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Charles B Wright, Younghee Kim, Tetsuhiro Yasuma, Shengjian Li, Benjamin J Fowler, Mark Ellsworth Kleinman, Jayakrishna Ambati, Bradley D Gelfand; Enhanced Alu RNA stability due to iron-mediated DICER1 impairment causes NLRP3 inflammasome priming. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2187. doi: https://doi.org/.
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Geographic atrophy (GA) is an untreatable advanced form of nonexudative age-related macular degeneration that results in loss of central vision via retinal pigment epithelium (RPE) cell death. We previously demonstrated that downregulation of DICER1 in the RPE resulted in the accumulation of toxic Alu RNA and cell death via the NLRP3 inflammasome in human GA eyes. Here we explored whether iron overload can cause Alu RNA accumulation via interference with the DICER1 cofactor Poly(C)-binding protein 2 (PCBP2).
Iron overload was induced by supplementing culture media of human RPE cells and by subretinal injection in wild-type mice. Northern blotting and fluorescence in situ hybridization were used to determine whether iron overload affected abundance and localization of endogenous Alu RNA and mouse B1/B2 RNA. Time-resolved degradation of a synthetic labeled Alu RNA was used to assess Alu RNA stability upon exposure to iron. To determine whether iron overload induced inflammasome priming, qRT-PCR was used to measure NLRP3 mRNA abundance. Binding affinity of Alu RNA to PCBP2 was assessed by pulldown assay, and the effect of PCBP2 on DICER1-medieated Alu RNA processing was measured by in vitro cleavage assays. Finally, to determine whether Alu RNA stability mediated inflammasome priming, cells were subjected to siRNA-mediated knockdown of PCBP2, in the presence of an Alu RNA antisense oligonucleotide compared to non-targeting controls.
Iron overload resulted in an increase in Alu RNA and B1/B2 RNA levels in human RPE cells and mouse retina, respectively, which was associated with increased stability of Alu RNA transcripts. Pulldown assays revealed iron-sensitive Alu RNA/PCBP2 binding. PCBP2 promoted dose-dependent, DICER1-dependent Alu RNA processing. Knockdown of PCBP2 expression resulted in higher Alu RNA stability and NLRP3 mRNA levels, which was prevented by Alu RNA targeted antisense oligonucleotide.
Alu RNA causes RPE cell death via the NLRP3 inflammasome, iron overload promotes Alu RNA accumulation. The addition of iron resulted in decreased Alu RNA binding by PCBP2, which enhances DICER1-mediated processing of Alu RNA. Alu RNA accumulation due to iron overload or PCBP2 targeting caused NLRP3 inflammasome priming. This work suggests that increased iron levels in human RPE might impair DICER1/Alu RNA metabolism in GA, thus contributing to RPE degeneration.
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