Image-based genome-wide siRNA screen identifies selective autophagy factors (original) (raw)
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01 December 2011
Two minor text corrections were made in paragraphs beginning 'Screening of a human siGenome library...' and 'However, a significant decrease...', respectively.
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Acknowledgements
We thank M. Vishwanath, S. Wei and B. Posner for assistance with high-throughput siRNA screening; W. Sun for information technology support; K. Scudder for assistance with image analysis algorithms; A. Diehl for expert medical illustration; V. Stollar, M. McDonald, R. Kuhn and R. Youle for helpful discussions and providing reagents; A. Bugde for assistance in the UTSW Live Cell Imaging Facility; and L. Mueller and T. Januszewski for assistance with electron microscopy. This work was supported by NIH grants AI109617 (B.L.), CA84254 (B.L.), UL1 RR024982 (G.X., Y.X.), AI062773 (R.J.X.), DK83756 (R.J.X.), DK086502 (R.J.X.) and DK043351 (R.J.X. and A.N.); NSF grant DMS-0907562 (G.X.); and the Center for Cancer Research, National Cancer Institute Intramural Research Program (Y.E.Z.).
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Author notes
- Anthony Orvedahl and Rhea Sumpter Jr: These authors contributed equally to this work.
Authors and Affiliations
- Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, 75390-9113, Texas, USA
Anthony Orvedahl, Rhea Sumpter Jr., Zhongju Zou, Qihua Sun & Beth Levine - Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, 75390-9113, Texas, USA
Anthony Orvedahl & Beth Levine - Center for Autophagy Research, University of Texas Southwestern Medical Center, Dallas, 75390-9113, Texas, USA
Rhea Sumpter Jr., Zhongju Zou, Qihua Sun & Beth Levine - Department of Clinical Sciences, University of Texas Southwestern Medical Center, Dallas, 75390-9113, Texas, USA
Guanghua Xiao, Christian V. Forst & Yang Xie - Center for Computational and Integrative Biology, Massachusetts General Hospital, Harvard Medical School, Boston, 02114, Massachusetts, USA
Aylwin Ng & Ramnik J. Xavier - Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School, Boston, 02114, Massachusetts, USA
Aylwin Ng & Ramnik J. Xavier - Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, 02142, Massachusetts, USA
Aylwin Ng & Ramnik J. Xavier - Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, 75390-9113, Texas, USA
Zhongju Zou & Beth Levine - Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, 20892, Maryland, USA
Yi Tang & Ying E. Zhang - Center for Systems Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada
Masahiro Narimatsu & Jeffrey L. Wrana - Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, 75390-9113, Texas, USA
Christopher Gilpin & Katherine Luby-Phelps - Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, 75390-9113, Texas, USA
Michael Roth - Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, 75390-9113, Texas, USA
Michael Roth, Yang Xie & Beth Levine - Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 3E1, Canada
Jeffrey L. Wrana
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- Anthony Orvedahl
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Contributions
A.O., R.S., M.N., M.R., J.L.W., Y.E.Z., K.L.-P., C.G. and B.L. designed the experiments. A.O., R.S., Z.Z. Q.S. and Y.T. performed the experiments. G.X., A.N., C.V.F., R.J.X. and Y.X. performed statistical and bioinformatic analyses. A.O., R.S. and B.L. wrote the manuscript. G.X. and A.N. contributed equally to the manuscript.
Corresponding author
Correspondence toBeth Levine.
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The authors declare no competing financial interests.
Supplementary information
Supplementary Information
The file contains Supplementary Methods, Supplementary References and Supplementary Figures 1-12 with legends. The original file posted online was corrupted and has been replaced on 23 November 2011. (PDF 8705 kb)
Supplementary Table 1
This table lists the primary data for the virus capsid/autophagosome colocalization screen. Shown are the z-scores for each replicate for each gene in the Dharmacon siRNA library. “NA” denotes insufficient numbers of green or red puncta per cell or total number of cells per well for analysis. (XLS 4656 kb)
Supplementary Table 2
This table lists the data for the virus capsid/autophagosome colocalization confirmation screen, using a customized library (from Dharmacon) composed of individual siRNAs from the pool of 4 siRNAs targeting each gene that scored “positive” in the primary co-localization screen. Genes with p-values of <0.05 for 2 or more individual siRNAs were considered confirmed colocalization hits. “NA” denotes insufficient numbers of green or red dots per cell or total number of cells per well for analysis (XLS 127 kb)
Supplementary Table 3
This table lists the results for each individual siRNA from a pool of 4 targeting each gene that scored positive in the primary screen for viral capsid/autophagosome colocalization, with respect to whether they scored positive in the confirmation screen of viral capsid/ autophagosome colocalization (C) screen, the secondary screen for survival of virus-infected cells (S) and the secondary screen for Parkin-mediated mitophagy (M). siRNA sequences are listed in column J. For each siRNA, this table also lists the number of 7-8mer miRNA seed sequences (positions 2-8 on mature miRNA) contained in each siRNA oligo (column K), the identity of such seed sequences (columns L-0), and the specific miRNAs that contain the seed sequences (columns P-S). The confirmed siRNAs in each screen are not enriched for siRNAs containing miRNA seed sequences (P=0.95 for colocalization screen; P=0.71 for cell survival screen; and P=0.97 for mitophagy screen) (XLS 200 kb)
Supplementary Table 4
This table lists the predicted targets (identified using TargetScan) for each miRNA seed sequence listed in Supplementary Table 3 (MS Excel spreadsheet, 302 KB). (XLS 302 kb)
Supplementary Table 5
This table lists the molecular function and biological process categories from Panther and Gene Ontology, and protein class and pathway assignments from Panther for the siRNA hits in the viral capsid/ autophagosome colocalization screen. Clusters listed correspond to graphical representation in Supplementary Figure 3a (MS Excel spreadsheet, 36 KB). (XLS 36 kb)
Supplementary Table 6
This table lists the data from the cell survival screen, using a using a customized library (from Dharmacon) composed of individual siRNAs from the pool of 4 siRNAs targeting each gene that scored “positive” in the primary colocalization screen. Genes with p-values of <0.05 for 2 or more individual siRNAs were considered to be confirmed cell survival factors during viral infection (MS Excel spreadsheet, 36 KB). (XLS 127 kb)
Supplementary Table 7
This table lists the data from the mitophagy screen, using a customized library (from Dharmacon) composed of individual siRNAs from the pool of 4 siRNAs targeting each gene that scored “positive” in the primary colocalization screen. Genes with p-values of <0.05 for 2 or more individual siRNAs were considered to be confirmed mitophagy factors. (XLS 127 kb)
Supplementary Table 8
This table includes the data in Figure 2a of the main text, with additional details for each gene including Locus ID, Gene Accession numbers, and Gene Annotations from Panther Molecular Function (MF), Panther Biological Process (BP), and UniProt. (XLS 112 kb)
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Orvedahl, A., Sumpter, R., Xiao, G. et al. Image-based genome-wide siRNA screen identifies selective autophagy factors.Nature 480, 113–117 (2011). https://doi.org/10.1038/nature10546
- Received: 06 September 2010
- Revised: 07 September 2011
- Accepted: 23 October 2011
- Published: 01 December 2011
- Issue Date: 01 December 2011
- DOI: https://doi.org/10.1038/nature10546