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. 2005 Dec;37(12):1323-32.
doi: 10.1038/ng1682. Epub 2005 Nov 20.

A genome-wide RNA interference screen in Drosophila melanogaster cells for new components of the Hh signaling pathway

Kent Nybakken  1 , Steven A Vokes, Ting-Yi Lin, Andrew P McMahon, Norbert Perrimon
Affiliations

A genome-wide RNA interference screen in Drosophila melanogaster cells for new components of the Hh signaling pathway

Kent Nybakken et al. Nat Genet. 2005 Dec.

Abstract

Members of the Hedgehog (Hh) family of signaling proteins are powerful regulators of developmental processes in many organisms and have been implicated in many human disease states. Here we report the results of a genome-wide RNA interference screen in Drosophila melanogaster cells for new components of the Hh signaling pathway. The screen identified hundreds of potential new regulators of Hh signaling, including many large protein complexes with pleiotropic effects, such as the coat protein complex I (COPI) complex, the ribosome and the proteasome. We identified the multimeric protein phosphatase 2A (PP2A) and two new kinases, the D. melanogaster orthologs of the vertebrate PITSLRE and cyclin-dependent kinase-9 (CDK9) kinases, as Hh regulators. We also identified a large group of constitutive and alternative splicing factors, two nucleoporins involved in mRNA export and several RNA-regulatory proteins as potent regulators of Hh signal transduction, indicating that splicing regulation and mRNA transport have a previously unrecognized role in Hh signaling. Finally, we showed that several of these genes have conserved roles in mammalian Hh signaling.

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Conflict of interest statement

COMPETING INTERESTS STATEMENT

The authors declare competing financial interests; see the Nature Genetics website for details.

Figures

Figure 1
Figure 1
Assay design and validation of the primary screen for new components of Hh signaling. (a) Outline of screen design and schematic representation of constructs used in the primary screen. Relevant portions of the vectors shown are shown in inset. Left boxes are promoter fragments and right boxes are coding regions; parent vectors are indicated at the upper right of each construct. (b) Verification of Hh assay in 384-well plate format. Clone 8 cells were assayed using the ptcΔ136-GL2 reporter and either Act5C-Hh–expressing vector (Hh) or empty Act5C vector (Act). Normalized luciferase values were averaged from 4 wells in a 384-well plate. The dsRNA used to treat the cells is indicated. smo and ci dsRNAs eliminated most reporter activity and resulted in a sixfold difference in normalized signal between cells treated with GFP dsRNA and cells treated with smo or ci dsRNAs. Treatment with dsRNA against the fu coding region reduced reporter signal by ~75%, whereas dsRNAs against the 5′ untranslated region (UTR) of fu reduced signal by 50%. Error bars in this figure and all subsequent figures indicate two standard deviations.
Figure 2
Figure 2
Primary screen results. (a) Correlation of replicate Z scores for the first five primary screening plates. The data in a and b were filtered to remove values greater than +10.0, which correspond to the artificially high normalized values found in some edge wells. (b) Scatter plot of all averaged Z scores from the primary screen. Red lines indicate −2.0 and +3.0 averaged Z score thresholds. Each screening plate contained four control wells, one of which contained smo dsRNA. These smo control wells formed the majority of the data points with Z scores near −5.0. (c) Functional classification of the 449 genes with Z scores less than −2.0 when reduced by RNAi in the Hh signaling assay. These genes are likely to function as positive regulators of Hh signaling. Numbers of genes in each category are indicated. (d) Functional classification of the 60 genes with Z scores greater than +3.0 when reduced by RNAi in the Hh signaling assay. These genes are likely to function as negative regulators of Hh signaling.
Figure 3
Figure 3
Vesicular trafficking genes are important in Hh signaling. (a) COP and Snap dsRNAs reduce Hh signaling by 40% or more in secondary assays. Scores were taken from the Hh (GL3) secondary assay and are presented as the average percent change from a GFP dsRNA control. dsRNAs targeting smo were included as a control. (b) Effects of COP and Snap dsRNAs on activation of reporter by Ci. Scores were taken from Ci secondary assay. dsRNAs and scale are the same as in a. (c) Effects of mouse Cop and Snap siRNAs on soluble Shh signaling. RNAi of mouse Copa (ortholog of αCOP), in particular, caused a greater reduction in reporter activity than even siRNA targeting mouse Smo.
Figure 4
Figure 4
Identification of new kinases affecting Hh signaling. (a) Reduction of Pitslre and Cdk9/CycT by RNAi substantially reduce Hh signaling, as assayed by ptcΔ136-GL3 reporter activity. dsRNAs targeting smo and fu were included as controls. (b) Effect of siRNAs targeting mouse Pitslre and Cdk9 on ShhN signaling in NIH3T3 cells. Cdk9 RNAi had variable effects on ShhN signaling but consistently increased reporter activity.
Figure 5
Figure 5
Splicing factors have a role in Hh signaling. (a) Degree to which splicing factors modulate Hh signaling. Scores were taken from the Hh (GL3) secondary assay and are presented as the average percent change from a GFP dsRNA control. crn, a gene involved in splicing in Drosophila and humans, is a strong positive regulator of the Hh signaling pathway. All three dsRNAs representing this gene in the dsRNA library were identified in the primary screen, although only two were tested in secondary assays. (b) siRNA against mouse Crnkl1 (ortholog of crn) reduced ShhN signaling by almost 50%.
Figure 6
Figure 6
PP2A and its regulatory B subunits regulate Hh signaling. (a) Effect of GFP, mts and cos dsRNA on reporter activity in cells expressing Hh or empty vector. All values were taken from the Hh (GL3) secondary assay and were normalized such that Hh-stimulated GFP dsRNA was 100. (b) Presumed structure of D. melanogaster PP2A holoenzyme, based on data from vertebrate PP2A studies. The catalytic subunit is bound to a regulatory A subunit. This minimal dimer is bound to a regulatory B subunit that is involved in targeting the PP2A to its substrates or proper subcellular location. The names of the four D. melanogaster B subunits are indicated. (c) Effects of dsRNAs targeting the catalytic, A and B subunits of PP2A on Hh signaling. dsRNA against the A subunit had mixed results, with one amplicon having no effect and the others reducing Hh signaling by 30–50% relative to GFP dsRNA. None of the four twins dsRNAs has an effect on reporter activity. dsRNAs against wdb resulted in a 40–50% reduction in reporter activity. dsRNAs against PP2A-B′ give mixed results depending on the particular dsRNA. Amplicons from the Drosophila RNAi Screening Center are indicated by DRSC amplicon numbers. RNAi against ci and smo was included as controls. (d) Overexpression of mts and Pka-C1 results in matching phenotypes. Equal amounts of each expression construct were cotransfected with reporters. Overexpression of both mts and Pka-C1 in the absence of Hh stimulation resulted in small but reproducible increases in reporter activity.
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References

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