{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"submitter":["Timms RT"],"funding":["European Research Council","Howard Hughes Medical Institute","NIA NIH HHS","Wellcome Trust"],"pagination":["1535-1545"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC10567573"],"repository":["biostudies-literature"],"omics_type":["Unknown"],"volume":["25(10)"],"pubmed_abstract":["Specificity within the ubiquitin-proteasome system is primarily achieved through E3 ubiquitin ligases, but for many E3s their substrates-and in particular the molecular features (degrons) that they recognize-remain largely unknown. Current approaches for assigning E3s to their cognate substrates are tedious and low throughput. Here we developed a multiplex CRISPR screening platform to assign E3 ligases to their cognate substrates at scale. A proof-of-principle multiplex screen successfully performed ~100 CRISPR screens in a single experiment, refining known C-degron pathways and identifying an additional pathway through which Cul2<sup>FEM1B</sup> targets C-terminal proline. Further, by identifying substrates for Cul1<sup>FBXO38</sup>, Cul2<sup>APPBP2</sup>, Cul3<sup>GAN</sup>, Cul3<sup>KLHL8</sup>, Cul3<sup>KLHL9/13</sup> and Cul3<sup>KLHL15</sup>, we demonstrate that the approach is compatible with pools of full-length protein substrates of varying stabilities and, when combined with site-saturation mutagenesis, can assign E3 ligases to their cognate degron motifs. Thus, multiplex CRISPR screening will accelerate our understanding of how specificity is achieved within the ubiquitin-proteasome system."],"journal":["Nature cell biology"],"pubmed_title":["Defining E3 ligase-substrate relationships through multiplex CRISPR screening."],"pmcid":["PMC10567573"],"funding_grant_id":["n/a","201387/Z/16/Z","R01 AG011085","947709"],"pubmed_authors":["Timms RT","Koren I","Leng Y","Elledge SJ","Tchasovnikarova IA","Mena EL","Li MZ"],"additional_accession":[]},"is_claimable":false,"name":"Defining E3 ligase-substrate relationships through multiplex CRISPR screening.","description":"Specificity within the ubiquitin-proteasome system is primarily achieved through E3 ubiquitin ligases, but for many E3s their substrates-and in particular the molecular features (degrons) that they recognize-remain largely unknown. Current approaches for assigning E3s to their cognate substrates are tedious and low throughput. Here we developed a multiplex CRISPR screening platform to assign E3 ligases to their cognate substrates at scale. A proof-of-principle multiplex screen successfully performed ~100 CRISPR screens in a single experiment, refining known C-degron pathways and identifying an additional pathway through which Cul2<sup>FEM1B</sup> targets C-terminal proline. Further, by identifying substrates for Cul1<sup>FBXO38</sup>, Cul2<sup>APPBP2</sup>, Cul3<sup>GAN</sup>, Cul3<sup>KLHL8</sup>, Cul3<sup>KLHL9/13</sup> and Cul3<sup>KLHL15</sup>, we demonstrate that the approach is compatible with pools of full-length protein substrates of varying stabilities and, when combined with site-saturation mutagenesis, can assign E3 ligases to their cognate degron motifs. Thus, multiplex CRISPR screening will accelerate our understanding of how specificity is achieved within the ubiquitin-proteasome system.","dates":{"release":"2023-01-01T00:00:00Z","publication":"2023 Oct","modification":"2024-11-14T18:02:58.064Z","creation":"2024-11-14T18:02:58.064Z"},"accession":"S-EPMC10567573","cross_references":{"pubmed":["37735597"],"doi":["10.1038/s41556-023-01229-2"]}}