Deltex and RING-UIM E3 ligases cooperate to create a ubiquitin-ADP-ribose hybrid mark on tankyrase, promoting its stabilization

ADP-ribosylation is a key post-translational modification that impacts diverse cellular pathways. The modification can occur as mono-ADP-ribose (MAR) or be extended into poly-ADPribose (PAR). Tankyrase, a PAR transferase, adds PAR to itself and other proteins to influence their function and stability by tagging them for proteasomal degradation via the PAR-binding E3 ligase RNF146. This degradation can be counteracted by RING-UIM E3 ligases RNF114 and RNF166, though the process is unclear. Here we identify a new mechanism that can regulate the balance between MAR and PAR on tankyrase to control degradation. We show that Deltex E3 ligases DTX2 and DTX3 catalyze monoubiquitylation of tankyrase in cells. This ubiquitylation occurs, not on a (canonical) lysine, but rather on mono-ADP-ribose, creating a monoubiquitin-MAR hybrid mark. RNF114 and RNF166 recognize this mark using a unique hybrid reader domain comprising two binding sites - one for MAR and one for ubiquitin - and further diubiquitylate it. This ubiquitylation of MAR, which occurs near the ADP-ribose addition site, prevents PAR formation, antagonizing the action of the PAR-binding E3 ligase RNF146 and stabilizing tankyrase. These findings reveal a novel interplay between ubiquitin, ADP-ribose, and E3 ligases in cellular signaling.