Fig 2: Endogenous RNF219 is implicated in cell cycle regulation. (A) Knock-down of RNF219 induces an increase in p27 protein level. HeLa cells were transfected with two siRNA against RNF219 (CDS and UTR) or CNOT3. RNF219, CNOT3 and p27 protein levels were analyzed by immunoblotting using RNF219-A, CNOT3 and p27 antibodies respectively. (B) Knock-down of RNF219 does not substantially affect p27 mRNA level. RNA was extracted in control (SCR) or in RNF219 depleted cells (CDS). RT-QPCR was performed using primers specific to p27/CDKN1b (Table S1) and was normalized on 18S mRNA level. p27/CDKN1b mRNA level was set to 1 in the control (SCR). Error bars represent SD, n = 3. ns P > 0.05 based on unpaired two-tailed Student’s t test. (C) Volcano plot showing differentially expressed (DE) genes of two replicates between siRNF219 CDS versus control SCR. Blue dots represent genes upregulated in RNF219 depleted cells, red represent downregulated genes, black signifying non-DE genes. Differential expression threshold is BH corrected p-value < 0.05 and |LogFC| > 0.5. (D) The top 10 downregulated (red) and upregulated (blue) GO Biological Process terms using gene set enrichment analysis comparing siRNF219 CDS versus SCR. All enrichments are significant with a BH corrected p-value < 0.001.

Fig 3: RNF219 mediated translational repression is CCR4-NOT dependent. (A) CNOT3 interacts with RNF219 tethered to the mRNA reporter. RNA-IPs were performed on extracts of cells transfected with the RL reporter and NHA-LacZ or NHA-RNF219 using CNOT3 antibody. The level of Renilla mRNA in the CNOT3-IP was highly enriched in NHA-RNF219 transfected cells as compared to NHA-LacZ transfected cells, as quantified by RTPCR. Results representative of two replicates are shown. (B) RNF219 interaction with the CCR4-NOT complex is necessary for RFN219 to repress RL reporter translation efficiently. (Top) RL activity was determined in the indicated conditions (NHA-LacZ, NHA-RNF219 and NHA-RNF219-Id). RL activity repression was determined as in Fig. 3B. Error bars represent SD, n = 3. (Bottom) Protein levels of NHA-LacZ, NHA-RNF219, NHA-RNF219-Id in HeLa cells transfected with the corresponding plasmids were analyzed by immunoblotting. (C) mRNA levels of samples in (B) were quantified as in Fig. 3D. Error bars represent SD, n = 3. (D) Knock down of CCR4-NOT scaffold CNOT1 affects NHA-RNF219 mediated repression. RL activity was determined in the indicated conditions (NHA-LacZ, NHA-RNF219 and NHA-RNF219-Id) in control cells (siSCR) or in siCNOT1 depleted cells (siCNOT1A and siCNOT1B are two siRNA targeting different regions of CNOT1 mRNA). (Right) RL activity repression was determined as in Fig. 3B. RL repression levels are relative to that of the control NHA-LacZ in the siSCR cells, set to 1. Error bars represent SD, n = 3. (Left) Protein levels. The arrow indicates the expected CNOT1 band. The lower is a non-specific band as it does not disappear in siCNOT1 treated cells.