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M. H. Farkas, K. Bujakowska, A. Krishan, J. J. Graziotto, E. F. Nandrot, S. S. Bhattacharya, E. A. Pierce; Characterization of Aberrant Splicing by Comparative Exon Microarray-Based Analysis in Mice With Targeted Mutations in Prpf3, Prpf8, and Prpf31. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3667.
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Mutations in pre-mRNA processing factors 3, 8, 31 (PRPF3, 8, 31), PAP-1, and SNRNP200 have been identified as causative agents for the inherited retinal degenerative disease Retinitis Pigmentosa (RP). These factors are ubiquitously expressed and required for proper processing of pre-mRNA by the spliceosome. To investigate how mutations in RNA splicing factors cause RP, we generated Prpf3, Prpf8, Prpf31 knockin mice with mutations known to cause RP in humans. We used exon microarray analyses to evaluate the extent of aberrant splicing in the retinas of these mice.
Retinas were harvested from homozygous Prpf3 and Prpf8 mice at 2 months and 9 months for heterozygous Prpf31 mice. Using the Affymetrix 1.0 ST exon array, we compared splicing between the mutant mice and their littermate controls. Secondary to this initial analysis we performed analyses to determine aberrant splicing common to all three models. Results were validated using qRT-PCR, as well as direct sequencing of exons identified to significantly change.
The initial analysis of the exon microarrays show significant changes in the splicing of several hundred transcripts in each of the mutant mice relative to their littermate controls. By comparing the results from all three models, we were able to focus the results to 53 transcripts that are in common. Of those 53 transcripts, 3 were known retinal disease genes. Using RT-PCR, we have validated 20 of the altered splicing events detected by the arrays, out of 37 tested to date. This includes 1 of the splicing changes detected in the transcripts from the three retinal degeneration disease genes. Furthermore, we were able to demonstrate that splicing alterations also occur in non-ocular tissues.
Determining the mechanism of RP as a result of mutations in RNA splicing factors is a difficult challenge. By analyzing the results of the three mouse models (Prpf3, 8, and 31) for the disease against each other, we were able to reduce the number of transcripts detected with significant splicing changes to a more manageable set for validation. Through these analyses, we were able to demonstrate that mutations in the PRPF3, 8, and 31 genes results in significant splicing changes in retina. These changes support our hypothesis that RNA splicing factor forms of RP are the result of aberrant splicing in genes required for proper retinal function.
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