We thank the Italian Neuroblastoma Foundation for the full financial support to this project.

NOTE: The throughput of such assay systems depends on the available HTS lab equipment. This protocol is facilitated by a Tecan Freedom EVO 200 robot, which performs liquid handling in 96-well format. Miniaturization to 384-well format is also possible. The robotic liquid handling system is positioned under a laminar flow hood in order to maintain aseptic conditions during all experimental steps. If no liquid-handling automation is available, the protocol can be readily adapted to low-throughput format.
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<ol>
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F., Parker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+properties+of+eukaryotic+mRNPs.">Principles and properties of eukaryotic mRNPs.</a> Mol. Cell. 54 (4), 547-558 (2014).</li><li>Fu, Y., Dominissini, D., Rechavi, G., He, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+regulation+mediated+through+reversible+m6A+RNA+methylation.">Gene expression regulation mediated through reversible m6A RNA methylation.</a> Nat. Rev. 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The authors declare that they have no competing financial interests.

This protocol describes a cell-based reporter-gene assay aiming at the identification of modulators that target 3&#8217; UTR-dependent post-transcriptional processes. It encompasses the primary screening and the counter-screen and, if needed, can be accompanied by a cytotoxicity assay. The outcome of the primary screening is a number of valuable candidate compounds, whose reproducibility and specificity is further validated in the counter-screening.
The identification of primary hits is based ...

For a long period transcriptional regulation of gene expression was thought to play a major if not exclusive role in controlling protein production. Accumulating evidence, however, indicates that post-transcriptional regulation contributes as much as, if not more than, transcriptional regulation to determine cellular protein abundance1,2. Post-transcriptional control of gene expression is much more complex and elaborate than was at first thought. In fact, all the various stages of post-transcriptional control have emerged to be regulated, including mRNA processing, localization, turnover, translation3 as well as the newly described reversible RNA methylation4. From a number of post-transcriptional regulation processes potentially affected, the assay described below focuses on those involving the mRNA 3&#39; untranslated region (3&#8217; UTR). 3&#8217; UTR broadly affects mRNA fate mainly via specific interaction of RNA-binding proteins and non-coding RNAs to its regulatory sequences and/or secondary structures5. Therefore, small molecules that alter these interactions or interfere with upstream signal transduction pathways will shift the balance that regulates protein abundance. This therapeutic approach is of particular importance for human pathologies for which evidence exists showing that disease-relevant genes are subjected to post-transcriptional regulation, which thus represents a potential target for pharmacological intervention. Therefore, systems allowing for screening of mRNA levels&#8217; modulators through interference with post-transcriptional control mechanisms in a high-throughput format can become valuable tools in the identification of potential novel treatments.
The described cell-based reporter gene assay allows for the identification of compounds able to modulate mRNA fate via mechanisms dependent on its 3&#8217; UTR. It consists of several principal components (Figure 1) and requires few preliminary steps to validate the feasibility of the assay. First of all, the reporter construct is designed to include the 3&#8217; UTR from a gene of interest. The 3&#8217; UTR is fused to the end of a reporter gene such as firefly luciferase (Figure 1). Any changes in post-transcriptional control mechanisms exerted through the inserted 3&#8217; UTR will alter the stability and/or translation efficiency of this chimeric transcript, resulting in varied luciferase levels. Therefore, changes in luciferase activity serve as indirect measure of affected post-transcriptional regulation of the gene of interest. The second component of the system is a control reporter construct, an identical expression plasmid that expresses the same reporter gene but does not contain any 3&#8217; UTR. The two reporter plasmids can be designed in the lab using conventional molecular biology protocols or purchased from commercial sources.
The selection of an appropriate cell line is critical for the assay construction. Which cell line is the optimal model depends on numerous factors ranging from clinical requirements like maximal resemblance to pathology to just practical issues like availability, transfectability, and growth characteristics. Importantly, prior to proceed one should make sure that fusion of 3&#8217; UTR to the reporter gene has an expected effect on the level of the chimeric transcript. The absence of significant difference in luciferase signal from the 3&#8217; UTR-bearing and control reporter constructs transiently transfected in the selected cells would indicate that the 3&#8217; UTR is not functional in this cellular model, prompting for search of alternative ones. For the high-throughput format, the 3&#8217; UTR-bearing and control luciferase reporter constructs should be stably integrated in the selected cell line. Using a stably integrated over transiently transfected cell line is preferable for several reasons. This will broaden the choice of a cellular model including hard-to-transfect cell lines, decrease the variability in the data arising from fluctuations in transfection efficiency, subside the costs. Moreover, upon transient transfection cells are very often overloaded with a DNA plasmid leading to the saturation of the system. In these settings a further increase in the reporter expression might be challenging, resulting in decreased sensitivity towards potential upregulating compounds.
Finally, when all the assay components are set up, it is critical before moving to the high-throughput format to determine the feasibility of the assay, i.e., if luciferase activity can be indeed up- and down-regulated specifically via the inserted 3&#8217; UTR. The best controls would be small molecules reported to modulate the stability or translatability of mRNA through the 3&#8217; UTR of interest. If having these is not possible, surrogate controls imitating the desired effect are accepted. These could be miRNAs or RNA-binding proteins known to exert their effects via binding to the 3&#8217; UTR of interest. Evaluation of such controls before the primary screening not only indicates the functionality of the developed assay, but also allows for estimation of its dynamic range, sensitivity and performance in the high-throughput format.
Using the described protocol we screened a 2,000-compound library6. Neuroblastoma, the most common extracranial solid tumor of infancy, was used as a biological model and the 3&#8217; UTR of the MYCN oncogene, whose amplification strongly predicts adverse outcome of neuroblastoma, as a target gene7. Figure 2 outlines the experimental layout. The screening was divided in three runs with the batch size of 27 plates screened in one run. The batch of 27 plates included Spectrum Collection library plates n.1-n.8+n.25 (first run), n.9-n.16+n.25 (second run) and n.16-n.24+n.25 (third run), each plate screened in triplicate. Thus, one library plate (n.25) was assayed within all three runs making possible inter-run comparison. The protocol below describes a single run of 9 library plates tested in triplicate.

