Decreased levels of the RNA splicing factor Prpf3 in mice and zebrafish do not cause photoreceptor degeneration.
OBJECTIVE
Pre-mRNA processing factor 3 (PRPF3) is a spliceosomal component essential for pre-mRNA processing. Mutations in PRPF3 have been implicated in retinitis pigmentosa (RP) 18 through an unknown mechanism. The authors created and characterized Prpf3 knockout mice and zebrafish to determine whether RP18 is a result of haploinsufficiency.


METHODS
Mice were produced from a Prpf3 gene trap cell line, and parameters of retinal function, structure, and RNA splicing were analyzed. The retinas of prpf3 insertional mutant zebrafish were also analyzed histologically.


RESULTS
Homozygous Prpf3 knockout mice do not survive to 14 days postfertilization (dpf), implying that this allele is required for early embryonic development. Homozygous Prpf3 knockout zebrafish die by 4dpf, well beyond the mid-blastula transition at which transcription activates. Zebrafish knockout embryos reveal abnormally high levels of cell death in the developing eye. Heterozygous Prpf3 knockout mice have less than the expected 50% reduction in Prpf3 at the mRNA and protein levels, implying compensatory expression from the wild-type allele. The heterozygous mice develop normally, with no changes in retinal function, no evidence for photoreceptor degeneration at up to 23 months of age, and no decrease in pre-mRNA splicing of transcripts mutated in other forms of RP in the retina. Similarly, heterozygous prpf3 knockout zebrafish develop normally and show no retinal degeneration up to 12 months of age.


CONCLUSIONS
These models suggest that RP18 is not a result of haploinsufficiency but instead arises from a toxic gain of function caused by missense mutations in PRPF3.
Graziotto
John J
JJ
F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, Philadelphia, Pennsylvania, USA.
Inglehearn
Chris F
CF
Pack
Michael A
MA
Pierce
Eric A
EA
D000818:Animals; D002460:Cell Line; D002678:Chimera; D017931:DNA Primers; D004596:Electroretinography; D053595:Embryonic Stem Cells / Q000502:physiology*; D051379:Mice; D012322:RNA Precursors / Q000378:metabolism*; D000072260:RNA Splicing Factors; D017949:Retinal Cone Photoreceptor Cells / Q000473:pathology*; D017948:Retinal Rod Photoreceptor Cells / Q000473:pathology*; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000172:deficiency / Q000378:metabolism*; D015027:Zebrafish
D017931:DNA Primers; C532027:Prpf3 protein, mouse; D012322:RNA Precursors; D000072260:RNA Splicing Factors; D017414:Ribonucleoprotein, U4-U6 Small Nuclear