Engineering an orthogonal ubiquitin transfer cascade with RING E3 RNF38 by phage display to reveal its regulation of nuclear transport.

RING E3 ubiquitin (UB) ligases rely on signature RING domains for mediating UB transfer to substrate proteins. The large number of RING E3s and their weak association with substrates pose a significant challenge in identifying the substrates of individual E3s, thereby hindering the elucidation of their biological functions. Here, we utilized phage display to engineer an "orthogonal UB transfer" (OUT) cascade with RING E3 RNF38, enabling the exclusive transfer of an engineered UB (xUB) to its substrates in the cell. The OUT screen revealed RNF38 substrates regulating nucleocytoplasmic transport (Ran, RanGAP1, and KPNA2), protein translation (HuR and Rack1), and endosomal sorting (VPS35). Furthermore, RNF38-catalyzed ubiquitination was found to induce the degradation of the substrate proteins and negatively affect the translocation of transcription factors E2F1 and phosphorylated STAT3 (p-STAT3) into the nucleus. Phage selection of the RNF38 RING library also revealed hotspot residues for E2 interaction, which may guide the engineering of orthogonal E2-E3 pairs with other RING E3s. Overall, our work discovered new roles of RNF38 in regulating nuclear transport and established an anchoring point for expanding OUT cascades within the large family of RING E3s for revealing their UB transfer targets and cellular functions.