<table border="0" cellpadding="0" cellspacing="0" width="786">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Name of Material/ Equipment</td>
			<td style="width:239px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Culture plate 96-well White</td>
			<td style="width:239px;">PerkinElmer</td>
			<td align="right" style="width:119px;">6005688</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">StorPlate 96-well U bottom (dilution plate)</td>
			<td style="width:239px;">PerkinElmer</td>
			<td align="right" style="width:119px;">6008190</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">100 ml disposable trough for reagents</td>
			<td style="width:239px;">Tecan</td>
			<td>10 613 048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">300 ml disposable robotic reservoir</td>
			<td style="width:239px;">VWR</td>
			<td style="width:119px;">PB12001301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Robot tips DITI 50&#956;L Sterile</td>
			<td style="width:239px;">Tecan</td>
			<td align="right" style="width:119px;">30038607</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Robot tips DITI 200&#956;L Sterile</td>
			<td style="width:239px;">Tecan</td>
			<td align="right" style="width:119px;">30038617</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrix 1.4 ml 2D Barcoded w, Flat Bottom Tubes</td>
			<td style="width:239px;">Thermo Scientific</td>
			<td align="right">3711</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">ONE-Glo Luciferase Assay System&#160;</td>
			<td style="width:239px;">Promega</td>
			<td style="width:119px;">E6120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">RPMI</td>
			<td style="width:239px;">Lonza</td>
			<td style="width:119px;">BE12-918F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">PBS-1X, w/o Ca++, Mg++</td>
			<td style="width:239px;">Lonza</td>
			<td style="width:119px;">BE17-516F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">L-Glutamine 200mM</td>
			<td style="width:239px;">Lonza</td>
			<td style="width:119px;">BE17-605E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">FBS</td>
			<td style="width:239px;">Lonza</td>
			<td style="width:119px;">DE14-801F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">DMSO</td>
			<td style="width:239px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8418</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">The Spectrum collection (compound library)</td>
			<td style="width:239px;">MicroSource Discovery Systems</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Tecan Freedom EVO200 robot&#160;</td>
			<td style="width:239px;">Tecan</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Tecan F200 multiplate reader&#160;</td>
			<td style="width:239px;">Tecan</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput screening for chemical modulators of post-transcriptionally regulated genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Using the described approach, we screened a 2,000-compound library for potential modulators of post-transcriptional control mechanisms exerted through the 3&#8217; UTR of the MYCN gene. Figure 3 depicts the results of the primary screening exemplified by a single library plate. Luciferase signal displayed as percentage of vehicle-treated controls was obtained by measuring luciferase activity in triplicate plates of CHP134-mycn3UTR cells treated with compounds of a single library plate. As expect...

Watch this Scientific Journal Video about High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes at JoVE.com

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

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9.1K visualizações.  University of Trento. Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3’ UTR.