Abstract
RNA–protein interactions are central to all gene expression processes and contribute to a variety of human diseases. Therapeutic approaches targeting RNA–protein interactions have shown promising effects on some diseases that are previously regarded as ‘incurable’. Here, we developed a fluorescent on-bead screening platform, RNA Pull-Down COnfocal NAnoscanning (RP-CONA), to identify RNA–protein interaction modulators in eukaryotic cell extracts. Using RP-CONA, we identified small molecules that disrupt the interaction between HuR, an inhibitor of brain-enriched miR-7 biogenesis, and the conserved terminal loop of pri-miR-7–1. Importantly, miR-7′s primary target is an mRNA of α-synuclein, which contributes to the aetiology of Parkinson’s disease. Our method identified a natural product quercetin as a molecule able to upregulate cellular miR-7 levels and downregulate the expression of α-synuclein. This opens up new therapeutic avenues towards treatment of Parkinson’s disease as well as provides a novel methodology to search for modulators of RNA–protein interaction.
Highlights
RNA-protein interactions coordinate the whole of RNA metabolism, including transcription, RNA processing, modification, translation and turnover [1]
The Lin28a-GFP rings were significantly reduced by polyclonal anti-Lin28a antibody, compared to anti-HuR antibody (Figure 5E, F). These results demonstrate that RNA Pull-Down COnfocal NAnoscanning (RP-CONA) is a robust technique to screen for RNA-protein interaction modulators and that it could be used with various confocal image scanning platforms
We identified quercetin, luteolin and gossypol as pri-miR-7-1/HuR inhibitors, which generated more than 50% inhibition at 100 μM concentration according to the relative mCherry/FITC signals compared to the DMSO control (Figure 6C)
Summary
RNA-protein interactions coordinate the whole of RNA metabolism, including transcription, RNA processing, modification, translation and turnover [1]. Dysregulated RNA metabolism can result in serious diseases including cancer [2] and neuropathological conditions [3]. Therapies targeting RNA-protein interactions have shown promising results and some of them are already in 13 clinical trials [4,5,6]. Among many types of RNAs, short non-coding microRNAs (miRNAs, miRs), that regulate gene expression by imperfect base-pairing to mRNA, hold great potential both as biomarkers [7] and therapeutics [8] in multiple human pathologies. The hairpin containing transcripts, known as primary miRNAs (pri-miRNAs), are processed by the Microprocessor complex to become stem-loop precursor miRNAs (pre-miRNAs) [9,10,11,12,13].
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