Identification of RNF114 as ADPr-Ub reader through non-hydrolysable ubiquitinated ADP-ribose.

Area of Science:

• Biochemistry
• Molecular Biology
• Cellular Biology

Background:

• Post-translational modifications like ubiquitination and ADP-ribosylation crosstalk in cellular processes.
• Hybrid modifications, such as ADP-ribose linked to ubiquitin, are implicated in bacterial infections and DNA damage responses.

Purpose of the Study:

• To investigate the ubiquitination of ADP-ribose by human E3 ligases.
• To elucidate the function of the RNF114 protein in recognizing and processing these hybrid modifications.

Main Methods:

• Preparation of a non-hydrolyzable ADP-ribose-ubiquitin (ADPr-Ub) probe.
• Proteomics to identify interacting proteins.
• Biophysical and biochemical assays to validate interactions.
• Domain deletion analysis of RNF114.
• Laser-induced DNA damage assays.

Main Results:

• RNF114 was identified as a protein interacting with the ADPr-Ub probe.
• RNF114 preferentially binds to ubiquitinated ADP-ribose over unmodified ubiquitin.
• RNF114 can elongate the ubiquitinated ADP-ribose with K11-linked ubiquitin chains.
• The tandem zinc finger (ZnF2+ZnF3) and ubiquitin-interacting motif (UIM) domains of RNF114 are critical for recognizing ubiquitinated ADP-ribose.
• These RNF114 domains are essential for recruiting the protein to DNA damage sites.

Conclusions:

• RNF114 plays a key role in processing ubiquitinated ADP-ribose modifications.
• The specific domains of RNF114 are crucial for this recognition and subsequent ubiquitination.
• RNF114's function in modifying ubiquitinated ADP-ribose is important for the DNA damage response pathway.