). Asterisks indicated inactivated X chromosome in female IMR90 cells and arrows point examples of macroH2A1.2 positive SAHF. i-j, Immunostaining for macroH2A1.2 in control and RAS-induced senescent IMR90 cells with and without Lys05 treatment (i), and the percentage of macroH2A1.2 foci positive cells were quantified (> 200 cells) (j). k-l, Immunostaining for macroH2A1.2 in proliferating (PD26) and replicative senescent (PD63) IMR90 cells with and without ATG7 knockdown (k), and the percentage of macroH2A1.2 foci positive cells were quantified (> 200 cells) (l). m, IMR90 cells expressing shControl or shADAR1 (#1) with or without rescue by ectopically expression of wildtype ADAR1 p110 or a deaminase inactive mutant E912A ADAR1 p110 were analyzed for expression of the indicated proteins. The experiment was repeated three times independently with similar results. Data represent the mean ± s.d. in f and h, or s.e.m. in d, j and l of three biologically independent experiments. Scale bars = 10 μm. P values were calculated using a two-tailed Student’s t-test. Source numerical data and unprocessed blots are available in source data.

Figure 4:. ADAR1 loss promotes senescence by upregulating p16^INK4a via SIRT1

a-e, IMR90 cells expressing shControl or shADAR1 with or without simultaneous shpRb (a) or shp16^INK4a (b) expression were analyzed for expression of the indicated proteins or stained for SA-β-Gal activity (c). Percentage of SA-β-Gal positive cells were quantified (> 200 cells) (d). The indicated cells were subjected to colony formation assay to determine senescence-associated growth arrest (e). Scale bars = 100 μm. The experiment was repeated three times independently with similar results. f, IMR90 cells expressing shControl or shADAR1s were analyzed for expression of the indicated proteins. The experiment was repeated three times independently with similar results. g-j, IMR90 cells expressing shControl or shADAR1 (#1) with or without ectopic V5-tagged SIRT1 expression were analyzed for expression of the indicated proteins (g). The indicated cells were stained for SA-β-Gal activity (h) and positive cells were quantified (> 200 cells) (i). The indicated cells were also subjected to colony formation (j). Scale bars = 100 μm. k-l, IMR90 cells expressing shControl or shADAR1 (#1) were subjected to RNA immunoprecipitation analysis using an anti-HuR antibody for SIRT1 mRNA (k), or treated with actinomycin D and chased for 2, 4 and 6 hours and expression of SIRT1 mRNA was determined by RT-qPCR (l). m, The indicated IMR90 cells were subjected to RNA immunoprecipitation analysis using an anti-HuR antibody for SIRT1 mRNA. n, IMR90 cells expressing shControl or shADAR1 (#1) were subjected to RNA immunoprecipitation analysis using an anti-ELF3a antibody for p16^INK4a mRNA. o, Polysome profiling assay was performed in IMR90 cells with or without ADAR1 knockdown. The association of p16^INK4a mRNA with polysomes or monosomes was quantified using RT-qPCR. The experiment was repeated two times with similar results. p, A model on how autophagy-mediated ADAR1 downregulation promotes senescence via upregulating p16^INK4a through SIRT1 post-transcriptionally. Data represent the mean ± s.d. in d, e, i, j, k, l and n, or s.e.m. in m and o of three biologically independent experiments unless otherwise stated. P values were calculated using a two-tailed Student’s t-test. Source numerical data and unprocessed blots are available in source data.

Figure 5:. Inhibition of lysosomal pathway reverses age-associated decline in ADAR1 expression

a-c, The cortex tissues from brains of young (4 months) and aged (24 months) mice were analyzed for expression of the indicated proteins by immunoblot (a). The intensity of Adar1 (b) and Sirt1 (c) immunoblot was quantified by NIH ImageJ software in young and aged mouse cortex tissues and normalized against a loading control β-actin expression. Correlation between Adar1 and Sirt1 protein expression was determined by Pearson correlation analysis (d). e-h, Expression of Adar1 and Sirt1 was determined by immunoblot in the ovary tissues harvested from young mice (4 months), and aged mice (20 months) treated with vehicle PBS control or Lys05 (10 mg/kg daily) for 2 weeks (e). The intensity of Adar1 (f) and Sirt1 (g) immunoblot was quantified by NIH ImageJ software in indicated groups and normalized against a loading control β-actin expression. Correlation between Adar1 and Sirt1 protein expression was determined by a two-sided Pearson correlation analysis (h). n=5 biologically independent mice per group. Data represent the mean ± s.d. in b and c, or s.e.m. in f and g of biologically independent experiments. P values were calculated using a two-tailed Student’s t-test in b, c, f and g, or a two-sided Pearson correlation analysis in d and h. Source numerical data and unprocessed blots are available in source data.