ABSTRACT
RNA-protein interactions are central to all gene expression processes and contribute to 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 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 novel methodology to search for RNA-protein interaction modulators.
Competing Interest Statement
The authors have declared no competing interest.
ABBREVIATIONS
- 3’-UTR
- 3 prime untranslated region
- ATP
- adenosine triphosphate
- CPC
- cetylpyridinium chloride
- CTL
- conserved terminal loop
- CV
- coefficient of variation
- DHTS
- dihydrotanshinone I
- DNA
- deoxyribonucleic acid
- DTT
- dithiothreitol
- EDTA
- ethylenediaminetetraacetic acid
- EMSA
- electrophoretic mobility gel shift assay
- FITC
- fluorescein isothiocyanate
- FP
- fluorescence polarisation
- FRET
- fluorescence resonance energy transfer
- HCS
- High Content Screening
- HuR
- human antigen R
- KO
- knockout
- miR, miRNA
- microRNA
- mRNA
- messenger RNA
- MSI2
- Musashi RNA binding protein 2
- OA
- oleic acid
- PBS
- phosphate-buffered saline
- PCR
- polymerase chain reaction
- PD
- Parkinson’s disease
- PMSF
- phenylmethanesulfonylfluoride
- pre-miR, pre-miRNA
- precursor microRNA pri-miR, pri-miRNA primary microRNA
- qRT-PCR
- real-time quantitative PCR
- RBP
- RNA binding protein
- RNA
- ribonucleic acid
- Ro
- Ro 08-2750
- RP-CONA
- RNA pull-down-Confocal Nanoscanning
- RRM
- RNA recognition motif
- SD
- standard deviation
- SEM
- standard error mean
- shRNA
- short hairpin RNA
- siRNA
- small interfering RNA
- SNpc
- substantia nigra pars compacta
- TNF-α
- tumour necrosis factor alpha
- α-Syn
- α-Synuclein