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 
and failure to properly regulate protein synthesis is a common feature of many diseases, including cancer 
. 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 5′ end of cellular mRNAs 
. 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 
. 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 
. 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 
. In these cases, translation initiation occurs efficiently independent of the cap structure and many of the associated translation initiation factors 
The subset of cellular mRNAs (~3%) that apparently can be translated efficiently when cap-dependent translation is generally compromised 
includes cell growth regulators that are critical in cancer 
. 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 
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 
. Cap-independent translation of the VEGF mRNA is mediated by two internal ribosome entry sequences (VEGF IRES-A and –B, respectively) located in the 5′ untranslated region (UTR) 
. 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 
. Thus, in many cancers tumorigenesis involves a switch enabling more cap-independent translation, which appears to be important for tumor progression 
. Accordingly, ‘druggable’ specific positive regulators of the translational activity of VEGF IRES (and perhaps other cellular IRESs) could potentially be attractive additional therapeutic targets in oncology. However, to our knowledge no cellular factors that specifically modulate VEGF IRES activity have yet been identified.
The discovery that RNAi works effectively in mammalian cells 
paved the way for high-throughput RNAi screening to address such questions in a global manner. Here, we present a novel, automated, easily scalable high-throughput screening approach that enables the identification of regulatory proteins that modulate the translational activity of a specific mRNA element contained in a transfected reporter mRNA. We took advantage of the well established protocol for solid-phase reverse transfection of cells on small interfering siRNA transfection mixes in coated 96-well plate format 
. This robust approach routinely achieves strong and specific knockdowns, but has so far been used predominantly for high-content microscopy applications. Here we significantly expand its range of use by optimizing its compatibility with automated transfection of a reporter mRNA of interest. Specifically, we present a robust method to: 1) quantitatively analyze IRES-dependent translation and 2) compare it with conventional cap-dependent translation activity using monocistronic reporter mRNAs. Our approach addresses the numerous caveats for studying IRES activity that are inherent to the commonly used DNA transfection method with bicistronic reporters, which has led to much confusion and contention in the IRES translation field 
. For this reason, we expect the method presented here to be a more sensitive screening tool with a much lower false positive rate.
In a proof-of-concept screen we assessed the potential role of 702 human kinases and 298 human phosphatases in VEGF IRES-driven translation. 91 genes that qualified as hits in the primary screen were selected for secondary validation assays, in which specificity for IRES-driven translation was examined by transfecting the original VEGF IRES-driven reporter mRNA and a cap-driven reporter mRNA. Ultimately, we identified three kinases specifically involved in IRES-, but not in cap-dependent translational regulation. For one of these, MAPK3, we show here that it is a bona fide novel, positive-acting, post-transcriptional regulator of VEGF production. In our view this provides a striking demonstration of the power of this method for identifying novel regulators of mRNA translation. Importantly, the method can be easily scaled up for genome-wide screens and could also be readily adapted for use with other mRNA elements of (clinical) interest.