Journal: Nature Communications
Article Title: RNF219 attenuates global mRNA decay through inhibition of CCR4-NOT complex-mediated deadenylation
doi: 10.1038/s41467-021-27471-6
Figure Lengend Snippet: a Schematic illustration of the domain architecture of RNF219; RING, really interesting new gene; SLiM, short linear motif. RNF219-C (amino acid residues 434–726) and RNF219-ΔSLiM are shown below. b Co-precipitation of RNF219 was examined by FLAG IP of the endogenous CCR4-NOT complex from FST-NOT1 HeLa cells following transfection with either control or NOT9-targeting siRNAs for 48 h. Proteins were analyzed by western blotting with antibodies as indicated; the asterisks denote non-specific bands. c Coomassie-stained polyacrylamide gel of in vitro pull-down assays with recombinant RNF219-C fused to maltose-binding protein (MBP) and a StrepII (Strep) affinity tag upon incubation with four different CCR4-NOT modules as indicated. d Co-IP of endogenous NOT2 and NOT9 with RNF219 using HeLa cells stably expressing 3xFLAG-SBP (3xFS)-RNF219-WT or 3xFS-RNF219-ΔSLiM. Precipitated proteins were detected by western blot analysis using antibodies as indicated. e Co-sedimentation analysis of CCR4-NOT subunits by 10–30% linear glycerol gradient fractionation and western blotting from HeLa cells stably expressing 3xFS-RNF219-WT or 3xFS-RNF219-ΔSLiM. The core CCR4-NOT complex elutes in fractions 7–14. Data from one representative experiment are shown (left panel), and the relative distribution of RNF219 and NOT7 along the gradient was quantified (right panel); values are presented as mean ± SD (WT n = 3; ΔSLiM n = 4; fraction 8, p = 0.0313; fraction 9, p = 0.0154; fraction 14, p = 0.0106). The asterisks denote non-specific bands. p values were calculated using a two-tailed, paired t test (* p < 0.05). Source data for panels ( b – e ) are provided as a Source Data file.
Article Snippet: The following primary antibodies were used: mouse monoclonal anti-FLAG M2 (1:1000, Sigma–Aldrich), rabbit polyclonal anti-histone H3 acetyl K27 (1:1000, Abcam), rabbit polyclonal anti-NOT10 (1:1000, Proteintech), rabbit monoclonal anti-NOT7 (1:1000, Cell Signaling), rabbit polyclonal anti-acetylated lysine (1:1000, Cell Signaling), rabbit polyclonal anti-RNF219 (1:500, Bethyl), rabbit polyclonal anti-FHL2 (1:1000, Abcam), rabbit polyclonal anti-TNKS1BP1 (1:1000, Bethyl), rabbit monoclonal anti-NOT2 (1:1000, Cell Signaling), rabbit polyclonal anti-NOT9 (1:1000, Proteintech), mouse monoclonal anti-SBP (1:1000, Santa Cruz), rat monoclonal anti-tubulin (1:1000, Abcam), rabbit monoclonal anti-NOT6 (1:1000, Cell Signaling), rabbit polyclonal anti-histone H3 (1:1000, Abcam), rabbit polyclonal anti-NOT1 (1:1000, Proteintech), rabbit polyclonal anti-CAF1a (1:1000, kindly provided by Ann-Bin Shyu, McGovern Medical School, University of Texas, Houston), rabbit monoclonal anti-NOT3 (1:1000, Cell Signaling), mouse monoclonal anti-ubiquitin (1:1000, Cell Signaling), rabbit monoclonal anti-CAPZA1 (1:1000, Abcam), rabbit monoclonal anti-CAPZB (1:1000, Abcam), rabbit polyclonal anti-GFP (1:1000, Abcam), mouse monoclonal anti-HDAC1 (1:1000, Santa Cruz).
Techniques: Transfection, Western Blot, Staining, In Vitro, Recombinant, Binding Assay, Incubation, Co-Immunoprecipitation Assay, Stable Transfection, Expressing, Sedimentation, Fractionation, Two Tailed Test
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