Supplementary Online Material

Combinatorial mapping of E3 ubiquitin ligases to their target substrates

Chase C. Suiter*✉, Diego Calderon*, David S. Lee, Melodie Chiu, Shruti Jain, Florence M. Chardon, Choli Lee, Riza M. Daza, Cole Trapnell, Ning Zheng, Jay Shendure✉


* These authors contributed equally
 ✉ Correspondence to:
(C.C.S.)
(J.S.)

Multiplex, single-cell CRISPRa screening in single cells to detect target gene upregulation 
An overview of the COMET workflow for assaying many potential E3-substrate interactions in a single experiment
Summary

E3 ubiquitin ligases (E3s) confer specificity of protein degradation through ubiquitination of substrate proteins. Yet the vast majority of the >600 human E3s have no known substrates. To identify proteolytic E3-substrate pairs at scale, we developed COMET (COmbinatorial Mapping of E3 Targets), a framework for testing the role of many E3s in degrading many candidate substrates within a single experiment. We applied COMET to SCF ubiquitin ligase subunits that mediate degradation of target substrates (6,716 F-box-ORF combinations) and E3s that degrade short-lived transcription factors (TFs) (26,028 E3-TF combinations). Our data suggest many E3-substrate relationships are complex rather than 1:1 associations. Finally, we leverage deep learning to predict the structural basis of E3-substrate interactions, and probe the strengths and limits of such models. Looking forward, we consider the practicality of transposing this framework, i.e., computational structural prediction of all possible E3-substrate interactions, followed by multiplex experimental validation.

AlphaFold predictions

All AlphaFold predictions analyzed in the manuscript are available below: