A case study of the reproducibility of transcriptional reporter cell-based RNAi screens in Drosophila

A second generation dsRNA library was used to re-assess factors that influence the outcome of transcriptional reporter-based whole-genome RNAi screens for the Wnt/Wingless (wg) and Hedgehog (hh)-signaling pathways

Bili Inhibits Wnt/β-Catenin Signaling by Regulating the Recruitment of Axin to LRP6

BACKGROUND: Insights into how the Frizzled/LRP6 receptor complex receives, transduces and terminates Wnt signals will enhance our understanding of the control of the Wnt/ss-catenin pathway. METHODOLOGY/PRINCIPAL FINDINGS: In pursuit of such insights, we performed a genome-wide RNAi screen in Drosophila cells expressing an activated form of LRP6 and a beta-catenin-responsive reporter. This screen resulted in the identification of Bili, a Band4.1-domain containing protein, as a negative regulator of Wnt/beta-catenin signaling. We found that the expression of Bili in Drosophila embryos and larval imaginal discs significantly overlaps with the expression of Wingless (Wg), the Drosophila Wnt ortholog, which is consistent with a potential function for Bili in the Wg pathway. We then tested the functions of Bili in both invertebrate and vertebrate animal model systems. Loss-of-function studies in Drosophila and zebrafish embryos, as well as human cultured cells, demonstrate that Bili is an evolutionarily conserved antagonist of Wnt/beta-catenin signaling. Mechanistically, we found that Bili exerts its antagonistic effects by inhibiting the recruitment of AXIN to LRP6 required during pathway activation. CONCLUSIONS: These studies identify Bili as an evolutionarily conserved negative regulator of the Wnt/beta-catenin pathway

Properties of dsRNAs and reporter genes can influence the sensitivity of the RNAi assay

The dynamic range of validation dsRNAs is smaller than that of the DRSC1.0 dsRNAs, which could potentially increase the rate of false negatives. The effects of dsRNA-mediated knockdown of known Wg-pathway regulators were tested by measuring their effect on the Wg reporter activity. DRSC1.0 and DRSC-v dsRNAs were compared in parallel. Knockdown of and using DRSC1.0 dsRNAs resulted in 90% and 99% reduction in Wg-reporter activity, respectively ((a), black bars). On the other hand, validation dsRNAs for and reduced reporter activity by only 58% and 90%, respectively ((a), grey bars), suggesting that the DRSC-v dsRNAs for some genes may not be as efficient in targeting their endogenous transcripts. Some of the validation dsRNAs ((b), grey and light grey bars) targeting known negative regulators did not produce robust effects on reporter activity compared to their DRSC1.0 counterparts ((b), black bars), including , , and one dsRNA in the case of . Two independent validation dsRNAs targeting the same gene could influence reporter activity to different extents (compare DRSC_v1 and DRSC-v2 dsRNAs for each target gene in (c)). Finally, the number of Tcf binding sites in the Wg responsive luciferase reporter gene can affect the robustness (fold change) upon induction by Wg. Reporter gene carrying 8 (white bar), 12 (grey bar) or 16 (black bar) sites were co-transfected with wg expressing cDNA. Increasing the number of Tcf binding sites increased the fold induction of the luciferase reporter upon addition of both Wg or ΔNLrp6 to induce the Wg pathway. All luciferase reporter assays were performed in 4 replicas and error bars represent the standard error between the four data points.<p><b>Copyright information:</b></p><p>Taken from "A case study of the reproducibility of transcriptional reporter cell-based RNAi screens in "</p><p>http://genomebiology.com/2007/8/9/R203</p><p>Genome Biology 2007;8(9):R203-R203.</p><p>Published online 28 Sep 2007</p><p>PMCID:PMC2375041.</p><p></p