RNAi screening comes of age: improved techniques and complementary approaches

Mohr, Stephanie E.; Smith, Jennifer A.; Shamu, Caroline E.; Neumüller, Ralph A.; Perrimon, Norbert

doi:10.1038/nrm3860

Cited by 309 publications

(249 citation statements)

References 105 publications

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“…For the type IA PI3K components that appeared to have positive roles in entry of inv ϩ HB101 or of L. monocytogenes, we performed additional RNAi experiments to address potential off-target effects of siRNAs. One common method of minimizing off-target effects is to verify that several different siRNA molecules recognizing distinct sequences in a given mRNA cause the same biological phenotype (56). Importantly, three different siRNAs directed against each of the nine human genes implicated in internalization of inv ϩ HB101 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Role of Host Type IA Phosphoinositide 3-Kinase Pathway Components in Invasin-Mediated Internalization of Yersinia enterocolitica

et al. 2016

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Many bacterial pathogens subvert mammalian type IA phosphoinositide 3-kinase (PI3K) in order to induce their internalization into host cells. How PI3K promotes internalization is not well understood. Also unclear is whether type IA PI3K affects different pathogens through similar or distinct mechanisms. Here, we performed an RNA interference (RNAi)-based screen to identify components of the type IA PI3K pathway involved in invasin-mediated entry of Yersinia enterocolitica, an enteropathogen that causes enteritis and lymphadenitis. The 69 genes targeted encode known upstream regulators or downstream effectors of PI3K. A similar RNAi screen was previously performed with the food-borne bacterium Listeria monocytogenes. The results of the screen with Y. enterocolitica indicate that at least nine members of the PI3K pathway are needed for invasin-mediated entry. Several of these proteins, including centaurin-␣1, Dock180, focal adhesion kinase (FAK), Grp1, LL5␣, LL5␤, and PLD2 (phospholipase D2), were recruited to sites of entry. In addition, centaurin-␣1, FAK, PLD2, and mTOR were required for remodeling of the actin cytoskeleton during entry. Six of the human proteins affecting invasin-dependent internalization also promote InlBmediated entry of L. monocytogenes. Our results identify several host proteins that mediate invasin-induced effects on the actin cytoskeleton and indicate that a subset of PI3K pathway components promote internalization of both Y. enterocolitica and L. monocytogenes. Many microbial pathogens exploit host signal transduction pathways in order to cause disease (1-4). One key pathway subverted by bacterial, viral, and protozoan pathogens involves a mammalian lipid kinase called type IA phosphoinositide 3-kinase (PI3K) (3). Type IA PI3K plays a critical role in internalization of several pathogens into host cells. These microbes include bacteria that cause anthrax (Bacillus anthracis) (5), respiratory infections (Pseudomonas aeruginosa and Chlamydia pneumoniae) (6, 7), and food-borne disease (Campylobacter jejuni and Listeria monocytogenes) (3,4,8). Type IA PI3K also promotes entry of Ebola virus (9), influenza A virus (10), and parasites causing Chagas' disease (Trypanosoma cruzi) (11) or toxoplasmosis (Toxoplasma gondii) (12). Overall, the mechanism by which this mammalian lipid kinase controls infection by these diverse pathogens is not well understood.Mammalian type IA PI3K is an heterodimeric enzyme composed of a 110-kDa catalytic subunit and a 85-kDa regulatory subunit (13). This PI3K is coupled to growth factor, cytokine, or cell adhesion receptors and controls a variety of processes, including cell growth, survival, and motility (13,14). Type IA PI3K promotes its biological effects through at least two mechanisms, the best understood of which involves lipid kinase activity. This PI3K produces phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ], a lipid second messenger. PI(3,4,5)P 3 is converted by phosphatases to phosphatidylinositol 3,4-bisphosphate [PI(3,4)P 2 ]. Together, PI(3,4,5...

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Section: Resultsmentioning

confidence: 99%

Role of Host Type IA Phosphoinositide 3-Kinase Pathway Components in Invasin-Mediated Internalization of Yersinia enterocolitica

et al. 2016

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“…We used RNAi to investigate the roles of Raptor and Rictor in the entry of Listeria. These two human proteins were each targeted by three different siRNAs in order to minimize the possibility of off-target effects (24). siRNAs were introduced into HeLa cells by transfection.…”

