title: High-throughput screening for chemical modulators of post-transcriptionally regulated genes.
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.
authors: Laboratory of Translational Genomics, Centre for Integrative Biology, University of Trento; Viktoryia.sidarovich@unitn.it.
authors: Viktoryia
authors: V
authors: Sidarovich
authors: High Throughput Screening Core Facility, Centre for Integrative Biology, University of Trento.
authors: Valentina
authors: V
authors: Adami
authors: Laboratory of Translational Genomics, Centre for Integrative Biology, University of Trento.
authors: Alessandro
authors: A
authors: Quattrone
mesh_terms: D020413:3' Untranslated Regions / Q000187:drug effects; D045744:Cell Line, Tumor; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter / Q000187:drug effects; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D000071447:N-Myc Proto-Oncogene Protein; D009447:Neuroblastoma / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics; D015513:Oncogene Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D012323:RNA Processing, Post-Transcriptional / Q000187:drug effects*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D054852:Small Molecule Libraries / Q000494:pharmacology*
chemical_list: D020413:3' Untranslated Regions; C459801:MYCN protein, human; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D012333:RNA, Messenger; D054852:Small Molecule Libraries; D008156:Luciferases