Dataset Information

RNA pull-down-Confocal Nanoscanning (RP-CONA) detects quercetin as pri-miR-7/HuR interaction inhibitor that decreases a-Synuclein levels

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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

DISEASE(S): Parkinson's Disease

SUBMITTER: Gracjan Michlewski

LAB HEAD: Gracjan Michlewski

PROVIDER: PXD025617 | Pride | 2022-02-17

REPOSITORIES: Pride

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Publications

RNA pull-down confocal nanoscanning (RP-CONA) detects quercetin as pri-miR-7/HuR interaction inhibitor that decreases α-synuclein levels.

Zhu Siran S Choudhury Nila Roy NR Rooney Saul S Pham Nhan T NT Koszela Joanna J Kelly David D Spanos Christos C Rappsilber Juri J Auer Manfred M Michlewski Gracjan G

Nucleic acids research 20210601 11

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 ...[more]

PMID: 34107032

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Project description:Although-synuclein is implicated in the pathogenesis of Parkinson’s disease and related disorders, it remains unclear whether specific conformations or levels of-synuclein assemblies are toxic and how they cause progressive loss of human dopaminergic neurons. To address this issue, we used iPSC-derived dopaminergic neurons with -synuclein triplication or controls where endogenous -synuclein was imprinted into synthetic or disease-relevant conformations. We used -synuclein fibrils generated de novo or amplified from homogenates of brains affected with Parkinson’s disease (n=3) or multiple system atrophy (n=5). We found that a 2.5-fold increase in -synuclein levels in -synuclein gene triplication neurons promoted seeded aggregation in a dose and time-dependent fashion, which was associated with a further increase in -synuclein gene expression. Progressive neuronal loss was observed only in -synuclein triplication neurons seeded with brain-amplified fibrils. Transcriptomic analysis and isogenic correction of -synuclein triplication revealed that intraneuronal-synuclein levels solely and sufficiently explained vulnerability to neuronal death. Proximity-dependent biotinylation in living cells identified 56 differentially interacting proteins with endogenously assembled -synuclein including evasion of Parkinson’s disease-associated deglycase DJ-1 by aggregates triggered with brain amplified fibrils. Knockout of DJ-1 and related glyoxalase-1 in cell lines increased -synuclein aggregation. Similarly, methylglyoxal treatment or CRISPR/Cas9 knockout of DJ-1 in iPSC-derived dopaminergic neurons enhanced fibril-induced aggregation and cell death. Thus, toxicity of -synuclein strains depends on aggregate burden, which is determined by monomer levels and conformation which dictates differential interactomes. Our results define parameters for iPSC-based modellingof -synuclein pathology using brain amplified fibrils and demonstrate how Parkinson’s disease-associated genes influence the phenotypic manifestation of strains in human neurons.

Dataset Information

RNA pull-down-Confocal Nanoscanning (RP-CONA) detects quercetin as pri-miR-7/HuR interaction inhibitor that decreases a-Synuclein levels

Publications

RNA pull-down confocal nanoscanning (RP-CONA) detects quercetin as pri-miR-7/HuR interaction inhibitor that decreases α-synuclein levels.

Similar Datasets

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