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ADAR1 controls apoptosis of stressed cells by inhibiting Staufen1-mediated mRNA decay

Abstract

Both p150 and p110 isoforms of ADAR1 convert adenosine to inosine in double-stranded RNA (dsRNA). ADAR1p150 suppresses the dsRNA-sensing mechanism that activates MDA5–MAVS–IFN signaling in the cytoplasm. In contrast, the biological function of the ADAR1p110 isoform, which is usually located in the nucleus, is largely unknown. Here, we show that stress-activated phosphorylation of ADAR1p110 by MKK6–p38–MSK MAP kinases promotes its binding to Exportin-5 and its export from the nucleus. After translocating to the cytoplasm, ADAR1p110 suppresses apoptosis in stressed cells by protecting many antiapoptotic gene transcripts that contain 3′-untranslated-region dsRNA structures primarily comprising inverted Alu repeats. ADAR1p110 competitively inhibits binding of Staufen1 to the 3′-untranslated-region dsRNAs and antagonizes Staufen1-mediated mRNA decay. Our study reveals a new stress-response mechanism in which human ADAR1p110 and Staufen1 regulate surveillance of a set of mRNAs required for survival of stressed cells.

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Figure 1: Stress-induced cytoplasmic localization of ADAR1p110.
Figure 2: Cytoplasmic localization of ADAR1p110 is regulated by p38 MAP kinase signaling.
Figure 3: Phosphorylation of ADAR1p110 at five sites.
Figure 4: Phosphorylation of ADAR1p110 promotes binding to Xpo5–Ran–GTP.
Figure 5: ADAR1p110 protects stressed cells against apoptosis.
Figure 6: ADAR1 protects mRNAs containing 3′-UTR dsRNA from SMD.
Figure 7: ADAR1 inhibits binding of Staufen1 to the 3′-UTR dsRNA structure.
Figure 8: Stress-response function of ADAR1p110 regulated by MKK6–p38–MSK1/2 MAP kinases.

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Acknowledgements

We thank Z. Paroo (University of Illinois), M. Cobb (University of Texas), S. Janiki (Wistar Institute), G. Dreyfuss (University of Pennsylvania), and L. Maquat (University of Rochester) for various recombinant-protein-expression constructs. We also thank J. Hayden for assistance with fluorescence microscopy, Y. Sakurai for technical assistance, and M. Murphy and J.M. Murray for critical reading of the manuscript. Research reported in this publication was supported by grants to K.N. from the National Institutes of Health (R01GM040536 and R01CA175058), the Macula Vision Research Foundation, and the Japan Society for the Promotion of Science (S13204). C.S. is supported as a member of the Roy and Dianna Vagelos Scholars Program in Molecular Life Sciences at the University of Pennsylvania. M.S. is supported in part by a fellowship from the Japan Society for the Promotion of Science (JSPS 2010-22). We also acknowledge services provided by the Animal, Bioinformatics, Genomics, Imaging, Protein Expression, and Proteomics Shared Facilities of The Wistar Cancer Center, which are supported by the National Cancer Institute (P30CA010815). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Authors and Affiliations

Authors

Contributions

M.S., Y.S., and K.N. designed the study. M.S. conducted fluorescence microscopy experiments, apoptosis analysis, and RIP experiments. M.S. and H.O. conducted ADAR1-Xpo5 interaction studies. M.S. and Y.S. performed deep-sequencing sample preparation, bioinformatics data analysis, and gene expression analysis. C.S. prepared mutant ADAR1 constructs. A.V.K., J.W., and L.C.S. performed bioinformatics analysis of sequencing data. E.S. conducted molecular modeling studies. H.-Y.T. and D.W.S. performed LC-MS/MS analysis and data interpretation. M.S., Y.S., and K.N. supervised research, reviewed data, and wrote and edited the paper. All authors agree to the contents of the final manuscript.

