Translation monitoring by puromycin labeling (SUnSET assay)

The assay was described in previous research.⁶⁸ The cells were grown to the time of interest, and puromycin was added to the culture at 10 μg/mL. 2 h later, the cells were collected for immunoblotting. And new translated proteins, which labeled by Puromycin, were detected by anti-Puromycin 12D10 monoclonal antibody.

The functional domains of Rim4 were mapped by Simple Modular Architecture Research Tool (SMART, http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1).

The full length Rim4 protein (entry: {"type":"entrez-protein","attrs":{"text":"P38741","term_id":"731605","term_text":"P38741"}}P38741) structure was predicted by AlphaFold (https://www.alphafold.ebi.ac.uk/). The structure of RRMs of Rim4 were predicted by Robetta program with the RoseTTAFold method (https://robetta.bakerlab.org/). All the predicted structures were downloaded as PDB files, visualized and analyzed by Chimera software (version 1.16) (https://www.cgl.ucsf.edu/chimera/).

The cellular distribution of Rim4 C terminus LCD was analyzed by LocTree (https://rostlab.org/services/loctree3/).