Fig. 3: RNF219 is a CCR4-NOT complex-associated RING-type E3 ligase.

a Amino acid sequence alignment of the RNF219 RING domain with other RING-type E3 ligases. Cysteine and histidine residues involved in zinc atom coordination are highlighted in red. Cysteines at position 18 and 21 were mutated to serine in RNF219-RINGmut, shown in green. b Protein stability of 3xFS-RNF219-WT and 3xFS-RNF219-RINGmut was assessed in transiently transfected HeLa cells following translation shut-off with 100 µM cycloheximide (CHX). Proteins were isolated at regular time intervals and detected by western blot analysis using α-FLAG and α-tubulin antibodies. 3xFS-RNF219-WT and -RINGmut protein half-lives are presented as mean ± SD (n = 3). c In vitro ubiquitination assay showing autoubiquitination of RNF219 within the endogenous CCR4-NOT complex. Native CCR4-NOT complexes were purified from HeLa cells expressing FST-NOT1, eluted with 3xFLAG peptide, and incubated with recombinant E1 enzyme (UBA1), E2 enzyme (UbcH5b) and ubiquitin in the absence or presence of 5 mM ATP. Reaction products were detected by western blot analysis as indicated. d In vitro ubiquitination assay showing autoubiquitination of RNF219 in the presence and absence of the CCR4-NOT complex. CCR4-NOT complexes were purified from CRISPR control or NOT9 KO HeLa cells stably expressing 3xFS-RNF219 by FLAG IP and used for in vitro ubiquitination assays as described in (c). The asterisk denotes a non-specific band. e Total protein lysates derived from two control and two RNF219 KO HeLa clones were analyzed by western blotting using antibodies as indicated. Source data for panels (b–e) are provided as a Source Data file.