Identification of RNF114 as ADPr-Ub reader through non-hydrolysable ubiquitinated ADP-ribose.
Kloet MS., Chatrin C., Mukhopadhyay R., van Tol BDM., Smith R., Rotman SA., Tjokrodirijo RTN., Zhu K., Gorelik A., Maginn L., Elliott PR., van Veelen PA., Ahel D., Ahel I., van der Heden van Noort GJ.
Crosstalk between the post-translational modification processes of ubiquitination and ADP-ribosylation occurs in DNA-damage- and immune-responses, in addition the physical linkage of ADP-ribose and ubiquitin is found during bacterial infection. Here, we study the ubiquitination of ADP-ribose mediated by human Deltex E3 ligases and the subsequent fate of the formed hybrid post-translational modification. We prepare a non-hydrolysable ADPr-Ub probe that we employ in a proteomics approach and identify RNF114 as an interacting protein. Using biophysical and biochemical experiments, we validate that RNF114 preferentially interacts with ubiquitinated ADP-ribose over non-modified ubiquitin. Subsequently, RNF114 can elongate the ubiquitinated ADP-ribose with a K11-linked ubiquitin chain. Using domain deletion analysis, we pinpoint the tandem zinc fingers and ubiquitin interacting motif (ZnF2 + ZnF3+UIM) domains of RNF114 to be crucial for recognising ubiquitinated ADP-ribose. Moreover, these domains are essential for the recruitment of RNF114 to the sites of laser-induced DNA damage.