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Defining E3 ligase-substrate relationships through multiplex CRISPR screening.

Accepted version
Peer-reviewed

Type

Article

Change log

Authors

Mena, Elijah L 
Leng, Yumei 
Li, Mamie Z 
Tchasovnikarova, Iva A 

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 Cul2FEM1B targets C-terminal proline. Further, by identifying substrates for Cul1FBXO38, Cul2APPBP2, Cul3GAN, Cul3KLHL8, Cul3KLHL9/13 and Cul3KLHL15, 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.

Description

Keywords

3101 Biochemistry and Cell Biology, 31 Biological Sciences

Journal Title

Nat Cell Biol

Conference Name

Journal ISSN

1465-7392
1476-4679

Volume Title

Publisher

Springer Science and Business Media LLC
Sponsorship
Wellcome Trust (201387/Z/16/Z)