An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput CRISPRi screens

  1. Robert W Coukos
  2. David Yao
  3. Mateo Lopez Sanchez
  4. Eric T Strand
  5. Meagan E Olive
  6. Namrata D Udeshi
  7. Jonathan S Weissman
  8. Steven A Carr
  9. Michael C Bassik  Is a corresponding author
  10. Alice Y Ting  Is a corresponding author
  1. Stanford University, United States
  2. Broad Institute of MIT and Harvard, United States
  3. Whitehead Institute, United States

Abstract

The trafficking of specific protein cohorts to correct subcellular locations at correct times is essential for every signaling and regulatory process in biology. Gene perturbation screens could provide a powerful approach to probe the molecular mechanisms of protein trafficking, but only if protein localization or mislocalization can be tied to a simple and robust phenotype for cell selection, such as cell proliferation or fluorescence-activated cell sorting (FACS). To empower the study of protein trafficking processes with gene perturbation, we developed a genetically-encoded molecular tool named HiLITR. HiLITR converts protein colocalization into proteolytic release of a membrane-anchored transcription factor, which drives the expression of a chosen reporter gene. Using HiLITR in combination with FACS-based CRISPRi screening in human cell lines, we identified genes that influence the trafficking of mitochondrial and ER tail-anchored proteins. We show that loss of the SUMO E1 component SAE1 results in mislocalization and destabilization of many mitochondrial tail-anchored proteins. We also demonstrate a distinct regulatory role for EMC10 in the ER membrane complex, opposing the transmembrane-domain insertion activity of the complex. Through transcriptional integration of complex cellular functions, HiLITR expands the scope of biological processes that can be studied by genetic perturbation screening technologies.

Data availability

Lead contact: Further information and requests for resources or reagents should be directed to the lead contact, Alice Ting (ayting@stanford.edu)Materials availability: Plasmids generated in the study have been deposited to Addgene or are available upon request (Supplementary File 1)Data and code availability: HiLITR screen sequencing data has been deposited to Dryad (doi:10.5061/dryad.tb2rbp00n). The original mass spectra and the protein sequence database used for searches have been deposited in the public proteomics repository MassIVE (http://massive.ucsd.edu) and are accessible at ftp://massive.ucsd.edu/MSV000087769/.

The following data sets were generated
    1. Yao D
    (2021) HiLITR CRISPR screens
    https://creativecommons.org/publicdomain/zero/1.0/.

Article and author information

Author details

  1. Robert W Coukos

    Genetics, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7307-8293
  2. David Yao

    Department of Genetics, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Mateo Lopez Sanchez

    Genetics, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1359-6969
  4. Eric T Strand

    Genetics, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1488-1043
  5. Meagan E Olive

    Broad Institute of MIT and Harvard, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Namrata D Udeshi

    Broad Institute of MIT and Harvard, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Jonathan S Weissman

    Department of Biology, Whitehead Institute, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Steven A Carr

    Broad Institute of MIT and Harvard, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Michael C Bassik

    Department of Genetics, Stanford University, Stanford, United States
    For correspondence
    bassik@stanford.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5185-8427
  10. Alice Y Ting

    Department of Biology, Stanford University, Stanford, United States
    For correspondence
    ayting@stanford.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8277-5226

Funding

National Institute of Mental Health (MH119353)

  • Alice Y Ting

NIH Office of the Director (1DP2HD084069-01)

  • Michael C Bassik

National Science Foundation (1656518)

  • David Yao

Stanford Bio-X

  • Robert W Coukos

National Institute of Standards and Technology

  • Robert W Coukos

National Human Genome Research Institute (2T32HG000044)

  • Robert W Coukos
  • David Yao

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Heedeok Hong, Michigan State University, United States

Publication history

  1. Received: April 6, 2021
  2. Preprint posted: April 12, 2021 (view preprint)
  3. Accepted: August 18, 2021
  4. Accepted Manuscript published: August 20, 2021 (version 1)
  5. Version of Record published: September 7, 2021 (version 2)

Copyright

© 2021, Coukos et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 4,030
    Page views
  • 705
    Downloads
  • 2
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Robert W Coukos
  2. David Yao
  3. Mateo Lopez Sanchez
  4. Eric T Strand
  5. Meagan E Olive
  6. Namrata D Udeshi
  7. Jonathan S Weissman
  8. Steven A Carr
  9. Michael C Bassik
  10. Alice Y Ting
(2021)
An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput CRISPRi screens
eLife 10:e69142.
https://doi.org/10.7554/eLife.69142
  1. Further reading

Further reading

    1. Biochemistry and Chemical Biology
    2. Neuroscience
    Jinli Geng, Yingjun Tang ... Xiaodong Liu
    Research Article Updated

    Dynamic Ca2+ signals reflect acute changes in membrane excitability, and also mediate signaling cascades in chronic processes. In both cases, chronic Ca2+ imaging is often desired, but challenged by the cytotoxicity intrinsic to calmodulin (CaM)-based GCaMP, a series of genetically-encoded Ca2+ indicators that have been widely applied. Here, we demonstrate the performance of GCaMP-X in chronic Ca2+ imaging of cortical neurons, where GCaMP-X by design is to eliminate the unwanted interactions between the conventional GCaMP and endogenous (apo)CaM-binding proteins. By expressing in adult mice at high levels over an extended time frame, GCaMP-X showed less damage and improved performance in two-photon imaging of sensory (whisker-deflection) responses or spontaneous Ca2+ fluctuations, in comparison with GCaMP. Chronic Ca2+ imaging of one month or longer was conducted for cultured cortical neurons expressing GCaMP-X, unveiling that spontaneous/local Ca2+ transients progressively developed into autonomous/global Ca2+ oscillations. Along with the morphological indices of neurite length and soma size, the major metrics of oscillatory Ca2+, including rate, amplitude and synchrony were also examined. Dysregulations of both neuritogenesis and Ca2+ oscillations became discernible around 2–3 weeks after virus injection or drug induction to express GCaMP in newborn or mature neurons, which were exacerbated by stronger or prolonged expression of GCaMP. In contrast, neurons expressing GCaMP-X were significantly less damaged or perturbed, altogether highlighting the unique importance of oscillatory Ca2+ to neural development and neuronal health. In summary, GCaMP-X provides a viable solution for Ca2+ imaging applications involving long-time and/or high-level expression of Ca2+ probes.

    1. Biochemistry and Chemical Biology
    2. Chromosomes and Gene Expression
    Radhika A Varier, Theodora Sideri ... Folkert Jacobus van Werven
    Research Article

    N6-methyladenosine (m6A) RNA modification impacts mRNA fate primarily via reader proteins, which dictate processes in development, stress, and disease. Yet little is known about m6A function in Saccharomyces cerevisiae, which occurs solely during early meiosis. Here we perform a multifaceted analysis of the m6A reader protein Pho92/Mrb1. Cross-linking immunoprecipitation analysis reveals that Pho92 associates with the 3’end of meiotic mRNAs in both an m6A-dependent and independent manner. Within cells, Pho92 transitions from the nucleus to the cytoplasm, and associates with translating ribosomes. In the nucleus Pho92 associates with target loci through its interaction with transcriptional elongator Paf1C. Functionally, we show that Pho92 promotes and links protein synthesis to mRNA decay. As such, the Pho92-mediated m6A-mRNA decay is contingent on active translation and the CCR4-NOT complex. We propose that the m6A reader Pho92 is loaded co-transcriptionally to facilitate protein synthesis and subsequent decay of m6A modified transcripts, and thereby promotes meiosis.