Fig 1: RNF219 binds to the CCR4-NOT complex. (A) (Left) FLAG and HA tandem affinity chromatography was performed on cytoplasmic S100 extracts prepared from HeLa S3 cells stably expressing FHA-RNF219 (S3-FHA-RNF219) or non-transduced HeLa S3 cells. Proteins were resolved by SDS-PAGE and visualized by silver staining. (Right) The identity of FHA-RNF219-associated proteins was determined by tandem mass spectrometry and percent peptide coverage by amino acid count for CCR4-NOT subunits detected are listed. (B) FLAG-immuno-precipitations from S3-FHA-RNF219 cell extracts confirm that FHA-RNF219 is associated with a number of CCR4-NOT subunits as determined by mass spectrometry. The presence of CCR4-NOT subunits CNOT3, CNOT2, CNOT7 in the IPs was analyzed by immunoblotting (IB). (C) Endogenous RNF219 was immuno-purified from HeLa cell extracts using a specific antibody against RNF219 (RNF219-B) or pre-immune IgG control (IgG-B-PI). The presence of the CCR4-NOT subunit CNOT1, CNOT2 and CNOT3 in the IP was analyzed by IB. RNF219 was detected with RNF219-A antibody (long exposure is shown in Fig. S2B). (D) RNF219 interaction with NOT module components CNOT2 and CNOT3 is CNOT1 dependent. FHA-RNF219 expressing HeLa cells were transfected with siRNA against CNOT1 (+) or control siRNA (−). IPs were performed using anti-FLAG agarose beads and were blotted for the indicated proteins. The arrow indicates the expected CNOT1 band. (E) Schematic representation of RNF219-Id, a CCR4-NOT complex interaction defective RNF219 allele. Amino acids 540–549 (ELDSMMSESD) were replaced by VE. (F) RNF219-Id interacts with the CCR4-NOT complex very inefficiently. 293T cells were transfected with the indicated constructs. Whole cell extracts were immuno-purified with anti-FLAG agarose beads and eluates immunoblotted with the indicated proteins. (G) RNF219-Id localizes both in the nucleus and in the cytoplasm similarly to WT RNF219. Immuno-fluorescence was performed with anti-HA antibody on HEK293T cells transfected with the indicated constructs.