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

Nature Genetics volume 37, pages 1323–1332 (2005)Cite this article

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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Figure 1: Assay design and validation of the primary screen for new components of Hh signaling.
Figure 2: Primary screen results.
Figure 3: Vesicular trafficking genes are important in Hh signaling.
Figure 4: Identification of new kinases affecting Hh signaling.
Figure 5: Splicing factors have a role in Hh signaling.
Figure 6: PP2A and its regulatory B subunits regulate Hh signaling.

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Acknowledgements

We thank R. Dasgupta, S. Armknecht, K. Kerr, S. Talala, J. Murphy, I. Flockhart, M. Booker, N. Ramadan, B. Mathey-Prevot and the entire staff of the Drosophila RNAi Screening Center at Harvard Medical School for assistance throughout the project; the Harvard Institute of Chemistry and Cell Biology screening center and its staff for use of equipment and advice; A. Kiger, M. Boutros and the Heidelberg Screening Consortium for their work in establishing the dsRNA collection; K. Richards, K. Kerr, L. Hrdlicka and C. Villalta for technical assistance; P. Bradley, H. Agaisse, R. Zhou and the rest of the Perrimon lab for advice; P. Beachy for the ptcΔ136 reporter construct; T. Kornberg and P. Aza-Blanc for the Hh and Ci plasmids; and P. Wolfe for assistance in identifying mammalian orthologs. This work was supported in part by a National Institutes of Health postdoctoral fellowship (K.N.), the Howard Hughes Medical Institute (N.P.), National Institutes of Health grants (T.-Y.L., N.P. and A.P.M.) and the Helen Hay Whitney Foundation (S.A.V.).

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Authors and Affiliations

  1. Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, 02115, Massachusetts, USA

    Kent Nybakken & Norbert Perrimon

  2. Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, 02138, Massachusetts, USA

    Steven A Vokes, Ting-Yi Lin & Andrew P McMahon

  3. Howard Hughes Medical Institute, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, 02115, Massachusetts, USA

    Norbert Perrimon

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Correspondence to Kent Nybakken or Norbert Perrimon.

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Competing interests

A.P.M. is a patent holder on licensed Hedgehog-related technologies and has stock options in Curis Corp., a biotechnology company pursuing the Hedgehog pathway.

Supplementary information

Supplementary Fig. 1

Secondary assays. (PDF 116 kb)

Supplementary Fig. 2

Phosphorylation of Fu and Cos2 are not affected by reduction in Cdk9, Pitslre, or mts. (PDF 133 kb)

Supplementary Table 1

Potential positive regulators of Hh signaling. (PDF 159 kb)

Supplementary Table 2

Potential negative regulators of Hh signaling. (PDF 103 kb)

Supplementary Table 3

Orthologs of potential Hh regulators. (PDF 141 kb)

Supplementary Table 4

Summary information and scores for genes tested in secondary assays. (PDF 225 kb)

Supplementary Methods (PDF 184 kb)

Supplementary Note (PDF 256 kb)

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Nybakken, K., Vokes, S., Lin, TY. et al. A genome-wide RNA interference screen in Drosophila melanogaster cells for new components of the Hh signaling pathway. Nat Genet 37, 1323–1332 (2005). https://doi.org/10.1038/ng1682

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