A Novel High Throughput Biochemical Assay to Evaluate the HuR Protein-RNA Complex Formation

<div><p>The RNA binding protein HuR/ELAVL1 binds to AU-rich elements (AREs) promoting the stabilization and translation of a number of mRNAs into the cytoplasm, dictating their fate. We applied the AlphaScreen technology using purified human HuR protein, expressed in a mammalian cell-based system, to characterize <i>in vitro</i> its binding performance towards a ssRNA probe whose sequence corresponds to the are present in TNFα 3’ untranslated region. We optimized the method to titrate ligands and analyzed the kinetic in saturation binding and time course experiments, including competition assays. The method revealed to be a successful tool for determination of HuR binding kinetic parameters in the nanomolar range, with calculated <i>Kd</i> of 2.5±0.60 nM, <i>k</i>_<i>on</i> of 2.76±0.56*10⁶ M^-1 min^-1, and <i>k</i>_<i>off</i> of 0.007±0.005 min^-1. We also tested the HuR-RNA complex formation by fluorescent probe-based RNA-EMSA. Moreover, in a 384-well plate format we obtained a Z-factor of 0.84 and an averaged coefficient of variation between controls of 8%, indicating that this biochemical assay fulfills criteria of robustness for a targeted screening approach. After a screening with 2000 small molecules and secondary verification with RNA-EMSA we identified mitoxantrone as an interfering compound with rHuR and TNFα probe complex formation. Notably, this tool has a large versatility and could be applied to other RNA Binding Proteins recognizing different RNA, DNA, or protein species. In addition, it opens new perspectives in the identification of small-molecule modulators of RNA binding proteins activity.</p> </div

Competition assays with unmarked RNA oligonucleotide and with rTTP protein.

<p><b>A</b>) The percentage of inhibition increased as function of untagged TNFα RNA probe (U-TNF) concentration. U-TNF was added to the reaction together with Bi-TNF probe and signals were detected at equilibrium. <i>Ki</i> values were determined from nonlinear regression fits of the data according to 1-site fit <i>Ki</i> model in GraphPad Prism®, version 5.0, by keeping constant the concentration (50 nM) and the <i>Kd</i> (2.5 nM) for the labeled Bi-TNF probe. Mean and standard deviation values derive from two independent experiments. <b>B</b>) Coomassie staining of purified and recovered Zeba^TM Spin desalted rTTP protein (25 ng) loaded on 15% SDS-PAGE. <b>C</b>) REMSA showing rTTP (0.1 µM) complexed with Cy-TNF RNA probe (0.5 µM reacted and loaded on native gel). <b>D</b>) Competitive AlphaScreen assay as a function of rTTP concentration. Equal amounts of BSA were independently reacted as negative control. <i>Ki</i> values were determined from nonlinear regression fits of the data according to 1-site fit <i>Ki</i> model in GraphPad Prism®, version 5.0, by keeping constant the concentration (50 nM) and the <i>Kd</i> (2.5 nM) for the labeled Bi-TNF probe. Mean and standard deviation values derive from two independent experiments with two rTTP protein preparations. <b>E</b>) Western blot showing rHuR purified from control (Mock; DMSO) and CsA [4 µM] stimulated HEK293T cells. After 3 hr of treatment the total amount of purified rHuR proteins (150 ng loaded on gel) was not affected, while the phosphorylation of the protein was clearly induced, as showed by an anti-phosphoserine antibody (P-SER). <b>F</b>) Saturation binding experiments comparing the binding capability of rHuR and phosphorylated rHuR (P-rHuR). Nonlinear regression fits of the data revealed an equilibrium dissociation constants equal to 3.1±0.55 nM for P-rHuR, not statistically relevant (<i>P value</i> = 0.59) with respect to the <i>Kd</i> of rHuR. Mean and standard deviation values derive from two independent experiments.</p

Characterization of the functional binding of rHuR to the AU-rich RNA substrate.

<p><b>A</b>) REMSA showing the binding capability of rHuR (0.5 µM) resulting in the presence of an higher molecular weight protein-RNA complex with respect to the free Cy-TNF RNA probe (0.5 µM). The supershift caused by the anti-HuR antibody (1 µg) indicates the presence of at least a ternary complex and the qualitative binding of rHuR. <b>B</b>) Saturation binding experiments. Equilibrium dissociation constants (<i>Kd</i>) were determined from nonlinear regression fits of the data according to a 1-site binding model in GraphPad Prism®, version 5.0. Mean and standard deviation values derive from four independent experiments with four rHuR protein preparations. <b>C</b>) Time course experiments. Association (<i>K</i>_<i>on</i>) and dissociation (<i>K</i>_<i>off</i>) rate constants were determined from nonlinear regression fits of the data according to association kinetic model of multiple ligand concentration in GraphPad Prism®, version 5.0. Mean and standard deviation values derive from two independent experiments with two rHuR protein preparations.</p

Purification of rHuR and optimization of the AlphaScreen assay.

