Abstract
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 in vitro 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 Kd of 2.5±0.60 nM, k on of 2.76±0.56*106 M-1 min-1, and k off 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.
Highlights
The stability of a specific mRNA is dependent upon both ciselements and trans-acting factors such as RNA binding proteins (RBPs)
We identified the formation of rHuR-RNA probe complex using nondenaturing and non crosslinked RNA-Electrophoresis Mobility Shift Assays (REMSAs) (Figure 2A) after mixing equimolar amount (0.5 μM) of protein and Cy3-tagged TNF probe (Cy-TNF)
We have recently applied the AlphaScreen technology to quantitatively discriminate the affinity of HuR towards RNA probes carrying AU-rich elements (AREs) consensus sequences by comparing the binding to TNFα and p62 mRNA probes [24]
Summary
The stability of a specific mRNA is dependent upon both ciselements and trans-acting factors such as RNA binding proteins (RBPs). High turnover mRNAs that form complexes with HuR (see review [7]) are usually studied by ribonucleo-immunoprecipitation coupled to immunoblotting/RT-PCR or by RNA-Electrophoresis Mobility Shift Assays (REMSAs). We decided to develop a biochemical tool, based on AlphaScreen technology, that could complement traditional biochemical methods in the rapid and sensitive evaluation of HuR-RNA interaction and of competition with other trans-acting factors (direct or indirect protein–protein interactions). To this aim, we exploited the affinity between HuR and the AU-rich region of the TNFα 3’ UTR mRNA for the development of our tool. We show that this tool can substitute standard REMSAs for quantitative evaluation of the protein-RNA association and the feasibility of the AlphaScreen assay for high throughput screening (HTS) applications
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