Letter

High-content genome-wide RNAi screens identify regulators of parkin upstream of mitophagy

Received:
Accepted:
Published online:

Abstract

An increasing body of evidence points to mitochondrial dysfunction as a contributor to the molecular pathogenesis of neurodegenerative diseases such as Parkinson’s disease1. Recent studies of the Parkinson’s disease associated genes PINK1 (ref. 2) and parkin (PARK2, ref. 3) indicate that they may act in a quality control pathway preventing the accumulation of dysfunctional mitochondria4,5,6,7,8. Here we elucidate regulators that have an impact on parkin translocation to damaged mitochondria with genome-wide small interfering RNA (siRNA) screens coupled to high-content microscopy. Screening yielded gene candidates involved in diverse cellular processes that were subsequently validated in low-throughput assays. This led to characterization of TOMM7 as essential for stabilizing PINK1 on the outer mitochondrial membrane following mitochondrial damage. We also discovered that HSPA1L (HSP70 family member) and BAG4 have mutually opposing roles in the regulation of parkin translocation. The screens revealed that SIAH3, found to localize to mitochondria, inhibits PINK1 accumulation after mitochondrial insult, reducing parkin translocation. Overall, our screens provide a rich resource to understand mitochondrial quality control.

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        Acknowledgements

        We thank P. Tuzmen for qRT–PCR assistance; C. Klumpp for automation; D. Maric for FACS sorting; C. Smith for microscopy assistance; H. Jaffe for mass spectrometry analyses; R. Fields (NINDS) for lentivirus assistance; N. Malik for iPS-derived neurons; the NINDS DNA Sequencing Facility; K. Mihara for the TOMM7 antibody and A. Koretsky and N. Caplen for support. Research was supported by the Japan Society for Promotion of Science Postdoctoral Fellowship for Research Abroad (K.Y.), the NIGMS Research Associate Program, the Intramural Research Program of the NIH, NINDS and the Trans-NIH RNAi initiative.

        Author information

          • Samuel A. Hasson
          •  & Lesley A. Kane

          These authors contributed equally to this work.

        Affiliations

        1. Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA

          • Samuel A. Hasson
          • , Lesley A. Kane
          • , Koji Yamano
          • , Chiu-Hui Huang
          • , Danielle A. Sliter
          • , Chunxin Wang
          • , Tara Hessa
          •  & Richard J. Youle

        2. Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20850, USA

          • Samuel A. Hasson
          • , Eugen Buehler
          • , Rajarshi Guha
          •  & Scott E. Martin

        3. NIH Center for Regenerative Medicine, Bethesda, Maryland 20892, USA

          • Sabrina M. Heman-Ackah

        Authors

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        Contributions

        S.A.H. and R.J.Y. conceived the project; S.A.H., L.A.K., K.Y., D.A.S., C.-H.H., T.H., C.W., S.E.M. and R.J.Y. designed experiments; S.A.H., L.A.K., K.Y., S.M.H.-A., C.-H.H., D.A.S., T.H. and C.W. performed experiments; S.A.H., E.B., R.G. and S.E.M. conducted bioinformatics; and S.A.H. performed the screens. S.A.H., L.A.K. and R.J.Y. wrote the manuscript.

        Competing interests

        The authors declare no competing financial interests.

        Corresponding author

        Correspondence to Richard J. Youle.

        All screening data has been deposited into PubChem under the assay IDs 651811 (non-pooled screen, with siRNA sequences) and 651810 (pooled screen).

        Extended data

        Extended data figures

        1. 1.

          Automated screening strategy and image analysis for the parkin translocation assay.

        2. 2.

          Segmentation examples and screen reproducibility.

        3. 3.

          Overview of data analysis workflow.

        4. 4.

          Activity distributions of parkin translocation data and seed sequence bias.

        5. 5.

          Knockdown analysis of candidate reconfirmation siRNA sets with only one active siRNA reagent and of the top-ranked genes from reconfirmation experiments.

        6. 6.

          TOMM7 knockdown and knockout supporting data.

        7. 7.

          TOMM7 modulates PINK1 accumulation in HCT116 cells and iPS-derived neurons.

        8. 8.

          HSPA1L and BAG4 supporting data.

        9. 9.

          Parkin and BAG4 binding and HSPA1L and BAG4 knockdown.

        10. 10.

          SIAH3 is a negative regulator of parkin translocation.

        Supplementary information

        PDF files

        1. 1.

          Supplementary Information

          This file contains Supplementary Methods which contain screening methodology and siRNA library details, information on all reagents used in the screen and follow up studies, complete explanation of how candidate genes were selected for various follow up analyses and a guide to interpreting Supplementary Table 10.

        Zip files

        1. 1.

          Supplementary Data

          This zipped file contains Supplementary Tables 1-9 and a Supplementary Table Guide.

        Excel files

        1. 1.

          Supplementary Data

          This file contains Supplementary Table 10 – see the Supplementary Information document for a guide to interpreting this.