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

Area of Science:

• Biochemistry
• Molecular Biology
• Post-Translational Modifications

Background:

• Ubiquitin (Ub) and ADP-ribose (ADPr) modifications regulate proteins.
• Deltex E3 ligases can ubiquitylate ADPr-containing molecules in vitro.
• Mono-ADPr ubiquitylation (MARUbylation) on PARP7, extended by K11-linked polyubiquitin, was previously observed.

Purpose of the Study:

• To elucidate the E3 ligases involved in PARP7 MARUbylation and subsequent polyubiquitin chain extension.
• To characterize the molecular mechanism of recognition for MARUbylated species.
• To identify novel E3 ligases that target MARUbe.

Main Methods:

• Cellular assays to determine E3 ligase activity on PARP7.
• Chemoenzymatic synthesis of a fluorescent Ub-ADPr probe.
• AlphaFold3 structure prediction to analyze protein-ligand interactions.
• Biochemical assays to test E3 ligase specificity.

Main Results:

• DTX2 was identified as the E3 ligase catalyzing MARUbylation on PARP7, dependent on PARP7 activity.
• RNF114 was identified as the E3 ligase responsible for K11-linked polyubiquitin extension on PARP7 MARUbylation sites.
• RNF114 utilizes a tandem Di19-UIM module as a MARUbe-binding domain (M-UBD) to recognize MARUbylated substrates.
• A family of MARUbe-Targeted Ligases (M-UTLs) with M-UBD domains was described.

Conclusions:

• DTX2 and RNF114 act sequentially to create a multilayered ubiquitylation signal on PARP7.
• RNF114's M-UBD provides a specific recognition mechanism for MARUbylated proteins.
• The discovery of M-UTLs expands the repertoire of E3 ligases involved in complex post-translational modification pathways.