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Tadeusz Kaczynski, Elizabeth D. Au, Maria E Sousa, Michael H Farkas; Localization of Coding and Noncoding RNAs in the Retinal Pigmented Epithelium. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1541.
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© ARVO (1962-2015); The Authors (2016-present)
RNA localization is emerging as an important layer of regulatory complexity that is relevant in both the healthy and disease states. Here, in order to gain insight into how transcript localization is altered by disease-related stressors in age-related macular degeneration (AMD), we mapped the RNA localization landscape of iPSC-derived retinal pigmented epithelium (iPSC-RPE) cells under normal and oxidative stress conditions.
Two distinct lines of iPSCs were differentiated into RPE. A subset of the RPE cells were subjected to 500 µM H2O2 for 3 hours to induce oxidative stress. The samples were then collected and fractionated. Total RNA was isolated from each fraction and used in the preparation of sequencing libraries, which were run on an Illumina NextSeq 500. Reads were aligned to the hg38 human genome build using STAR, quantified using RSubread in conjunction with the Gencode v29 transcriptome database, and validated using RNA fluorescence in situ hybridization. Potential adenosine to inosine (A-to-I) editing sites were found using SPRINT and validated with Sanger sequencing.
Using stringent classification parameters, we found that, under normal conditions, mRNAs and lncRNAs occupied a wide range of localization patterns, with 3,868 nuclear enriched transcripts, 2,355 cytoplasmic enriched transcripts, and 46,720 without specific localization. In agreement with previous studies, mRNAs tended toward equal distribution between the fractions, while lncRNAs tended to be enriched in the nucleus. However, when subjected to oxidative stress, the transcriptome became more segregated, increasing to 9,402 nuclear and 5,345 cytoplasmic transcripts. Our analysis also revealed that, under stress conditions, A-to-I RNA editing was upregulated in both the nuclear and cytoplasmic fractions by 1.4 and 6.8 fold, respectively – a finding which may help explain the observed localization shift.
To better our understanding of AMD, we have undertaken a high-throughput approach to study the link between oxidative stress and RNA localization in the RPE. Our findings have revealed a dramatic shift in RNA localization in the RPE transcriptome in response to oxidative stress. We have also investigated the mechanisms contributing to this localization shift. Furthermore, our mapping of lncRNA localization opens the way forward for future functional analyses of lncRNAs in RPE.
This is a 2020 ARVO Annual Meeting abstract.
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