Identification of Hedgehog Pathway Components by RNAi in Drosophila Cultured Cells

Abstract

Classical genetic screens can be limited by the selectivity of mutational targeting, the complexities of anatomically based phenotypic analysis, or difficulties in subsequent gene identification. Focusing on signaling response to the secreted morphogen Hedgehog (Hh), we used RNA interference (RNAi) and a quantitative cultured cell assay to systematically screen functional roles of all kinases and phosphatases, and subsequently 43% of predicted Drosophila genes. Two gene products reported to function in Wingless (Wg) signaling were identified as Hh pathway components: a cell surface protein (Dally-like protein) required for Hh signal reception, and casein kinase 1alpha, a candidate tumor suppressor that regulates basal activities of both Hh and Wg pathways. This type of cultured cell-based functional genomics approach may be useful in the systematic analysis of other biological processes.

Activation of the hedgehog-signaling pathway in human cancer and the clinical implications

“…Following translation, Hh proteins enter the secretory pathway and undergo autoprocessing and lipid modifications that produce a signaling peptide modified at its both ends by palmitoyl (N terminus) and cholesteryl (C terminus) adducts (Lee et al, 1994;Porter et al, 1995Porter et al, , 1996Buglino and Resh, 2008). The movement of Hh proteins is regulated by several molecules, such as the transmembrane transporter-like protein Dispatched (Disp) (11)(12)(13)(14) and metalloproteases (Dierker et al, 2009) for release of Hh from secreting cells, the heparan sulfate proteoglycans Dally-like (Dlp) and Dally (Lum et al, 2003;Beckett et al, 2008) or their regulators (Baena-Lopez et al, 2008) for extracellular transport of Hh protein as well as enzymes such as Sulfateless and Tout velu for heparan sulfate biosynthesis (Bellaiche et al, 1998;Toyoda et al, 2000;Koziel et al, 2004).…”

Regulation of Hedgehog signaling: a complex story

“…Ci 75 is produced through proteolytic cleavage of full-length Ci in cells receiving little or no Hh stimulation [32]. Cleavage of Ci occurs in a proteasome-dependent manner, and requires phosphorylation by Protein Kinase A (PKA), Glycogen Synthase Kinase 3β (GSK3β) and Casein Kinase 1δ (CK1δ) [56][57][58][59][60]. Also involved in production of Ci 75 is the F-box protein Supernumerary limbs (Slimb), which has been proposed to target phosphorylated full-length Ci for ubi-quitination [61].…”

“…For example, the Smo mutants SmoC and FFS, which lack the domains one would expect to interact with a Gprotein, are still capable of propagating Hh signaling [48]. Additionally, reducing the expression of all known Drosophila heterotrimeric GTP binding proteins, through use of RNA interference (RNAi), had little effect on Hh responses in cultured cells [56,75]. This lack of compelling evidence for G-protein involvement in the Hh pathway led multiple groups to look for direct interactions between Smo and HSC components.…”

Use of RNA interference to dissect the roles of trans-acting factors in alternative pre-mRNA splicing

“…In contrast to mammalian cells, where expensive synthetic siRNAs or vectors that express shRNAs are used, RNAi in cultured Drosophila cells can be performed by simply adding long (~400-500 bps) dsRNAs that can be enzymatically synthesized in the lab to the culture media [10]. The simplicity of this system has allowed several genome-wide RNAi screens to be performed in Drosophila culture cells and have led to the identification factors involved in biological processes ranging from cell viability to cell morphology [6,7,2,3]. In this chapter, we will describe in detail the RNAi approach we use to identify and analyze RNA binding proteins involved in controlling alternative splicing.…”