This work was supported by The Wellcome Trust, Glaxosmithkline (GSK) and the Scottish Institute for Cell Signalling (now part of the MRC Protein Phosphorylation and Ubiquitylation unit).

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.
<ol>
	<li>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 (DM...

<ol>
	<li>Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+function+of+ubiquitin-like+proteins.">Origin and function of ubiquitin-like proteins.</a> Nature. 458, 422-429 (2009).</li><li>Schulman, B. A., Harper, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin-like+protein+activation+by+E1+enzymes:+the+apex+for+downstream+signalling+pathways.">Ubiquitin-like protein activation by E1 enzymes: the apex for downstream signalling pathways.</a> Nat Rev Mol Cell Biol. 10, 319-331 (2009).</li><li>Siomi, H., Siomi, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+road+to+reading+the+RNA-interference+code.">On the road to reading the RNA-interference code.</a> Nature. 457, 396-404 (2009).</li><li>Kaelin, W. G., Ratcliffe, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+sensing+by+metazoans:+the+central+role+of+the+HIF+hydroxylase+pathway.">Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway.</a> Mol Cell. 30, 393-402 (2008).</li><li>Melvin, A., Mudie, S., Rocha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chromatin+remodeler+ISWI+regulates+the+cellular+response+to+hypoxia:+role+of+FIH.">The chromatin remodeler ISWI regulates the cellular response to hypoxia: role of FIH.</a> Mol Biol Cell. 22, 4171-4181 (2011).</li><li>Bhinder, B., Djaballah, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+analyzing+actives+in+random+RNAi+screens:+introducing+the+&amp;#34;H+Score&amp;#34;+for+hit+nomination+&amp;+gene+prioritization.">A simple method for analyzing actives in random RNAi screens: introducing the &amp;#34;H Score&amp;#34; for hit nomination &amp; gene prioritization.</a> Comb Chem High Throughput Screen. 15, 686-704 (2012).</li><li>Bett, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+P-body+component+USP52/PAN2+is+a+novel+regulator+of+HIF1A+mRNA+stability.">The P-body component USP52/PAN2 is a novel regulator of HIF1A mRNA stability.</a> Biochem J. 451, 185-194 (2013).</li><li>Zhang, J. H., Chung, T. D., Oldenburg, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays.">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> J Biomol Screen. 4, 67-73 (1999).</li><li>Birmingham, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+methods+for+analysis+of+high-throughput+RNA+interference+screens.">Statistical methods for analysis of high-throughput RNA interference screens.</a> Nat Methods. 6, 569-575 (2009).</li><li>Stagg, H. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TRC8+E3+ligase+ubiquitinates+MHC+class+I+molecules+before+dislocation+from+the+ER.">The TRC8 E3 ligase ubiquitinates MHC class I molecules before dislocation from the ER.</a> J Cell Biol. 186, 685-692 (2009).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNF146+is+a+poly(ADP-ribose)-directed+E3+ligase+that+regulates+axin+degradation+and+Wnt+signalling.">RNF146 is a poly(ADP-ribose)-directed E3 ligase that regulates axin degradation and Wnt signalling.</a> Nat Cell Biol. 13, 623-629 (2011).</li><li>Jackson, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+profiling+reveals+off-target+gene+regulation+by+RNAi.">Expression profiling reveals off-target gene regulation by RNAi.</a> Nat Biotechnol. 21, 635-637 (2003).</li><li>Birmingham, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3'+UTR+seed+matches,+but+not+overall+identity,+are+associated+with+RNAi+off-targets.">3' UTR seed matches, but not overall identity, are associated with RNAi off-targets.</a> Nat Methods. 3, 199-204 (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whither+RNAi.">Whither RNAi.</a> Nat Cell Biol. 5, 489-490 (2003).</li></ol>

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.&#160; 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.</...

Modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) represents an expansive biochemical system that regulates diverse biological pathways and stress responses.&#160; The covalent attachment of UBLs to their target proteins can have various outcomes regulating the stability, localization, function or interactome of the substrate1.&#160; 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.&#160; For modification to occur, the carboxylate group of the UBL diglycine motif is first activated by an E1 activating enzyme to form a high-energy thiol that is transferred to the active-site cysteine of an E2 conjugating enzyme.&#160; The E2 then interacts with a substrate-bound E3 ligase to mediate transfer of the UBL onto (usually) a target lysine residue creating a branched chain (isopeptide) linkage2.&#160; Successive rounds of modification can occur to build isopeptide chains onto the substrate, which for ubiquitin can occur through any of its seven lysines, or through its N-terminal methionine to create linear ubiquitin chains.&#160; These modifications form discrete topologies with diverse purposes such as creating new interaction motifs and targeting proteins for degradation prior to UBL removal by specialist proteases.&#160; In the case of ubiquitin there are two E1 enzymes, 30-40 E2 conjugating enzymes, at least 600 E3 ligases and approximately 100 deubiquitylating enzymes (DUBs).&#160; While the pathways are less expansive for the other 10 or so UBLs, the overall ubiquitome complexity affords huge diversity in the biological outcome of a particular UBL modification. &#160;However, while major advances in UBL biology have been made, the precise cellular roles of the majority of these ubiquitome components remain unknown.
The use of short interfering ribonucleic acid (siRNAs) has emerged as a powerful tool in reverse genetics due to the ability of siRNAs to specifically target cellular mRNAs for destruction, allowing the role of individual genes to be examined in different biological contexts3. Whole genome screens have been used to identify and validate new regulators of many cellular processes, and have created a wealth of useful data accessible to the wider scientific community.&#160; However, while whole genome screens have proven extremely useful, targeted screens are becoming increasingly popular as they are cheaper, faster, involve less data management and report only on members of the genome in which the investigator is most interested.&#160; Therefore, to better understand which cellular processes UBL family components are involved in, we have compiled a human siRNA library targeting all known and predicted components of the ubiquitome.&#160; This includes the UBLs, E1 activating enzymes, E2 conjugating enzymes, E3 ligases, ubiquitin-binding domain (UBD)-containing proteins and DUBs.&#160; This library can be used to screen against a wide range of reporter cell lines of distinct biological problems, thus allowing the unbiased identification of novel UBL components governing these pathways.
The following protocol describes how to perform a rigorous targeted siRNA ubiquitome screen to identify novel regulators of the HIF1A-dependent response to hypoxia.&#160; Under normal oxygen tension, HIF1A is subject to prolyl hydroxylation that causes it to be recognized and targeted for degradation by the Von Hippel Lindau (VHL) E3 ligase complex4.&#160; Hypoxia inhibits prolyl hydroxylation leading to the stabilization of HIF1A and its subsequent binding to hypoxia response elements (HREs) to drive gene expression. Here, we describe a screen using U20S osteosarcoma cells stably expressing firefly luciferase under the control of three tandem copies of the Hypoxia Response Element (U20S-HRE cells)5. &#160;This protocol can be adapted for any biological pathway if a robust read-out of reporter activity is achievable and can be coupled with appropriate positive and negative controls.

