Automated High-Throughput RNAi Screening in Human Cells Combined with Reporter mRNA Transfection to Identify Novel Regulators of Translation Claudia M. Casanova 1,2 , Peter Sehr 2 , Kerstin Putzker 2 , Matthias W. Hentze 2 , Beate Neumann 2 , Kent E. Duncan 3 *, Christian Thoma 1,2 * 1 Department of Medicine II, University Hospital of Freiburg, Freiburg, Germany, 2 EMBL, Heidelberg, Germany, 3 Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Abstract 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. Citation: Casanova CM, Sehr P, Putzker K, Hentze MW, Neumann B, et al. (2012) Automated High-Throughput RNAi Screening in Human Cells Combined with Reporter mRNA Transfection to Identify Novel Regulators of Translation. PLoS ONE 7(9): e45943. doi:10.1371/journal.pone.0045943 Editor: Sung Key Jang, Pohang University of Science and Technology, Republic of Korea Received June 25, 2012; Accepted August 23, 2012; Published September 27, 2012 Copyright: ß 2012 Casanova et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a grant from the Deutsche Forschungsgemeinschaft to C.T. (TH788/3-1). C.T. is a recipient of a Heisenberg-Fellowship from the Deutsche Forschungsgemeinschaft (TH788/2-1 and TH788/2-2). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: christian.thoma@uniklinik-freiburg.de (CT); kent.duncan@zmnh.uni-hamburg.de (KD) Introduction mRNA translation by the ribosome is the ultimate step in the expression of the ,20,000 human genes that encode proteins. Regulation of this event- ‘translational control’- ensures that the right amount of each protein is synthesized in the right place within an organism or cell at the right time. Translational control of gene expression plays a crucial role in adaptive cellular responses to external stimuli [1] and failure to properly regulate protein synthesis is a common feature of many diseases, including cancer [2]. Under normal physiological conditions, translation initiates via a ‘cap-dependent’ mode, in which recruitment of the small ribosomal subunit to the mRNA involves the 7-methyl- guanosine (‘cap’) structure, located at the 59 end of cellular mRNAs [3,4]. This interaction is mediated by the cytoplasmic cap-binding complex eIF4F, which enables recruitment of other translation initiation factors, scanning to the start codon, and joining of the large ribosomal subunit for translational elongation and protein synthesis [3,4,5]. Cap-dependent initiation appears to be the dominant mode for most cellular mRNAs under most conditions, and is the target of a wide variety of regulatory mechanisms [1]. However, certain viral RNAs and some mammalian mRNAs can use alternatives to cap-dependent initiation and thus are translated efficiently under conditions in which cap-dependent translation is repressed, such as apoptosis, mitosis, hypoxia, and cellular stress [6,7]. In these cases, trans- lation initiation occurs efficiently independent of the cap structure and many of the associated translation initiation factors [6]. The subset of cellular mRNAs (,3%) that apparently can be translated efficiently when cap-dependent translation is generally compromised [8] includes cell growth regulators that are critical in cancer [2,9,10]. One important example of major clinical relevance is the mRNA encoding vascular endothelial growth factor-A (VEGF-A, henceforth referred to as ‘VEGF’). As a key regulator of tumor angiogenesis, VEGF plays a crucial role in cancer progression for essentially all solid tumors [11,12] and consequently is a major oncology drug target. VEGF is also important for development and maintenance of the nervous system and both VEGF and regulators of VEGF signaling are of great therapeutic interest in neurodegenerative disease and acute neurological disorders, including cerebral ischemia/stroke [13,14]. Cap-independent translation of the VEGF mRNA is mediated by two internal ribosome entry sequences (VEGF IRES-A and –B, respectively) located in the 59 untranslated region (UTR) [15]. Cancer-relevant cellular stress conditions, such as hypoxia, can activate cap-independent translation mediated by these and other IRESs, while simultaneously repressing cap-dependent translation [16,17,18]. Thus, in many cancers tumorigenesis involves a switch enabling more cap-independent translation, which appears to be important for tumor progression [2]. Accordingly, ‘druggable’ specific positive regulators of the translational activity of VEGF IRES (and perhaps other cellular IRESs) could potentially be PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e45943