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Correspondence to Kazuko Nishikura.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Cellular localization of wild-type and phosphorylation mutants of mCherry-ADAR1p150 in A172 cells.

(a) UV irradiation did not affect overall cytoplasmic localization of ADAR1p150 in transiently transfected A172 human glioblastoma cells. (b) Fractional move to stress-granules was noted in A172 cells as reported previously (Weissbach and Scadden RNA 18, 462-71, 2012), as confirmed by co-localization of ADAR1p150 with a stress-granule marker, TIA1. TIA1-EGFP was a kind gift of John L. Goodier. (c) Cytoplasmic localization of ADAR1p150 was unaffected by phosphorylation site mutations. Scale bars, 20 μm.

Supplementary Figure 2 Cellular distribution of endogenous ADAR1p150 and p110 proteins in unirradiated and UV-irradiated A172 cells.

Nuclear and cytoplasmic fractions of control and UV-irradiated A172 cells were examined by western blotting analysis. Histone H3 and β-Actin were used as nuclear and cytoplasmic markers, respectively. UV irradiation increased significantly the cytoplasmic ADAR1p110, which was reversed by SB203580 p38 inhibitor.

Supplementary Figure 3 Phosphorylation of ADAR1p110 does not affect its A-to-I RNA-editing activity or its Dicer-promoting activity.

(a) In vitro A-to-I editing assay was carried out using HAT-ADAR1p110-WT, -T/S-to-D, or –T/S-to-A recombinant proteins and 32P-ATP labeled c-myc dsRNA substrate as described previously (Wagner et al. Proc. Natl. Acad. Sci. 86, 2647-51, 1989). A-to-I conversion was quantitated using TLC assay. (b) In vitro Dicing assay was carried out using 5’ 32P-labeled pre-let-7a substrate RNA, and results were quantitated as described previously (Ota et al. Cell 153, 575-89, 2013). (a, b) Data are mean ± S.D. (n = 3, technical replicates); significant differences were identified by two-tailed Student’s t-tests: ***P < 0.001, n.s., not significant. Source data are available online in Supplementary Data Set 1.

Supplementary Figure 4 UV-irradiation-induced cytoplasmic localization of ADAR1p110 depends on Xpo5.

(a) UV irradiation induced cytoplasmic localization of ADAR1p110-WT is inhibited by knockdown of Xpo5 in A172 cells. (b) Cytoplasmic localization of T/S-to-D phosphomimetic mutant of ADAR1p110 is also inhibited in un-irradiated A172 cells by knockdown of Xpo5. (c) Nuclear localization of T/S-to-A phosphorylation inhibitory mutant of ADAR1p110 is inhibited in un-irradiated A172 cells by knockdown of TRN1. Scale bars, 20 μm.

Supplementary Figure 5 Apoptosis induced in ADAR1-knockdown and UV-irradiated cells.

