Automated High-Throughput RNAi Screening in Human Cells Combined with Reporter mRNA Transfection to Identify Novel Regulators of Translation

EMBL, Heidelberg, Germany.
PLoS ONE (Impact Factor: 3.23). 09/2012; 7(9):e45943. DOI: 10.1371/journal.pone.0045943
Source: PubMed


Proteins that promote angiogenesis, such as vascular endothelial growth factor (VEGF), are major targets for cancer therapy. Accordingly, proteins that specifically activate expression of factors like VEGF are potential alternative therapeutic targets and may help to combat evasive resistance to angiogenesis inhibitors. VEGF mRNA contains two internal ribosome entry sites (IRESs) that enable selective activation of VEGF protein synthesis under hypoxic conditions that trigger angiogenesis. To identify novel regulators of VEGF IRES-driven translation in human cells, we have developed a high-throughput screening approach that combines siRNA treatment with transfection of a VEGF-IRES reporter mRNA. We identified the kinase MAPK3 as a novel positive regulator of VEGF IRES-driven translation and have validated its regulatory effect on endogenous VEGF. Our automated method is scalable and readily adapted for use with other mRNA regulatory elements. Consequently, it should be a generally useful approach for high-throughput identification of novel regulators of mRNA translation.

Download full-text


Available from: Beate Neumann, Apr 13, 2015
  • Source
    • "Using a high-throughput screening approach that combined siRNA treatment and transfection of a VEGF-A IRES reporter mRNA, Casanova and collaborators identified the kinase MAPK3 as a novel positive regulator. In addition, they have validated its regulatory effect on endogenous VEGF-A (105). The DEAD-box RNA helicase 6 (DDX6) has also been identified as an IRES trans-acting factor in the MCF-7 cell line. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Vascular Endothelial Growth Factor A (VEGF-A) is a potent secreted mitogen crucial for physiological and pathological angiogenesis. Post-transcriptional regulation of VEGF-A occurs at multiple levels. Firstly, alternative splicing gives rise to different transcript variants encoding diverse isoforms that exhibit distinct biological properties with regard to receptor binding and extra-cellular localization. Secondly, VEGF-A mRNA stability is regulated by effectors such as hypoxia or growth factors through the binding of stabilizing and destabilizing proteins at AU-rich elements located in the 3'-untranslated region. Thirdly, translation of VEGF-A mRNA is a controlled process involving alternative initiation codons, internal ribosome entry sites (IRESs), an upstream open reading frame (uORF), miRNA targeting and a riboswitch in the 3' untranslated region. These different levels of regulation cooperate for the crucial fine-tuning of the expression of VEGF-A variants. This review will be focused on our current knowledge of the complex post-transcriptional regulatory switches that modulate the cellular VEGF-A level, a paradigmatic model of post-transcriptional regulation.
    Full-text · Article · Jul 2013 · Nucleic Acids Research
  • [Show abstract] [Hide abstract]
    ABSTRACT: Abstract The use of small molecules to modulate cellular processes is a powerful approach to investigate gene function as a complement to genetic approaches. The discovery and characterization of compounds that modulate translation initiation, the rate-limiting step of protein synthesis, is important both to provide tool compounds to explore this fundamental biological process and to further evaluate protein synthesis as a therapeutic target. While most messenger ribonucleic acids (mRNAs) recruit ribosomes via their 5' cap, some viral and cellular mRNAs initiate protein synthesis via an alternative "cap-independent" mechanism utilizing internal ribosome entry sites (IRES) elements, which are complex mRNA secondary structures, localized within the 5' nontranslated region of the mRNA upstream of the AUG start codon. This report describes the design of a functional, high throughput screen of small molecules miniaturized into a 1,536-well format and performed using the luciferase reporter gene under control of the viral Cardiovirus encephalomyocarditis virus (EMCV) IRES element to identify nontoxic compounds modulating translation initiated from the EMCV IRES. One activating compound, validated in a dose response manner, has previously been shown to bind the glucocorticoid receptor (GR). Subsequent testing of additional GR modulators further supported this as the possible mechanism of action. Detailed characterization of this compound activity supported the notion that this was due to an effect at the level of translation.
    No preview · Article · Aug 2013 · Assay and Drug Development Technologies
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Hypoxia is known to be a major factor in the induction of angiogenesis during tumor development but its role in lymphangiogenesis remains unclear. Blood and lymphatic vasculatures are stimulated by the vascular endothelial family of growth factors - the VEGFs. In this review, we investigate the role of hypoxia in the molecular regulation of synthesis of the lymphangiogenic growth factors VEGF-A, VEGF-C, and VEGF-D. Gene expression can be regulated by hypoxia at either transcriptional or translational levels. In contrast to strong induction of DNA transcription by hypoxia-inducible factors (HIFs), the majority of cellular stresses such as hypoxia lead to inhibition of cap-dependent translation of mRNA and downregulation of protein synthesis. Here, we describe how initiation of translation of VEGF mRNA is induced by hypoxia through an internal ribosome entry site (IRES)-dependent mechanism. Considering the implications of the lymphatic vasculature for metastatic dissemination, it is crucial to understand the molecular regulation of lymphangiogenic growth factors by hypoxia to obtain new insights into cancer therapy.
    Preview · Article · May 2014
Show more