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Neel A. Gupta, Matthew J. Brooks, Harsha Rajasimha, Norimoto Gotoh, Linn Gieser, Rafael Villasmil, Tiziana Cogliati, Anand Swaroop; Analysis of Alternative Splicing and Alternative Promoter Use in Global RNA Expression Profiles During Mouse Photoreceptor Aging. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4562.
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Alternative splicing and/or alternative promoter usage is frequent in a majority of multi-exon human genes and is an important mechanism for generating protein diversity and regulating cell-type specific expression. Furthermore, mutations in splicing factors have been associated with blinding retinal diseases. Rod photoreceptors are among the first neurons to die in normal aging and in many retinal degenerative diseases. We sought to determine the precise extent of alternative splicing and promoter usage in transcriptional regulation during rod photoreceptor aging.
Enhanced GFP expression was directed specifically to rod photoreceptors using a 2.5-kb Nrl promoter segment. Retinal cells were flow-sorted to isolate rod photoreceptors and RNA was extracted at 3, 6, 9, 12, and 18 months. Total RNA (15 ng) was used with the TruSeq mRNA-seq Sample Preparation Kit (Illumina, San Diego, CA) to construct cDNA libraries. High-throughput deep sequencing was performed in two independent samples per time point using Illumina GAIIx. The sequencing produced 76 base-pair, single-end, directional reads. The sequencing data was analyzed for alternative splicing and alternative promoter usage using the open-source application AltAnalyze.
We identified numerous genes that contain dynamically spliced exons as well as alternative promoter and 3’ UTR usage. GO analysis of differentially expressed and alternatively spliced genes revealed a number of pathways altered during aging. We also validated a select group of alternatively spliced exons using qRT-PCR.
Our study represents the first use of RNA-Seq data to thoroughly examine splicing and transcript-level changes in mouse rod photoreceptors during the aging process. Our results provide the foundation for, and insights into, transcriptional regulatory networks that might mediate the adaptive process during aging of rod photoreceptors.
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