siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Summary

Here, we describe a methodology to perform a targeted siRNA “ubiquitome” screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.  This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Abstract

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.  To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA “ubiquitome” library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.  Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.  An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening.  The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.  Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Introduction

Modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) represents an expansive biochemical system that regulates diverse biological pathways and stress responses.  The covalent attachment of UBLs to their target proteins can have various outcomes regulating the stability, localization, function or interactome of the substrate¹.  The enzymatic steps underlying UBL modification were first established for ubiquitin, and now serve as a paradigm for modification with most UBLs, including SUMO, NEDD8, ISG15 and FAT10.  For modification to occur, the carboxylate group of the UBL diglycine motif is first activated by an E1 activa....

Protocol

1. Assay Development Stage

Note: prior to initiating the siRNA screen, an assay development stage is critical to set out important parameters for screening with the reporter cell line. It is essential to invest significant effort at this stage as this will underpin the future success of the screen.

1. To characterize the hypoxia-responsiveness of U20S-HRE reporter cell line, grow 2 x 75 cm² flasks of U20S-HRE cells to 80-90% confluence in Dulbeccos Modified Eagle Medium (DMEM) supplemented with 10% FBS.
2. Aspirate the media from one flask of cells, wash twice with 10 ml PBS and detach cells by adding 2 ml 0.05% t....

Results

Prior to screening, the hypoxia-responsiveness of U20S-HRE cells is established.  U20S-HRE cells express a reporter construct consisting of firefly luciferase fused downstream of three tandem copies of the hypoxia response element, which is bound by the HIF1A/HIF1B heterodimer upon exposure to hypoxia (Figure 1A).  Cells are placed in a hypoxia workstation for a range of times to establish which hypoxia exposure produces the most effective response for screening.  U20S cells express low levels of HIF2A, .......

Discussion

The use of genome-wide siRNA screens in mammalian cells has proven to be extremely valuable in identifying novel regulators of distinct biological pathways. Here, we have described the use of a targeted ubiquitome siRNA screen to identify regulators of the HIF1A-mediated cellular response to hypoxia.  Targeted screens are becoming increasingly attractive as they are generally cheaper, quicker, easier to manage and report only on the pathway components in which the investigators are interested ^{7, 10, 11}.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Automated Liquid Dispenser	Fluid-X	XPP-721	http://www.fluidx.eu/BIOTRACK/xpp-721-liquid-handling-system.html
White Walled Assay Plate	Greiner Bio One	655083	http://www.greinerbioone.com/en/row/articles/catalogue/article/37_11/13221/
Clear Plate Film	Perkin Elmer	1450-461	http://www.perkinelmer.co.uk/Catalog/Product/ID/1450-461
siRNA library	Thermo Scientific	On-Target Plus	http://www.thermoscientificbio.com/rnai-and-custom-rna-synthesis/sirna/on-targetplus-sirna/search-gene/
Transfection reagent	Invitrogen	Lipofectamine RNAimax	http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Protein-Expression-and-Analysis/Transfection-Selection/lipofectamine-rnaimx.html
Reduced Serum Medium	Invitrogen	Optimem	http://products.invitrogen.com/ivgn/product/31985062?ICID=search-product
DMEM	Invitrogen	41965-039	http://products.invitrogen.com/ivgn/product/41965039#
FBS	Invitrogen	16000-044	https://products.invitrogen.com/ivgn/product/16000044?ICID=search-product#
Tryspin-EDTA	Invitrogen	25300-054	https://products.invitrogen.com/ivgn/product/25300054?ICID=search-product#