Genome-wide base editor screen identifies regulators of protein abundance in yeast
Proteins are key molecular players in a cell, and their abundance is extensively regulated not just at the level of gene expression but also post-transcriptionally. Here, we describe a genetic screen in yeast that enables systematic characterization of how protein abundance regulation is encoded in...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2022-11-01
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| Series: | eLife |
| Subjects: | |
| Online Access: | https://elifesciences.org/articles/79525 |
| _version_ | 1828157111378378752 |
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| author | Olga T Schubert Joshua S Bloom Meru J Sadhu Leonid Kruglyak |
| author_facet | Olga T Schubert Joshua S Bloom Meru J Sadhu Leonid Kruglyak |
| author_sort | Olga T Schubert |
| collection | DOAJ |
| description | Proteins are key molecular players in a cell, and their abundance is extensively regulated not just at the level of gene expression but also post-transcriptionally. Here, we describe a genetic screen in yeast that enables systematic characterization of how protein abundance regulation is encoded in the genome. The screen combines a CRISPR/Cas9 base editor to introduce point mutations with fluorescent tagging of endogenous proteins to facilitate a flow-cytometric readout. We first benchmarked base editor performance in yeast with individual gRNAs as well as in positive and negative selection screens. We then examined the effects of 16,452 genetic perturbations on the abundance of eleven proteins representing a variety of cellular functions. We uncovered hundreds of regulatory relationships, including a novel link between the GAPDH isoenzymes Tdh1/2/3 and the Ras/PKA pathway. Many of the identified regulators are specific to one of the eleven proteins, but we also found genes that, upon perturbation, affected the abundance of most of the tested proteins. While the more specific regulators usually act transcriptionally, broad regulators often have roles in protein translation. Overall, our novel screening approach provides unprecedented insights into the components, scale and connectedness of the protein regulatory network. |
| first_indexed | 2024-04-11T23:23:58Z |
| format | Article |
| id | doaj.art-115487ded45c4826b4212d8523d001be |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-11T23:23:58Z |
| publishDate | 2022-11-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-115487ded45c4826b4212d8523d001be2022-12-22T03:57:23ZengeLife Sciences Publications LtdeLife2050-084X2022-11-011110.7554/eLife.79525Genome-wide base editor screen identifies regulators of protein abundance in yeastOlga T Schubert0https://orcid.org/0000-0002-2613-0714Joshua S Bloom1https://orcid.org/0000-0002-7241-1648Meru J Sadhu2Leonid Kruglyak3https://orcid.org/0000-0002-8065-3057Department of Human Genetics, University of California, Los Angeles, Los Angeles, United States; Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, United States; Howard Hughes Medical Institute, University of California, Los Angeles, Los Angeles, United States; Institute for Quantitative and Computational Biology, University of California, Los Angeles, Los Angeles, United States; Department of Environmental Systems Science, Swiss Federal Institute of Technology (ETH), Zürich, Switzerland; Department of Environmental Microbiology, Swiss Federal Institute of Aquatic Science and Technology (Eawag), Dübendorf, SwitzerlandDepartment of Human Genetics, University of California, Los Angeles, Los Angeles, United States; Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, United States; Howard Hughes Medical Institute, University of California, Los Angeles, Los Angeles, United States; Institute for Quantitative and Computational Biology, University of California, Los Angeles, Los Angeles, United StatesDepartment of Human Genetics, University of California, Los Angeles, Los Angeles, United States; Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, United States; Howard Hughes Medical Institute, University of California, Los Angeles, Los Angeles, United States; Institute for Quantitative and Computational Biology, University of California, Los Angeles, Los Angeles, United StatesDepartment of Human Genetics, University of California, Los Angeles, Los Angeles, United States; Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, United States; Howard Hughes Medical Institute, University of California, Los Angeles, Los Angeles, United States; Institute for Quantitative and Computational Biology, University of California, Los Angeles, Los Angeles, United StatesProteins are key molecular players in a cell, and their abundance is extensively regulated not just at the level of gene expression but also post-transcriptionally. Here, we describe a genetic screen in yeast that enables systematic characterization of how protein abundance regulation is encoded in the genome. The screen combines a CRISPR/Cas9 base editor to introduce point mutations with fluorescent tagging of endogenous proteins to facilitate a flow-cytometric readout. We first benchmarked base editor performance in yeast with individual gRNAs as well as in positive and negative selection screens. We then examined the effects of 16,452 genetic perturbations on the abundance of eleven proteins representing a variety of cellular functions. We uncovered hundreds of regulatory relationships, including a novel link between the GAPDH isoenzymes Tdh1/2/3 and the Ras/PKA pathway. Many of the identified regulators are specific to one of the eleven proteins, but we also found genes that, upon perturbation, affected the abundance of most of the tested proteins. While the more specific regulators usually act transcriptionally, broad regulators often have roles in protein translation. Overall, our novel screening approach provides unprecedented insights into the components, scale and connectedness of the protein regulatory network.https://elifesciences.org/articles/79525protein regulationCRISPR screenbase editorgene regulatory networkGAPDH isoenzymesras/PKA |
| spellingShingle | Olga T Schubert Joshua S Bloom Meru J Sadhu Leonid Kruglyak Genome-wide base editor screen identifies regulators of protein abundance in yeast eLife protein regulation CRISPR screen base editor gene regulatory network GAPDH isoenzymes ras/PKA |
| title | Genome-wide base editor screen identifies regulators of protein abundance in yeast |
| title_full | Genome-wide base editor screen identifies regulators of protein abundance in yeast |
| title_fullStr | Genome-wide base editor screen identifies regulators of protein abundance in yeast |
| title_full_unstemmed | Genome-wide base editor screen identifies regulators of protein abundance in yeast |
| title_short | Genome-wide base editor screen identifies regulators of protein abundance in yeast |
| title_sort | genome wide base editor screen identifies regulators of protein abundance in yeast |
| topic | protein regulation CRISPR screen base editor gene regulatory network GAPDH isoenzymes ras/PKA |
| url | https://elifesciences.org/articles/79525 |
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