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J. J. Graziotto, C. F. Inglehearn, E. A. Pierce; Exon Microarray-Based Analysis of Splicing in Prpf3 and Prpf8 Knockin Mice. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3993. doi: https://doi.org/.
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The pre-mRNA processing factors PRPF3 and PRPF8 have been implicated in Retinitis Pigmentosa 18 and 13, respectively. These factors are ubiquitous and are required for spliceosomal function. We generated Prpf3 and Prpf8 knockin mice with mutations known to cause disease in humans to study how mutations in these splicing factors lead to retina specific disease. We used exon microarrays to investigate the hypothesis that mutations in PRPF3 and PRPF8 lead to retina-specific alterations in RNA splicing.
We produced homozygous knockin mouse models bearing the Prpf3-T494M and Prpf8-H2309P mutations. We used the Affymetrix Mouse Exon 1.0 ST array to directly look for changes in splicing in the retinas of these animals. The exon arrays contain multiple probes per exon and allow the detection of alternative splicing events. We compared splicing in retinal RNA from age-matched homozygous knockin Prpf3 or Prpf8 mice with littermate control wild-type mice.
The exon microarrays show significant differences in splicing of several transcripts in knockin retina vs. wildtype controls. The changes were identified in both retina specific and non-retina specific transcripts, and include alternative 3’ or 5’ ends and alternatively spliced exons. Some transcripts have the same change for both Prpf3 and Prpf8 retina, while others only differ for one and not the other. We are using RT-PCR to verify the changes detected by the microarrays, and to identify the exact alterations for verified transcripts.
The pathway to disease for RP13 and RP18 is currently unknown. Our results show for the first time that mutations in the RNA splicing factors PRPF3 and PRPF8 may alter the splicing of specific transcripts in the retina.
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