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Michael Farkas, Elizabeth D Au, Maria E Sousa, Eric Pierce; Transcriptome analyses reveal minimal aberrant splicing in mouse models of RNA splicing factor retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4993.
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Mutations in genes that encode RNA splicing factors are the second most common cause of the dominant form of retinitis pigmentosa (RP). Currently, 5 splicing factors (PRPF3, PRPF6, PRPF8, PRPF31, and SNRNP200) have been identified to cause RP. It is unclear how mutations in these constituents of the spliceosome, a ubiquitously expressed macromolecule required for splicing in all tissues, cause retina-specific disease. Evidence suggests that aberrantly spliced transcripts underlie the disease, but whether these are retina specific or occur globally, but only affect the retina remains to be elucidated.
To investigate the potential mechanisms, we used RNA-Seq to analyze the transcriptomes of the neural retina, retinal pigment epithelium (RPE), brain, and skeletal muscle of 5 biological replicates from Prpf3, Prpf8, and Prpf31 mutant mice. We aligned data to the genome using the RNA-Seq Unified Mapper, and determined feature counts for transcripts, exons, introns, and splice junctions. Differential expression (DE) was investigated using the T-test Derived Differential expression algorithm. Using a combination of DE and splice junction data, aberrantly spliced transcripts were identified.
The RNA-seq data provided deep coverage of each tissue’s transcriptome and statistical power for DE analysis. Hundreds to thousands of features were identified as differentially expressed between Prpf-mutant mice and littermate controls in all tissues. Determination of aberrant splicing events revealed a significantly smaller (24-174 events) set of candidates. Of particular interest, abundant aberrant splicing was detected in the transcript for Rgr, but only in the RPE, although it is also expressed in Müeller cells. Further, of the 6 aberrant isoforms generated, all are predicted to maintain an open reading frame (ORF) and potentially code for a functional protein.
This is the first study to interrogate the transcriptomes of tissues affected in RNA splicing factor forms of RP. The data suggest that while expression is affected in all tissues, there are few aberrantly spliced transcripts. Aberrations in retina-specific transcripts, such as Rgr, may be the underlying cause of disease. Furthermore, since the ORFs are maintained, we must consider all mechanisms of disease including gain-of-function, dominant negative, and loss-of-function.
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