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

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

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

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

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

Proteome-Wide Characterization of the RNA-Binding Protein RALY-Interactome Using the in Vivo-Biotinylation-Pulldown-Quant (iBioPQ) Approach

RALY is a member of the heterogeneous nuclear ribonucleoproteins, a family of RNA-binding proteins generally involved in many processes of mRNA metabolism. No quantitative proteomic analysis of RALY-containing ribonucleoparticles (RNPs) has been performed so far, and the biological role of RALY remains elusive. Here, we present a workflow for the characterization of RALY’s interaction partners, termed iBioPQ, that involves in vivo biotinylation of biotin acceptor peptide (BAP)-fused protein in the presence of the prokaryotic biotin holoenzyme synthetase of BirA so that it can be purified using streptavidin-coated magnetic beads, circumventing the need for specific antibodies and providing efficient pulldowns. Protein eluates were subjected to tryptic digestion and identified using data-independent acquisition on an ion-mobility enabled high-resolution nanoUPLC-QTOF system. Using label-free quantification, we identified 143 proteins displaying at least 2-fold difference in pulldown compared to controls. Gene Ontology overrepresentation analysis revealed an enrichment of proteins involved in mRNA metabolism and translational control. Among the most abundant interacting proteins, we confirmed RNA-dependent interactions of RALY with MATR3, PABP1 and ELAVL1. Comparative analysis of pulldowns after RNase treatment revealed a protein–protein interaction of RALY with eIF4AIII, FMRP, and hnRNP-C. Our data show that RALY-containing RNPs are much more heterogeneous than previously hypothesized

Additional file 1: of Cancer cell metabolic plasticity allows resistance to NAMPT inhibition but invariably induces dependence on LDHA

Table S2. List of RT-PCR primer sequences. (XLS 34 kb

Additional file 3: of Cancer cell metabolic plasticity allows resistance to NAMPT inhibition but invariably induces dependence on LDHA

Table S1. List of gene expression from genome wide analysis in CCRF-CEM cells. (XLSX 44303 kb

Additional file 3: of EIF2A-dependent translational arrest protects leukemia cells from the energetic stress induced by NAMPT inhibition

Effects of CHS-828 and chemotherapeutics on protein translation. A) Jurkat cells were treated for 48 h with or without (Mock) the indicated concentration of CHS-828. Caspase 3/7 activity was quantified (using 5 μM of Camptothecin for 4 h as a positive control of apoptosis) and relative ATP levels were determined and then normalized to the number of viable cells. The levels of total AMPK, p-AMPK, total EIF2A and p-EIF2A, total 4EBP1, p-4EBP1 were evaluated by WB. Histogram shows the densitometric analysis of p-AMPK and p-EIF2A (* indicates p-value <0.05). Mean and SD of a biological triplicate. B) Jurkat cells were treated with the indicated concentration of drugs for 48 h and cell viability was measured by Cell Titer Glo. Data are represented as mean and SD of three independent experiments. C) Click-it chemistry based on the incorporation of an aminoacid analog (AHA) was used to monitor protein synthesis. Jurkat cells were treated for 48 h with or without (Mock) the indicated concentration of FK866, Rapamycin (RAPA), Doxorubicin (DOXO), Cisplatin (CIS) and Dexamethasone (DEXA). The histogram quantifies the % of AHA positive cells (active protein-synthesizing cells) in the viable cell population. Flow-cytometry experiments were carried out on two biological replicates and statistics were based on acquisition of 20000 events/sample. D) Jurkat cells were treated as in C and the level of p-EIF2A and p-4EBP1 was evaluated. Histogram shows the densitometric analysis of p-EIF2A (* indicates p-value <0.05). Mean and SD of a biological triplicate. E) Primary B-CLL cells were treated for 48 h with or without 30 nM FK866 in the presence or absence of 1 mM NA. Histogram shows the densitometric analysis of p-AMPK/AMPK. (PDF 691 kb

Autophagy Activation Clears ELAVL1/HuR-Mediated Accumulation of SQSTM1/p62 during Proteasomal Inhibition in Human Retinal Pigment Epithelial Cells

<div><p>Age-related macular degeneration (AMD) is the most common reason of visual impairment in the elderly in the Western countries. The degeneration of retinal pigment epithelial cells (RPE) causes secondarily adverse effects on neural retina leading to visual loss. The aging characteristics of the RPE involve lysosomal accumulation of lipofuscin and extracellular protein aggregates called “drusen”. Molecular mechanisms behind protein aggregations are weakly understood. There is intriguing evidence suggesting that protein SQSTM1/p62, together with autophagy, has a role in the pathology of different degenerative diseases. It appears that SQSTM1/p62 is a connecting link between autophagy and proteasome mediated proteolysis, and expressed strongly under the exposure to various oxidative stimuli and proteasomal inhibition. ELAVL1/HuR protein is a post-transcriptional factor, which acts mainly as a positive regulator of gene expression by binding to specific mRNAs whose corresponding proteins are fundamental for key cellular functions. We here show that, under proteasomal inhibitor MG-132, ELAVL1/HuR is up-regulated at both mRNA and protein levels, and that this protein binds and post-transcriptionally regulates <i>SQSTM1/p62</i> mRNA in ARPE-19 cell line. Furthermore, we observed that proteasomal inhibition caused accumulation of SQSTM1/p62 bound irreversibly to perinuclear protein aggregates. The addition of the AMPK activator AICAR was pro-survival and promoted cleansing by autophagy of the former complex, but not of the ELAVL1/HuR accumulation, indeed suggesting that SQSTM1/p62 is decreased through autophagy-mediated degradation, while ELAVL1/HuR through the proteasomal pathway. Interestingly, when compared to human controls, AMD donor samples show strong SQSTM1/p62 rather than ELAVL1/HuR accumulation in the drusen rich macular area suggesting impaired autophagy in the pathology of AMD.</p></div