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Revital Bronstein, Sudeep Mehrotra, Elizabeth E. Capowski, Leah Foltz, Michael H Farkas, David M Gamm, Dennis O Clegg, Eric A Pierce; Mutations in splicing factors that cause Retinitis Pigmentosa (RP) do not affect splicing in a global manner. Invest. Ophthalmol. Vis. Sci. 2019;60(9):379.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in 7 RNA splicing factor genes PRPF3, PRPF4, PRPF6, PRPF8, PRPF31, SNRNP200, DHX38 cause RP. Together mutations in these genes are the second most common cause of dominant RP. We and others have previously shown that mutations in the PRPF3, PRPF8, and PRPF31 genes disrupt RPE function in vivo and in vitro. To further investigate how mutations in these ubiquitously expressed genes lead to retina-specific disease, we sequenced RNA isolated from tissues of mice or human cells with mutations in these three splicing factors
Five tissues, RPE, retina, brain, muscle and kidney were harvested from mice harboring mutations in the Prpf3, Prpf8 or Prpf31 genes and littermate controls. Induced pluripotent stem cells (iPSC) from patients with mutations in the PRPF8 and PRPF31 genes and corrected control cells were differentiated into retinal pigment epithelial (RPE) cells and optic vesicles (OV). RNA was isolated and RNA-seq analysis was performed using established methods. Sequence reads were aligned to the appropriate genome using STAR. Differential gene expression was evaluated using the software tools DESeq2 and edgeR. Differential splicing was assessed using JunctionSeq and MAJIQ. Internal control ‘spike-in’ sequences (Sequins) were utilized to determine analysis parameters.
Between 12K-19K genes passed the filters that were determined by the sequin controls in each of the mouse tissues and human cells. These genes represent a large portion of the corresponding genomes. In contrast, only 0.2-1.8% of the filtered genes were found to be differentially spliced in the mutant mouse tissues or human cells. Similarly, 3-14% of the filtered genes were found to be differentially expressed. The Prpf31 mouse retina was an outlier with 30% differentially expressed genes. The differentially spliced and expressed genes showed a strong tissue/cell specificity bias, with little overlap between cell or tissue types.
The relatively small number of genes with altered splicing detected in RNA processing factor mutant tissues and cells indicates that mutations in these genes do not disrupt splicing globally. This and the tissue specific nature of the splicing alterations identified suggest that mutations in RNA splicing factor genes cause retinal disease via the alteration of splicing or expression of specific transcripts required for retinal and RPE function.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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