Journal: Nucleic Acids Research
Article Title: Dom34 mediates targeting of exogenous RNA in the antiviral OAS/RNase L pathway
Figure Lengend Snippet: RNase L interacts with Dom34 and acts together to eliminate exogenous mRNA. ( A ) HeLa cells were transfected with a combination of either pCMV-5×Myc-Dom34 (lanes 1, 2, 5, 6, 9 and 10) or pCMV-5×Myc-eRF1 (lanes 3, 4, 7, 8, 11 and 12) and either pCMV-5×Flag (1, 3, 5, 7, 8 and 11) or pCMV-5×Flag-RNase L (2, 4, 5, 6, 10 and 12). The cells were lysed in lysis buffer A for 30 min on ice. The lysates were clarified by centrifugation for 10 min at 20,400 × g, and the supernatants were rotated with anti-Flag M2 agarose in the presence of 1 μg/ml RNase A as needed at 10°C for 1 h. The immunoprecipitates (lanes 5–12) and inputs (lanes 1–4, 10% of the amount immunoprecipitated) were analyzed by western blotting with the indicated antibodies. ( B ) Cell extracts prepared from HeLa cells and HeLa cells expressing Flag-tagged RNase L were used for co-immunoprecipitation. The immunoprecipitates (lanes 3 and 4) and inputs (lanes 1 and 2, 2% of the amount immunoprecipitated) were analyzed by western blotting with the indicated antibodies. ( C ) Either recombinant 6×His-S2-Flag (lanes 1 and 3) or 6×His-S2-Flag-RNase L (lanes 2 and 4) was incubated with 6×His-S2-Myc-Dom34 in buffer A at 10°C for 1 h. The immunoprecipitates (lanes 3 and 4) and inputs (lanes 1 and 2, 10% of the amount immunoprecipitated) were analyzed by western blotting with the indicated antibodies. ( D ) HeLa cells were transfected with siRNA against either luciferase (control), Dom34, RNase L or Dom34/RNase L. At 24 h after siRNA transfection, the cells were treated with IFN-α/β (25 U/ml) for 24 h and the cells were infected with EMCV (MOI = 1 for 1 h). The cells were cultured in growth medium over time. EMCV–RNA and β-actin mRNA were analyzed by northern blotting. The levels of EMCV–RNA were quantified and normalized to the levels of β-actin mRNA, and the normalized levels of the 7-h time point of the control were defined as 1 (mean ± SEM, n = 3). ( E ) HeLa cells were transfected with siRNA against either luciferase (control), Dom34, RNase L or Dom34/RNase L. At 48 h after siRNA transfection, the cells were further transfected with 5×Flag-EGFP mRNA for 3 h, and cultured in growth medium over time. 5×Flag-EGFP mRNA and 28S rRNA were analyzed by northern blotting (upper panel) and ethidium bromide staining (lower panel), respectively. The leftmost five lanes, which analyzed 2-fold dilutions of total RNA, show that the conditions used for ethidium bromide staining are semi-quantitative. The levels of 5×Flag-EGFP mRNA that were normalized to the levels of 28S rRNA were quantified, where the normalized levels from the 0-h time point were defined as 100% (mean ± SEM, n = 4). The half-lives of 5×Flag-EGFP mRNA were calculated (average t 1/2 ± SEM, n = 4).
Article Snippet: For immunoprecipitation assay of endogenous proteins, HeLa or HeLa/5×Flag-RNase L cells were lysed in buffer D (20 mM Tris–HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM DTT, 10% glycerol, 0.25% sodium deoxycholate, 5 μg/ml RNase A and 1×protease inhibitor ocktail (nacalai tesque)) on ice for 30 min.
Techniques: Transfection, Lysis, Centrifugation, Immunoprecipitation, Western Blot, Expressing, Recombinant, Incubation, Luciferase, Infection, Cell Culture, Northern Blot, Staining