A Combined Ex Vivo and In Vivo RNAi Screen for Notch Regulators in Drosophila Reveals an Extensive Notch Interaction Network

Abstract

Notch signaling plays a fundamental role in cellular differentiation and has been linked to human diseases, including cancer. We report the use of comprehensive RNAi analyses to dissect Notch regulation and its connections to cellular pathways. A cell-based RNAi screen identified 900 candidate Notch regulators on a genome-wide scale. The subsequent use of a library of transgenic Drosophila expressing RNAi constructs enabled large-scale in vivo validation and confirmed 333 of 501 tested genes as Notch regulators. Mapping the phenotypic attributes of our data on an interaction network identified another 68 relevant genes and revealed several modules of unexpected Notch regulatory activity. In particular, we note an intriguing relationship to pyruvate metabolism, which may be relevant to cancer. Our study reveals a hitherto unappreciated diversity of tissue-specific modulators impinging on Notch and opens new avenues for studying Notch regulation and function in development and disease.

Regulation of Notch Signaling by an Evolutionary Conserved DEAD Box RNA Helicase, Maheshvara in Drosophila melanogaster

“…Genome-wide studies have revealed a complex network of genetic circuits involved in context-dependent regulation of Notch signaling. There are multiple modifiers and regulators of the Notch signaling pathway, depending on the tissue type and developmental context, and more recently the involvement of RNA-binding proteins in regulation of Notch signaling has been appreciated (Hall et al 2004;Saj et al 2010;Jung et al 2013).…”

“…Finetuning of Notch signaling is mediated by a vast number of Notch regulators or modifiers. Although Notch signaling has been extensively studied, identification of additional modifiers and regulators will help us to better understand the intricate regulation of this fundamental signaling pathway (Hall et al 2004;Saj et al 2010).…”

“…Finetuning of Notch signaling is mediated by a vast number of Notch regulators or modifiers. Although Notch signaling has been extensively studied, identification of additional modifiers and regulators will help us to better understand the intricate regulation of this fundamental signaling pathway (Hall et al 2004;Saj et al 2010).The RNA-binding protein of the DEAD box family constitutes the largest group of RNA helicases with signature motif DEAD (amino acid code for Asp, Glu, Ala, and Asp). They are known to unwind RNA in an ATP-dependent manner and can modulate RNA-RNA as well as RNA-protein interactions, which may influence the expression, localization, and stability of the target RNAs (Linder et al 1989;Liu et al 2008).…”

Analysis of neural stem cell self-renewal and differentiation by transgenic RNAi in Drosophila

“…Currently, three independent sources of genome-wide UAS-RNAi transgenic lines are available [Vienna Drosophila RNAi Center (VDRC), Transgenic RNAi Project (TRiP) at the Harvard Medical School, and NGI-FLY RNAi Resources in Japan], such that altogether about 90% of the annotated genes in the Drosophila genome can now be targeted by these lines [67]. With the availability of these lines, genome-wide transgenic RNAi screens have been successfully used to study Notch signaling [68,69], muscle morphogenesis and functions [70], host-pathogen interactions [71], and self-renewal of neuroblasts [12,13]. Taken together, these studies show that even very complicated biological mechanisms can be systematically analyzed at the genome-wide level by transgenic RNAi.…”

Genome-Wide RNAi Screen for the Discovery of Gene Function, Novel Therapeutical Targets and Agricultural Applications

“…Therefore new constructs expressing small hairpin RNAs were generated to produce second generation of transgenic RNAi Drosophila stains at Harvard medical school (Table. 2). In the past several years, a number of genome-wide RNAi screens in Drosophila have been conducted to study the major signaling pathways [51], as well as many important disease models [52][53][54][55].…”