Abstract
Purpose :
The exon-skipping isoform of RGR opsin (NM_001012722.1, RGR-d) has been reported to be associated with human age-related macular degeneration (AMD) and AMD-like pathology in gene-editing mouse models. RGR-d is considered an abnormal protein that undergoes different intracellular processing from the normal RGR protein. Here, we studied the expression and subcellular localization of RGR and RGR-d protein.
Methods :
ARPE-19 cells were transfected with FLAG labeled lentiviral vectors expressing RGR or RGR-d and cell lines with stable expression were selected by puromycin. Cells were treated with 4 μM MG132, a 26S proteasome inhibitor, for 0, 4, 12, and 20 h. The expression of RGR and RGR-d was detected by real-time PCR and western blot. After the cells were treated with 2 μM MG132 for 10 h, double-labeling immunofluorescence was used to detect the co-localization of RGR/RGR-d and the markers of organelles or autophagy-lysosomal pathway, including calnexin, GM130, LAMP-2, and Rab7.
Results :
After the transfection, the mRNA expression of both RGR and RGR-d were hundreds of times higher than that of the negative control. The protein expression of RGR was significantly elevated. But inconsistent with the mRNA expression, RGR-d expression was barely detected by western blot. After the cells were treated with MG132, the protein expression of RGR-d was detected after 4 h, and gradually increased after 12 h and 20 h, indicating an intracellular proteasome degradation of RGR-d. MG132 of a low concentration was then used for the enrichment of intracellular RGR and RGR-d protein. Double-labeling immunofluorescence showed that compared with RGR, RGR-d had less co-localization with calnexin, a marker of the endoplasmic reticulum (ER), and better co-localization with LAMP-2 and Rab7, markers of the autophagy-lysosomal pathway. Neither RGR nor RGR-d had significant co-localization with GM130, a marker of the Golgi apparatus.
Conclusions :
In transfected cells, RGR-d was normally transcribed but was almost completely degraded through the ubiquitin-proteasome pathway after translation, while the full-length RGR was normally expressed at both mRNA and protein levels. With the treatment of MG132, RGR-d was less likely to undergo the post-translational modification in the ER and Golgi apparatus and was prone to lysosomal degradation. The findings may provide insights into the processing of abnormal RGR-d protein in the aged population.
This is a 2021 ARVO Annual Meeting abstract.