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Integrative Genomics-Based Discovery of Novel Regulators of the Innate Antiviral Response

Fig 2

RNAi screens validate a role for the novel RLR candidates in RIG-I-mediated IFNβ induction.

(A) Flow chart of the RNAi validation screens. 187 candidate RLR genes were screened for RIG-I pathway activity in three different RNAi screens. In screens 1 and 2, HeLa cells stably expressing an IFNβ promoter-controlled firefly luciferase (Fluc) reporter were stimulated with a 5’-ppp-containing RIG-I RNA ligand. The 57 hits (15 up, 42 down) with the largest effect on IFNβ induction upon siRNA knockdown in screen 1 (stringent Z-score <-2 or >2) were tested again in screen 2 with a different set of siRNAs. The 19 top hits from screen 2 were then picked for screen 3, which is similar to the first two screens except that it measures IFNβ mRNA levels using quantitative real-time qRT-PCR. (B) Correlation between the negative control-based robust Z-scores of RNAi screens 1 and 2. The 57 top hits with Z-scores <-2 or >2 in screen 1 were tested again in screen 2 (purple data points). N.T., non-transfected; SCR, scrambled. (C) Overview of the 19 novel RIG-I pathway genes with the largest effects on IFNβ induction in screens 1 and 2 (Z-score <-2 in both screens). Black data points correspond to genes whose knockdown also causes a reduction in IFNβ mRNA levels in screen 3. (D) RNAi screen 3. 13 of the 19 top hits from screens 1 and 2 also reduce RIG-I-mediated IFNβ mRNA production (black bars). Experiments were performed in triplicate (n = 3). Bars (mean±SEM) display the fold induction of IFNβ mRNA (corrected for actin mRNA levels) compared to the mock-treated control. Statistical significance was assessed by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test, comparing the values for each of the 19 test genes to the combined negative control conditions (scrambled and LGP2, red bars). ** P < 0.01; *** P < 0.001. (E) Correlation between the in silico integrated RLR score and the probability of experimental confirmation in RNAi screen 1. The dark purple line represents all 94 hits with Z-score <-1.25 or >1.25; the light purple line represents the top 57 hits with Z-score <-2 or >2. The 187 experimentally tested genes were rank-ordered based on the RLR score and precision was calculated sequentially as the fraction of validated hits among all tested genes having a certain RLR score or higher.

Fig 2