<p><b>A</b>) Representative 15% SDS-PAGE and Coomassie staining of purified rHuR protein (80 ng) recovered after Zeba^TM Spin Desalting Columns dialyzation and western blot on the same sample (20 ng) using a polyclonal anti-HuR antibody. <b>B</b>) Bi-TNF RNA probe and rHuR protein double titration to determine optimal ligand concentrations with the AlphaScreen anti-c-Myc-Acceptor and streptavidin-Donor beads of the c-Myc detection kit (PerkinElmer), resulting in 1 nM and 50 nM for rHuR and Bi-TNF, respectively. <b>C</b>) Bi-TNF titration with 1 nM of rHuR. “Hooking effect” is shown for concentrations over 50 nM of RNA ligand (as the point at 100 nM in the log scale). Mean and standard deviation values derive from four independent experiments with four rHuR protein preparations.</p

Robustness of the miniaturized AlphaScreen assay and screening of a drug library.

<p><b>A</b>) rHuR and Bi-TNF positive or Bi-TNFneg negative controls were reacted at optimized nanomolar concentrations in a final volume of 20 µl in 384-wells Optiplates. Relative coefficient of variations and Z-factor value are reported. <b>B</b>) Plot of progressive Z-score values of 2000 compounds according to their interference to rHuR-RNA complex formation assay. <b>C</b>) Representative REMSA showing the effect of compounds, selected after counter screening assay, on rHuR-RNA complex formation. Lane 1: Bi-TNF probe only; Lane 2: rHuR-Bi-TNF; Lane 3-9: 1-Aspartame, 2-Cephradine, 3-Clomiphene citrate, 4-Cetylpyridinium chloride, 5-Diloxanide furoate, 6-Gentian violet, 7-Hydroquinone; Lane 10: Bi-TNF probe only; Lane 11-18: 8-Tilmicosin, 9-Nonoxynol-9, 10-Orlistat, 11-Protoveratrine, 12-Raloxifene hydrochloride, 13-Salsalate, 14-Switenolide diacetate, 15-Tetrandrine; Lane 19: Bi-TNF probe only; Lane 20: 16-Mitoxantrone hydrochloride. Compounds (0.5 µM) were added to a binding reaction containing, as in Line 2, 0.2 µM rHuR and 0.5 µM Bi-TNF.</p

Additional file 2: of TrkA is amplified in malignant melanoma patients and induces an anti-proliferative response in cell lines

Figure S1Genomic copy number levels of CDKN2A in primary MM.Figure S2Bioinformatic analysis of TrkA mRNA expression and copy number in public database of MM cell lines and tumor samples.Figure S3 Expression of TrkA in MM cell lines.Figure S4Dose-response activation of AKT and MAPK following stimulation of NGF-TrkA signaling in MM cell lines.Figure S5Morphological and quantitative analysis of MM cells in response to NGF-TrkA signaling.Figure S6 Cell proliferation and apoptosis analysis of MM cells in the absence of active NGF-TrkA signaling following MAPK and AKT pathway inhibition.Figure S7Proliferation of MM cells in the absence of active NGF-TrkA signaling following MAPK and AKT pathway inhibition.Figure S8 Analysis of MAPK downstream target expression following NGF-TrkA signaling in MM cells.Figure S9 Analysis of MAPK activation and p21cip1 expression in MM cells in the absence of NGF-TrkA signaling following inhibition of MAPK pathway in MM cells.Figure S10 Morphological analysis of MM cells in the absence of NGF-TrkA signaling activation and following inhibition of MAPK pathway

MSC morphology and flow cytometry analysis after QMR stimulation.

<p>A) The images were obtained after 10 minutes of QMR stimulation at Day 5 (first cycle of treatment) and at B) Day 12 (second cycle of treatment). Scale bar = 100 μm. Total magnification = 100X. One representative experiment was shown. C) Five colour combination of monoclonal antibodies was used to verify MSC identity according to the above listed surface markers of a representative sample. Grey line = unstained control (CTL-). Blue line = sham-exposed control (CTL). Green line = QMR setting 80. Red line = QMR setting 40.</p

Cellular viability and proliferation after QMR treatment.

<p>A) Histograms represent the % of cellular viability after two cycles of QMR treatment at the different settings compared to the sham-exposed controls determined by flow cytometry. Data were shown as mean ± SD of three independent experiments; B) Percentages of cellular proliferation on the controls were obtained by WST-1 assay after 72 hours. Data were represented as mean ± SD of n = 6 independent experiments. No statistical differences were found between conditions.</p

Relative gene expressions using quantitative real-time PCR.

<p>Expression of 8 genes selected by cDNA microarray was illustrated after n = 6 independent experiments using TBP as representative reference gene; mean ± SD; * p<0.05; ** p<0.01.</p

Selected genes for testing DNA microarray outcomes in quantitative real-time PCR.

<p>Selected genes for testing DNA microarray outcomes in quantitative real-time PCR.</p