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Benjamin J. Fowler, Valeria Tarallo, Sami Dridi, Hiroki Kaneko, Bradley Gelfand, Judit Baffi, Mark Kleinman, Jayakrishna Ambati; Retinal Pigmented Epithelial Cell Cytotoxicity Due To Deficient Processing Of Alu RNA, A Novel DICER Substrate. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2328. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Dicer is an enzyme that is integral to the regulation of gene expression and cellular defense against foreign double-stranded RNA (dsRNA). Recent work from our laboratory shows that geographic atrophy due to macular degeneration is a result of decreased levels of Dicer protein and increased levels of a repetitive element (Alu) RNA. In the present study, we sought to elucidate the protective role of Dicer in modulating the accumulation of cytotoxic Alu RNA in retinal pigmented epithelium (RPE).
Alu RNA was isolated from the RPE of donor patients with geographic atrophy, reverse transcribed, cloned and sequenced. An RNA identical to this Alu sequence was synthesized and subjected to a Dicer cleavage assay. C57Bl/6J mice received intravitreous injections of Alu RNA, Dicer-cleaved Alu RNA, or control PBS, and fundoscopic photography was performed at one week. Two primary isolates of human RPE cells were cultured with the same Alu products or control, and analyzed for cell viability. Under the same experimental conditions, quantitative PCR was performed for 84 genes related to pro- and anti-apoptotic pathways (n=3-4 per isolate). Data were analyzed using class comparison and multi-dimensional scaling to determine significant changes (p<0.05) in apoptotic gene response.
The pathogenic RNA isolated from diseased human eyes is a bona fide Alu element with unique secondary structure. Treatment of hRPE isolates and intravitreous injection of mice with full length Alu RNA, but not Dicer-cleaved Alu, decreased RPE cell viability and induced geographic atrophy, respectively. Class comparison and multi-dimensional scaling analyses revealed a distinctive expression profile of apoptotic genes in Alu-treated cells.
These results afford a previously unrecognized cytoprotective function for Dicer in processing endogenous single-stranded RNA. Additional studies are needed to further elucidate the mechanism of Alu-induced cell death, and regulation of Alu expression in geographic atrophy.
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