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. 2015 Jul 14;43(1):41–51. doi: 10.1016/j.immuni.2015.06.015

Figure 3.

Figure 3

H830 and cap1 2′O-Methylation of RNA Prevent Recognition of Endogenous RNAs

(A) RIG-I(WT) and RIG-I(H830A) were expressed in HEK293blue cells in the absence of exogenous RIG-I stimuli and IP10 was monitored at indicated times (24, 48, 72 hr) after transfection. Data from three independent experiments with technical duplicates are depicted as mean values + SEM.

(B) Flag-tagged RIG-I-CTD was overexpressed in HEK293blue cells. Endogenous RNA binding to immune-precipitated RIG-I-CTD was extracted and used for stimulation of RIG-I(WT)- and RIG-I(H830A)-expressing HEK293blue cells. Before stimulation, RNAs were treated with tobacco acid pyrophosphatase (TAP) or alkaline phosphatase (AP) or left untreated (nt). IP10 induction values 20 hr after stimulation from three independent experiments with technical duplicates are depicted as mean values + SEM. TAP hydrolyzes and inactivates free and capped, AP-only free triphosphate (ppp). Right panel: Ethidium bromide-stained agarose gel of RIG-I(WT)-CTD-bound RNAs. Untreated (nt), treated with TAP, or AP.

(C) Fibroblasts isolated from human nasal conchas were transfected with control siRNA (control siRNA pool) or siRNA against hMTr1 (siRNA hMTr1 pool) and harvested after 70 hr. RNA was isolated and IFN-β mRNA induction relative to TBP-1 mRNA expression was determined by real-time PCR. One representative of two experiments in triplicates is shown. Error bars indicate SD.

(D) IFN-β mRNA induction by siRNAs against hMTr1 in wild-type (WT) or RIG-I-deficient (Ddx58−/−) A549 cells were treated with indicated siRNAs, primed with 1,000 U/ml IFN-α, and assessed for IFN-β mRNA induction by qPCR 72 hr after siRNA transfection. Data from three independent experiments with technical duplicates are depicted as mean values + SEM.

(E) IP10 induction in RIG-I(WT)- or RIG-I(H830A)-expressing HEK293blue cells 72 hr after treatment with control siRNA (control siRNA pool) or siRNA against hMTr1 (siRNA hMTr1 pool); linear range limit, 31 pg/ml.

Data from three independent experiments with technical duplicates are depicted as mean values ± SEM (B, D, E). For statistics, two-way ANOVA and Bonferroni post-test was applied: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Knockdown efficiencies were determined by immunoblot (Figure S3A) of hMTr1 and real-time PCR (Figures S3B and S3C).