(a) Fluorescence microscopic detection of apoptosis induced in ADAR1 knocked down and UV-irradiated A172 cells in comparison to the cells treated with an inducer of apoptosis, Camptothecin (20 μM, 48 hrs). Apoptosis was evaluated by staining with AnnexinV-Alexa594 and also by CellEventTM Caspase-3/7 Green system (Themo Fischer), which utilizes a fluorogenic substrate for activated Caspase-3 and -7. Scale bars, 200 μm. (b) Apoptosis induced in UV-irradiated U2OS cells was evaluated by ApoTox-Glo Triplex apoptosis assay (Promega). ADAR1p150 specific knockdown did not cause UV-induced apoptosis. Knockdown of Staufen1 or UPF1 suppressed apoptosis induced by ADAR1 knockdown. Positive apoptosis control: un-irradiated U2OS cells treated with Camptothecin (20 μM) for 48 hrs. (c) The relative expression levels of FLAG-ADAR1p110 and endogenous ADAR1 (p150 and p110) in UV-irradiated A172 cells transiently transfected with FLAG-ADAR1p110 expression constructs were examined by western blotting analysis. Expression levels of the exogenous FLAG-ADAR1p110 proteins are about equal to those of endogenous ADAR1p110 proteins (compare lane 1 to lane 4 and 5). (d) The expression levels of ADAR1p150 and p110 as well as two ISGs, IFIT1 and MDA5, in A172 cells were examined by western blotting analysis of cell extracts. Note that UV-irradiation did not affect the expression of ADAR1 (both p150 and p110). Furthermore, ADAR1 knockdown alone did not induce the expression of IFIT1 or MDA5 in both un-irradiated and UV-irradiated cells. However, transfection of poly(I:C) dsRNA (Invivogen) for 12 hrs increased the expression of ADAR1p150 as well as IFIT1 and MDA5 in a dose dependent manner, indicating that the dsRNA sensing mechanism mediated by the MDA5-MAVS-IFN pathway remains intact in this cell line. (e) Apoptosis induced in UV-irradiated A172 cells was evaluated by ApoTox-Glo Triplex apoptosis assay (Promega). Knockdown of Staufen1 and/or UPF1 did not induce but suppressed apoptosis in UV irradiated and ADAR1 knocked down cells. (b, e) Data are mean ± S.D. (n = 4, cell cultures); significant differences were identified by two-tailed Student’s t-tests:***P < 0.001. Source data are available online in Supplementary Data Set 1.

Supplementary Figure 6 Secondary structures of 3′-UTR Alu dsRNAs.

The secondary structures of CASC5 (a) and ATM (b) 3’UTR Alu dsRNA predicted by mfold are shown. A-to-I editing sites detected by RNA-seq data analysis are indicated by red arrows with editing frequency. Primers used for RIP analysis are indicated as black arrows.

Supplementary Figure 7 Colocalization of Staufen1-EGFP and mCherry-ADAR1p110 in the cytoplasm of unirradiated and UV-irradiated A172 cells.

Staufen1 localizes in the cytoplasm of A172 cells regardless of UV irradiation. Scale bars, 20 μm.

Supplementary Figure 8 Quantitative RT–PCR analysis of pre-mRNAs.

Pre-mRNA levels of four SMD target genes containing 3’UTR Alu dsRNAs were analyzed by qRT-PCR. The expression of CCNG1 pre-mRNAs was slightly increased by ADAR1 knockdown. The pre-mRNA levels of the remaining SMD target genes were unchanged. Data are mean ± S.D. (n = 3 for HPRT1, 4 for other genes, technical replicates); significant differences were identified by two-tailed Student’s t-tests: *P < 0.05, **P < 0.01, n.s., not significant. Source data are available online in Supplementary Data Set 1.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 1699 kb)

Supplementary Table 1

RNA-seq analysis for gene transcripts reduced by ADAR1 knockdown & rescued by Staufen1 knockdown. (XLSX 2338 kb)

Supplementary Table 2

RNA-seq analysis for ISG Transcripts (XLSX 1139 kb)

Supplementary Table 3

qRT-PCR analysis for 52 gene transcripts decreased by ADAR1 knockdown (XLSX 29 kb)

Supplementary Table 4

GO enrichment analysis (XLSX 21 kb)

Supplementary Table 5

Genes analyzed for RIP and qRT-PCR (XLSX 491 kb)

Supplementary Table 6

DNA and RNA oligos used in this study (XLSX 15 kb)

Supplementary Table 7

Antibodies used in this study (XLSX 10 kb)

Supplementary Data Set 1

Excel data source for all bar graphs (XLSX 364 kb)

Supplementary Data Set 2

PDF files for all western blotting original gel images (PDF 39102 kb)

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Sakurai, M., Shiromoto, Y., Ota, H. et al. ADAR1 controls apoptosis of stressed cells by inhibiting Staufen1-mediated mRNA decay. Nat Struct Mol Biol 24, 534–543 (2017). https://doi.org/10.1038/nsmb.3403

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