Reagent and Data Resources for Investigation of RNA Binding Protein Functions in<i>Drosophila melanogaster</i>Cultured Cells

Mohr, Stephanie E.; Hu, Yanhui; Rudd, Kirstin; Buckner, Michael; Gilly, Quentin; Foster, Blake; Sierzputowska, Katarzyna; Comjean, Aram; Ye, Bing; Perrimon, Norbert

doi:10.1534/g3.115.019364

Cited by 7 publications

(8 citation statements)

References 35 publications

(50 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…An RNAi screen in S2R+ cells showed depletion of multiple CPA complex components increases ATP levels (Mohr et al., 2015). CDK8 is implicated in lipid metabolism, and KNS1 (DOA ortholog in yeast) in the regulation of protein homeostasis (Lee et al, 2012; Zhao et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

The TORC1-Regulated CPA Complex Rewires an RNA Processing Network to Drive Autophagy and Metabolic Reprogramming

Tang

Chen

et al. 2018

Cell Metabolism

Self Cite

View full text Add to dashboard Cite

Nutrient deprivation induces autophagy through inhibiting TORC1 activity. We describe a novel mechanism in Drosophila by which TORC1 regulates RNA processing of Atg transcripts and alters ATG protein levels and activities via the cleavage and polyadenylation (CPA) complex. We show that TORC1 signaling inhibits CDK8 and DOA kinases, which directly phosphorylate CPSF6, a component of the CPA complex. These phosphorylation events regulate CPSF6 localization, RNA binding, and starvation-induced alternative RNA processing of transcripts involved in autophagy, nutrient, and energy metabolism, thereby controlling autophagosome formation and metabolism. Similarly, we find that mammalian CDK8 and CLK2, a DOA ortholog, phosphorylate CPSF6 to regulate autophagy and metabolic changes upon starvation, revealing an evolutionarily conserved mechanism linking TORC1 signaling with RNA processing, autophagy, and metabolism.

show abstract

Section: Discussionmentioning

confidence: 99%

The TORC1-Regulated CPA Complex Rewires an RNA Processing Network to Drive Autophagy and Metabolic Reprogramming

Tang

Chen

et al. 2018

Cell Metabolism

Self Cite

View full text Add to dashboard Cite

show abstract

“…The dsRNA sequence for knocking-down Par3 was selected according to an RNAi database DRSC (DRSC25558) (Mohr et al, 2015).…”

Section: Methodsmentioning

confidence: 99%

Reconstruction of Par-dependent polarity in apolar cells reveals a dynamic process of cortical polarization

Kono

Yoshiura

Fujita

et al. 2019

eLife

View full text Add to dashboard Cite

Cellular polarization is fundamental for various biological processes. The Par network system is conserved for cellular polarization. Its core complex consists of Par3, Par6, and aPKC. However, the general dynamic processes that occur during polarization are not well understood. Here, we reconstructed Par-dependent polarity using non-polarized Drosophila S2 cells expressing all three components endogenously in the cytoplasm. The results indicated that elevated Par3 expression induces cortical localization of the Par-complex at the interphase. Its asymmetric distribution goes through three steps: emergence of cortical dots, development of island-like structures with dynamic amorphous shapes, repeating fusion and fission, and polarized clustering of the islands. Our findings also showed that these islands contain a meshwork of unit-like segments. Furthermore, Par-complex patches resembling Par-islands exist in Drosophila mitotic neuroblasts. Thus, this reconstruction system provides an experimental paradigm to study features of the assembly process and structure of Par-dependent cell-autonomous polarity.

show abstract

“…Cell-based screens require an arrayed dsRNA library (see Figure 3 , Table 3 ). Long dsRNA can be synthesized by in vitro transcription of PCR amplicons from complementary DNA (cDNA) or genomic DNA ( Clemens et al 2000 ; Kiger et al 2003 ; Boutros et al 2004 ; Falschlehner et al 2010 ; Mohr et al 2015 ). Synthesized dsRNA libraries are subsequently spotted as aqueous solutions into microtiter plates suitable for the specific readout ( e.g.…”

Section: Design Of High-throughput Screening Assaysmentioning

confidence: 99%

“…In this review, building on a number of previous reviews ( Echeverri and Perrimon 2006 ; Echeverri et al 2006 ; Boutros and Ahringer 2008 ; Mohr et al 2010 , 2015 ; Perrimon et al 2010 ; Mohr and Perrimon 2012 ; Mohr 2014 ), we will first describe different methodological options for RNAi screening in Drosophila . We will discuss how to design assays, which reagent resources are available, how to conduct RNAi screens in cells and in vivo , how to analyze data from high-throughput screens, what unintended effects can occur in screens, and how to independently confirm results.…”

mentioning

confidence: 99%

RNA Interference (RNAi) Screening inDrosophila

Heigwer¹,

Port²,

Boutros

2018

Genetics

102

View full text Add to dashboard Cite

In the last decade, RNA interference (RNAi), a cellular mechanism that uses RNA-guided degradation of messenger RNA transcripts, has had an important impact on identifying and characterizing gene function. First discovered in Caenorhabditis elegans, RNAi can be used to silence the expression of genes through introduction of exogenous double-stranded RNA into cells. In Drosophila, RNAi has been applied in cultured cells or in vivo to perturb the function of single genes or to systematically probe gene function on a genome-wide scale. In this review, we will describe the use of RNAi to study gene function in Drosophila with a particular focus on high-throughput screening methods applied in cultured cells. We will discuss available reagent libraries and cell lines, methodological approaches for cell-based assays, and computational methods for the analysis of high-throughput screens. Furthermore, we will review the generation and use of genome-scale RNAi libraries for tissue-specific knockdown analysis in vivo and discuss the differences and similarities with the use of genome-engineering methods such as CRISPR/Cas9 for functional analysis.

show abstract

Reagent and Data Resources for Investigation of RNA Binding Protein Functions inDrosophila melanogasterCultured Cells

Cited by 7 publications

References 35 publications

The TORC1-Regulated CPA Complex Rewires an RNA Processing Network to Drive Autophagy and Metabolic Reprogramming

The TORC1-Regulated CPA Complex Rewires an RNA Processing Network to Drive Autophagy and Metabolic Reprogramming

Reconstruction of Par-dependent polarity in apolar cells reveals a dynamic process of cortical polarization

RNA Interference (RNAi) Screening inDrosophila

Contact Info

Product

Resources

About