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William Samuel, R Kutty, Todd Duncan, Cynthia Jaworski, Toshifumi Hara, Ashish Lal, T. Michael Redmond; Translational Regulation of RPE65 Expression by microRNA. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6322.
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© ARVO (1962-2015); The Authors (2016-present)
The retinal pigment epithelium (RPE) performs functions critical to the process of vision. In particular, RPE65, retinol isomerohydrolase, plays an important role in the regeneration of chromophore 11-cis retinal. Expression of both RPE65 mRNA and protein is high in fresh RPE cells. However, RPE65 protein expression is lost in RPE primary culture while RPE65 mRNA remains. MicroRNAs (miRNAs) can regulate gene expression in eukaryotes by targeting mRNA for translational repression by binding to the 3’ UTR region. We wished to determine whether miRNAs play a role in the translational repression of RPE65 in RPE primary cultures by binding to the 3’UTR of RPE65.
The full-length 3’-UTR of human RPE65 was cloned into psiCHECK2 reporter vector 3’ to the Renilla Luciferase coding region. The possible binding of miRNAs to target sites in the 3’-UTR of human RPE65 was identified by TargetScan. RPE65 3’-UTR reporter construct was transiently transfected into human RPE cells (ARPE-19) grown in 24-well cell culture plates. Human miRIDIAN miRNA mimics (Dharmacon) based on TargetScan were also transfected along with human RPE65 3’-UTR construct. psiCHECK2 was used as a control vector. The luciferase assay was performed after 48 hr to identify miRNAs that target RPE65 3’-UTR.
Several miRNAs were predicted to bind to the 3’-UTR of human RPE65 by TargetScan bioinformatics analysis. These included miR-210, miR-181 and miR-374. We have found these miRNAs to be expressed in bovine RPE primary cultures. The human RPE65 3’-UTR reporter construct showed luciferase activity when the reporter vector was transfected into ARPE-19 cells. Of tested miRNA mimics, miR-210 decreased the luciferase activity of the reporter when co-transfected with the human RPE65 3’-UTR reporter construct. In addition, a moderate decrease in the luciferase activity of RPE65 3’-UTR reporter construct was also observed with miR-181a among the miR-181 family.
We show that miR-210 and 181a decrease the luciferase activity of the RPE65 3’-UTR reporter construct in ARPE-19 cells, suggesting that these miRNAs could potentially regulate the expression of RPE65 by translational repression by binding to its 3’-UTR. These results may explain why RPE65 protein disappears in RPE primary cultures, though its mRNA persists.
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