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

<div><p>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.</p> </div

MAPK3 specifically regulates endogenous VEGF expression without affecting mRNA stability.

<p>(a) Determination of endogenous VEGF by ELISA<b>.</b> Error bars represent standard deviations calculated from 3 independent experiments, each performed at least in duplicates. Statistical analysis of differences between controls and experimental samples were performed with the Microsoft Excel unpaired, type 2, Student’s t test. (b) Endogenous VEGF and MAPK3 mRNA levels measured by qRT-PCR. The expression ratio of the indicated endogenous mRNAs (VEGF and MAPK3) in MAPK3 siRNA-treated cells relative to scramble siRNA treated cells (%) is shown. Statistical analysis of differences between controls and experimental samples were performed with the Microsoft Excel unpaired, type 2, Student’s t test. A p-value of >0.5 and <0.05 was determined for VEGF and MAPK3, respectively. Relative VEGF and MAPK3 mRNA levels, standard deviations and statistical significance were calculated with REST 2009 software as described in “Material and Methods”.</p

Secondary screen of 91 candidate regulators of VEGF IRES-dependent translation.

<p> (a) Workflow of the secondary screen. Cells were reverse-transfected with individual siRNAs designated as hits from the primary screen (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045943#pone.0045943.s003" target="_blank">Table S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045943#pone.0045943.s004" target="_blank">S2</a>). This was followed 48 hours later by three assays which were designed, respectively, to assess: 1) <i>specificity</i> of the effects for IRES-driven translation and 2) to determine effects on cell <i>viability</i> that might confound the other analyses. (b) Summary of secondary screening data.</p

Workflow of the high-throughput siRNA screening approach to identify novel regulators of mRNA translation.

<p>The strategy features automated generation of lyophilized siRNA-coated 96 well plates, followed by two independent RNA transfections. Plates are first coated with siRNAs transfection mixes, cells are then seeded onto siRNA-coated plates (solid-phase reverse transfection) and incubated for two days to allow reduction of the protein levels of the targeted genes. Subsequently, a second RNA transfection is performed to introduce the reporter mRNA of interest. Finally, the effects of each siRNA knock-down are measured.</p

MAPK3 specifically regulates VEGF IRES-dependent translation.

<p> (a) HeLa cells were reverse transfected with the indicated siRNAs. This was followed 48 h later by reporter mRNA transfection for IRES-driven translation (left panel) and cap-driven translation (right panel). Luciferase activity was analyzed and the corresponding relative translation rate is indicated. (b) Quantification of ATP content as an indicator of cell viability. (c) Physical stability of the reporter VEGF IRES luciferase mRNA analyzed by Northern blotting. (d) Western blot of MAPK3 in siRNA-treated cells or control cells treated with scrambled siRNA. Beta-actin serves as a control for loading/transfer efficiency.</p