<table border="0" cellpadding="0" cellspacing="0" width="790">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:288px;">Automated Liquid Dispenser</td>
			<td style="width:147px;">Fluid-X</td>
			<td style="width:200px;">XPP-721</td>
			<td style="width:156px;">http://www.fluidx.eu/BIOTRACK/xpp-721-liquid-handling-system.html</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Automated cell counter</td>
			<td>Nexcelom Bioscience</td>
			<td>Cellometer Auto T4</td>
			<td>http://www.nexcelom.com/Cellometer-Auto-T4/index.html</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Hypoxia Workstation</td>
			<td>Ruskin</td>
			<td>In vivo2 300</td>
			<td>http://www.ruskinn.com/products/invivo2300</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Automated Cell Dispenser</td>
			<td>Thermo Scientific</td>
			<td>Matrix Wellmate</td>
			<td>http://www.matrixtechcorp.com/automated/pipetting.aspx?id=11</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Plate Shaker</td>
			<td>Heidolph</td>
			<td>Titramax 1000</td>
			<td>http://www.heidolph-instruments.co.uk/products/shakers-mixers/platform-shakers/vibrating/titramax/titramax-1000/</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Luminometer</td>
			<td>Perkin Elmer</td>
			<td>Envision 2104 Multilabel Reader</td>
			<td>http://www.perkinelmer.co.uk/Catalog/Family/ID/EnVision%20Multilabel%20Plate%20Readers</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">White Walled Assay Plate</td>
			<td>Greiner Bio One</td>
			<td>655083</td>
			<td>http://www.greinerbioone.com/en/row/articles/catalogue/article/37_11/13221/</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Clear Plate Film</td>
			<td>Perkin Elmer</td>
			<td>1450-461</td>
			<td>http://www.perkinelmer.co.uk/Catalog/Product/ID/1450-461</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Name of the Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">siRNA library</td>
			<td>Thermo Scientific</td>
			<td>On-Target Plus</td>
			<td>http://www.thermoscientificbio.com/rnai-and-custom-rna-synthesis/sirna/on-targetplus-sirna/search-gene/</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Transfection reagent</td>
			<td>Invitrogen</td>
			<td>Lipofectamine RNAimax</td>
			<td>http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Protein-Expression-and-Analysis/Transfection-Selection/lipofectamine-rnaimx.html</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Reduced Serum Medium</td>
			<td>Invitrogen</td>
			<td>Optimem</td>
			<td>http://products.invitrogen.com/ivgn/product/31985062?ICID=search-product</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DMEM</td>
			<td>Invitrogen</td>
			<td>41965-039</td>
			<td>http://products.invitrogen.com/ivgn/product/41965039#</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">FBS</td>
			<td>Invitrogen</td>
			<td>16000-044</td>
			<td>https://products.invitrogen.com/ivgn/product/16000044?ICID=search-product#</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tryspin-EDTA</td>
			<td>Invitrogen</td>
			<td>25300-054</td>
			<td>https://products.invitrogen.com/ivgn/product/25300054?ICID=search-product#</td>
		</tr>
	</tbody>
</table>

sirna screening to identify ubiquitin and ubiquitin-like system regulators of biological pathways in cultured mammalian cells

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.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; 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.&#160; 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.&#160; 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. &#160;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.&#160; 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.

Prior to screening, the hypoxia-responsiveness of U20S-HRE cells is established.&#160; 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).&#160; Cells are placed in a hypoxia workstation for a range of times to establish which hypoxia exposure produces the most effective response for screening.&#160; U20S cells express low le...

Watch this Scientific Journal Video about siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells at JoVE.com

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

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

sirna-screening-to-identify-ubiquitin-ubiquitin-like-system

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JoVE Journal

Biology

11.3K vistas.  University of Dundee.  Aquí se describe una metodología para llevar a cabo un siRNA dirigidos pantalla "ubiquitome" para identificar nuevos ubiquitina y reguladores-ubiquitina de la respuesta celular mediada HIF1A a la hipoxia. Esto puede ser adaptado a cualquier vía biológica, donde una lectura robusta de la actividad del indicador está disponible. 