Section: Resultsmentioning

confidence: 99%

Host Serine/Threonine Kinases mTOR and Protein Kinase C-α Promote InlB-Mediated Entry of Listeria monocytogenes

et al. 2017

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The bacterial pathogen Listeria monocytogenes causes foodborne illnesses resulting in gastroenteritis, meningitis, or abortion. Listeria induces its internalization into some human cells through interaction of the bacterial surface protein InlB with the host receptor tyrosine kinase Met. InlB-dependent entry requires localized polymerization of the host actin cytoskeleton. The signal transduction pathways that act downstream of Met to regulate actin filament assembly or other processes during Listeria uptake remain incompletely characterized. Here, we demonstrate important roles for the human serine/threonine kinases mTOR and protein kinase C-␣ (PKC-␣) in InlB-dependent entry. Experiments involving RNA interference (RNAi) indicated that two multiprotein complexes containing mTOR, mTORC1 and mTORC2, are each needed for efficient internalization of Listeria into cells of the human cell line HeLa. InlB stimulated Met-dependent phosphorylation of mTORC1 or mTORC2 substrates, demonstrating activation of both mTOR-containing complexes. RNAi studies indicated that the mTORC1 effectors 4E-BP1 and hypoxia-inducible factor 1␣ (HIF-1␣) and the mTORC2 substrate PKC-␣ each control Listeria uptake. Genetic or pharmacological inhibition of PKC-␣ reduced the internalization of Listeria and the accumulation of actin filaments that normally accompanies InlB-mediated entry. Collectively, our results identify mTOR and PKC-␣ to be host factors exploited by Listeria to promote infection. PKC-␣ controls Listeria entry, at least in part, by regulating the actin cytoskeleton downstream of the Met receptor.KEYWORDS InlB, Listeria monocytogenes, Met receptor, protein kinase C, actin, mTOR L isteria monocytogenes is a foodborne pathogen capable of causing serious infections resulting in meningitis or abortions (1). Critical for disease is the ability of Listeria to induce its internalization into nonphagocytic mammalian cells in the intestine, liver, or placenta (2). One of the major pathways of internalization of Listeria into human cells is mediated by interaction of the bacterial surface protein InlB with the host receptor tyrosine kinase Met (3). InlB-dependent entry involves remodeling of the host plasma membrane through actin polymerization (4, 5). Substantial progress in characterizing InlB-mediated uptake has been made, resulting in the identification of several host signal transduction pathways that control actin polymerization and perhaps other physiological processes during entry (4). Despite this progress, a detailed understanding of the molecular mechanism of InlB-dependent internalization of Listeria remains incomplete.The human type IA phosphoinositide 3-kinase (PI3K) pathway is one of the major signaling networks controlling InlB-mediated entry (4,(6)(7)(8). Recent RNA interference (RNAi) screens identified 13 components of the type IA PI3K pathway involved in InlB-dependent internalization of Listeria (9, 10). One host protein demonstrated in this work to play an important role in Listeria entry is mTOR (mammalian ...

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“…CRISPR-Cas9-based screens, like other genetic screens, can lead to false discovery due to off-target effects and false-positive hits (28,29). Thus, we performed individual validation experiments to verify the phenotype-modifying effects of genetic perturbations identified as hits from our screen.…”

Section: Significancementioning

confidence: 99%

Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM

Wong

Choi

Cui

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

222

171

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The orchestrated action of genes controls complex biological phenotypes, yet the systematic discovery of gene and drug combinations that modulate these phenotypes in human cells is labor intensive and challenging to scale. Here, we created a platform for the massively parallel screening of barcoded combinatorial gene perturbations in human cells and translated these hits into effective drug combinations. This technology leverages the simplicity of the CRISPR-Cas9 system for multiplexed targeting of specific genomic loci and the versatility of combinatorial genetics en masse (Combi-GEM) to rapidly assemble barcoded combinatorial genetic libraries that can be tracked with high-throughput sequencing. We applied CombiGEM-CRISPR to create a library of 23,409 barcoded dual guide-RNA (gRNA) combinations and then perform a high-throughput pooled screen to identify gene pairs that inhibited ovarian cancer cell growth when they were targeted. We validated the growth-inhibiting effects of specific gene sets, including epigenetic regulators KDM4C/BRD4 and KDM6B/BRD4, via individual assays with CRISPR-Cas-based knockouts and RNA-interference-based knockdowns. We also tested small-molecule drug pairs directed against our pairwise hits and showed that they exerted synergistic antiproliferative effects against ovarian cancer cells. We envision that the CombiGEM-CRISPR platform will be applicable to a broad range of biological settings and will accelerate the systematic identification of genetic combinations and their translation into novel drug combinations that modulate complex human disease phenotypes.CRISPR-Cas | CombiGEM | multifactorial genetics | genetic perturbations | high-throughput screening

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RNAi screening comes of age: improved techniques and complementary approaches

Cited by 309 publications

References 105 publications

Role of Host Type IA Phosphoinositide 3-Kinase Pathway Components in Invasin-Mediated Internalization of Yersinia enterocolitica

Role of Host Type IA Phosphoinositide 3-Kinase Pathway Components in Invasin-Mediated Internalization of Yersinia enterocolitica

Host Serine/Threonine Kinases mTOR and Protein Kinase C-α Promote InlB-Mediated Entry of Listeria monocytogenes

Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM

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