An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput CRISPRi screens
- Stanford University, United States
- Broad Institute of MIT and Harvard, United States
- 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/.
-
HiLITR CRISPR screenshttps://creativecommons.org/publicdomain/zero/1.0/.
Article and author information
Author details
Mateo Lopez Sanchez
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.
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.
Download links
Further reading
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
African trypanosomes are the causative agents of neglected tropical diseases affecting both humans and livestock. Disease control is highly challenging due to an increasing number of drug treatment failures. African trypanosomes are extracellular, blood-borne parasites that mainly rely on glycolysis for their energy metabolism within the mammalian host. Trypanosomal glycolytic enzymes are therefore of interest for the development of trypanocidal drugs. Here, we report the serendipitous discovery of a camelid single-domain antibody (sdAb aka Nanobody) that selectively inhibits the enzymatic activity of trypanosomatid (but not host) pyruvate kinases through an allosteric mechanism. By combining enzyme kinetics, biophysics, structural biology, and transgenic parasite survival assays, we provide a proof-of-principle that the sdAb-mediated enzyme inhibition negatively impacts parasite fitness and growth.
-
- Biochemistry and Chemical Biology
N 6,2’-O-dimethyladenosine (m6Am) is a modified nucleotide located at the first transcribed position in mRNA and snRNA that is essential for diverse physiological processes. m6Am mapping methods assume each gene uses a single start nucleotide. However, gene transcription usually involves multiple start sites, generating numerous 5’ isoforms. Thus, gene-level annotations cannot capture the diversity of m6Am modification in the transcriptome. Here, we describe CROWN-seq, which simultaneously identifies transcription-start nucleotides and quantifies m6Am stoichiometry for each 5’ isoform that initiates with adenosine. Using CROWN-seq, we map the m6Am landscape in nine human cell lines. Our findings reveal that m6Am is nearly always a high stoichiometry modification, with only a small subset of cellular mRNAs showing lower m6Am stoichiometry. We find that m6Am is associated with increased transcript expression and provide evidence that m6Am may be linked to transcription initiation associated with specific promoter sequences and initiation mechanisms. These data suggest a potential new function for m6Am in influencing transcription.
