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

Summary

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.

Abstract

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' untranslated region (3’ 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’ 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’ 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’ UTR.

Introduction

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 abundance^1,2. 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....

Protocol

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.

Primary Screening

1. Day 1: Prepare and Seed Cells

NOTE: Seed cells to yield 80% conflue....

Results

Using the described approach, we screened a 2,000-compound library for potential modulators of post-transcriptional control mechanisms exerted through the 3’ 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 expected, th.......

Discussion

This protocol describes a cell-based reporter-gene assay aiming at the identification of modulators that target 3’ 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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Culture plate 96-well White	PerkinElmer	6005688
StorPlate 96-well U bottom (dilution plate)	PerkinElmer	6008190
100 ml disposable trough for reagents	Tecan	10 613 048
300 ml disposable robotic reservoir	VWR	PB12001301
Robot tips DITI 50 μl Sterile	Tecan	30038607
Robot tips DITI 200 μl Sterile	Tecan	30038617
Matrix 1.4 ml 2D Barcoded w, Flat Bottom Tubes	Thermo Scientific	3711
ONE-Glo Luciferase Assay System	Promega	E6120
RPMI	Lonza	BE12-918F
PBS-1x, w/o Ca2+, Mg2+	Lonza	BE17-516F
L-Glutamine 200 mM	Lonza	BE17-605E
FBS	Lonza	DE14-801F
DMSO	Sigma-Aldrich	D8418
The Spectrum collection (compound library)	MicroSource Discovery Systems	N/A
Tecan Freedom EVO200 robot	Tecan	N/A
Tecan F200 multiplate reader	Tecan	N/A