Advertisement

Genome-Wide RNAi Screening to Dissect the TGF-β Signal Transduction Pathway

  • Xiaochu Chen
  • Lan Xu
Part of the Methods in Molecular Biology book series (MIMB, volume 1344)

Abstract

Thetransforming growth factor-β (TGF-β) family of cytokines figures prominently in regulation of embryonic development and adult tissue homeostasis from Drosophila to mammals. Genetic defects affecting TGF-β signaling underlie developmental disorders and diseases such as cancer in human. Therefore, delineating the molecular mechanism by which TGF-β regulates cell biology is critical for understanding normal biology and disease mechanisms. Forward genetic screens in model organisms and biochemical approaches in mammalian tissue culture were instrumental in initial characterization of the TGF-β signal transduction pathway. With complete sequence information of the genomes and the advent of RNA interference (RNAi) technology, genome-wide RNAi screening emerged as a powerful functional genomics approach to systematically delineate molecular components of signal transduction pathways. Here, we describe a protocol for image-based whole-genome RNAi screening aimed at identifying molecules required for TGF-β signaling into the nucleus. Using this protocol we examined >90 % of annotated Drosophila open reading frames (ORF) individually and successfully uncovered several novel factors serving critical roles in the TGF-β pathway. Thus cell-based high-throughput functional genomics can uncover new mechanistic insights on signaling pathways beyond what the classical genetics had revealed.

Key words

Growth factor-β (TGF-β) Embryonic development Adult tissue homeostasis Drosophila Mammals 

Notes

Acknowledgements

We would like to thank the DRSC at the Harvard Medical School for providing critical reagents, instrumentations, technical advice, and other resources that greatly facilitated our screening. The authors work was funded by the NIH (RO1 CA108509).

References

  1. 1.
    Raftery LA, Twombly V, Wharton K, Gelbart WM (1995) Genetic screens to identify elements of the decapentaplegic signaling pathway in Drosophila. Genetics 139:241–254PubMedCentralPubMedGoogle Scholar
  2. 2.
    Sekelsky JJ, Newfeld SJ, Raftery LA, Chartoff EH, Gelbart WM (1995) Genetic characterization and cloning of Mothers against dpp, a gene required for decapentaplegic function in Drosophila melanogaster. Genetics 139:1347–1358PubMedCentralPubMedGoogle Scholar
  3. 3.
    Macias-Silva M, Abdollah S, Hoodless PA, Pirone R, Attisano L, Wrana JL (1996) MADR2 is a substrate of the TGFß receptor and phosphorylation is required for nuclear accumulation and signaling. Cell 87:1215–1224CrossRefPubMedGoogle Scholar
  4. 4.
    Kretzschmar M, Liu F, Hata A, Doody J, Massagué J (1997) The TGF-ß mediator Smad1 is directly phosphorylated and functionally activated by the BMP receptor kinase. Genes Dev 11:984–995CrossRefPubMedGoogle Scholar
  5. 5.
    Souchelnytskyi S, Tamaki K, Engstrom U, Wernstedt C, ten Dijke P, Heldin CH (1997) Phosphorylation of Ser465 and Ser467 in the C terminus of Smad2 mediates interaction with Smad4 and is required for transforming growth factor-beta signaling. J Biol Chem 272(44):28107–28115CrossRefPubMedGoogle Scholar
  6. 6.
    Raftery LA, Sutherland DJ (1999) TGF-beta family signal transduction in Drosophila development: from Mad to Smads. Dev Biol 210(2):251–268CrossRefPubMedGoogle Scholar
  7. 7.
    Shi Y, Massagué J (2003) Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 113(6):685–700CrossRefPubMedGoogle Scholar
  8. 8.
    Sharp PA, Zamore PD (2000) Molecular biology. RNA interference. Science 287(5462):2431–2433CrossRefPubMedGoogle Scholar
  9. 9.
    Hannon GJ (2002) RNA interference. Nature 418(6894):244–251CrossRefPubMedGoogle Scholar
  10. 10.
    Xu L, Yao X, Chen X, Lu P, Zhang B, Ip YT (2007) Msk is required for nuclear import of TGF-{beta}/BMP-activated Smads. J Cell Biol 178:981–994PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    Chen X, Xu L (2010) Specific nucleoporin requirement for Smad nuclear translocation. Mol Cell Biol 30(16):4022–4034, PMCID: 2916443PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Clemens JC, Worby CA, Simonson-Leff N, Muda M, Maehama T, Hemmings BA et al (2000) Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc Natl Acad Sci U S A 97(12):6499–6503PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Armknecht S, Boutros M, Kiger A, Nybakken K, Mathey-Prevot B, Perrimon N (2005) High-throughput RNA interference screens in Drosophila tissue culture cells. Methods Enzymol 392:55–73CrossRefPubMedGoogle Scholar
  14. 14.
    Ramadan N, Flockhart I, Booker M, Perrimon N, Mathey-Prevot B (2007) Design and implementation of high-throughput RNAi screens in cultured Drosophila cells. Nat Protoc 2(9):2245–2264CrossRefPubMedGoogle Scholar
  15. 15.
    Flockhart I, Booker M, Kiger A, Boutros M, Armknecht S, Ramadan N et al (2006) FlyRNAi: the Drosophila RNAi screening center database. Nucleic Acids Res 34(Database issue):D489–D494, PMCID: 1347476PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Bunch TA, Grinblat Y, Goldstein LS (1988) Characterization and use of the Drosophila metallothionein promoter in cultured Drosophila melanogaster cells. Nucleic Acids Res 16(3):1043–1061PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Echeverri CJ, Beachy PA, Baum B, Boutros M, Buchholz F, Chanda SK et al (2006) Minimizing the risk of reporting false positives in large-scale RNAi screens. Nat Methods 3(10):777–779CrossRefPubMedGoogle Scholar
  18. 18.
    Perrimon N, Mathey-Prevot B (2007) Matter arising: off-targets and genome-scale RNAi screens in Drosophila. Fly (Austin) 1(1):1–5CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Program in Molecular MedicineUniversity of Massachusetts Medical SchoolWorcesterUSA
  2. 2.Blueprint MedicinesCambridgeUSA

Personalised recommendations