RNF114 and RNF166 exemplify reader-writer E3 ligases that extend K11 polyubiquitin onto sites of MARUbylation

Abstract Ubiquitin (Ub) cooperates with other post-translational modifications to provide a tiered opportunity for protein regulation. Deltex E3 ligases were previously implicated in ubiquitylation of ADP-ribose (ADPr)-containing macromolecules in vitro, generating a noncanonical mono-ADPr-Ub ester (MARUbe). We previously identified mono-ADPr ubiquitylation (MARUbylation) on PARP7 in cells, which was extended with K11-linked polyUb, suggesting an intricately regulated, multilayered post-translational modification. Here, we show that the Deltex DTX2 ubiquitylates ADPr modifications on PARP7 in cells, which depends on PARP7 catalytic activity. We further identify RNF114 as the E3 ligase responsible for K11-linked polyUb extension on sites of PARP7 MARUbylation. Using a chemoenzymatic approach, we developed a fluorescent Ub-ADPr probe and find that RNF114 explicitly recognizes MARUbylated species. We used AlphaFold3 to examine the mechanisms of Ub-ADPr recognition and K11-linked polyUb extension by RNF114. We identify a tandem Di19-UIM module in RNF114 as a MARUbe-binding domain (M-UBD), thus providing a reader function that interfaces with K11-specific writer activity. Finally, we describe a small family of M-UBD-containing E3 ligases that demonstrate preference for Ub-ADPr, which we call MARUbe-Targeted Ligases (M-UTLs).