Abstract
Purpose:
The rd12 allele of mouse Rpe65 has a nonsense mutation in exon 3 of the gene. The rd12 allele exerts a dominant-negative effect on vision that extends beyond the loss of visual function caused by a lack of Rpe65 enzymatic activity. The purpose of this study is to attempt to define a possible mechanism-of-action for the rd12 allele.
Methods:
Optokinetic tracking (OKT) and electroretinography (ERG) were used to assess the visual acuity and retinal function of C57BL/6J (+/+), Rpe65 KO (KO/KO), tvrm148 (tvrm148/tvrm148), rd12 (rd12/rd12), rd12/+, KO/+, tvrm148/+, KO/rd12, and KO/tvrm148 mice. Immunoblotting with an antibody specific to the N-terminus of Rpe65 was performed to detect protein, and qRT-PCR was used to detect steady-state mRNA levels of genes of interest. RNAfold was used to predict the centroid structures of the Rpe65 mRNAs from the +, tvrm148, and rd12 alleles. RNA immunoprecipitation (RIP) was used to detect Rpe65 mRNA binding to eIF4E.
Results:
Mice heterozygous for the rd12 allele lost 21% of their visual acuity by P210 while mice heterozygous for other mutations did not. Mice homozygous for the rd12 allele lost 100% visual acuity by P120, while mice homozygous for KO or tvrm148 alleles did not lose 100% visual acuity until P210. KO/rd12 mice had visual function loss that resembled rd12 homozygote mice. qRT-PCR for all Rpe65 exon boundaries showed the rd12 allele has no splicing defects in the mutant mRNA. No other coding mutations were found. Structural prediction of the mRNA predicted the rd12 allele mRNA did not adopt a different secondary structure than wild type. qRT-PCR showed the same level of wild type and mutant mRNA in the cytoplasm, but RIPs showed the mutant mRNA was not bound by eIF4E, a translation initiation factor found in polysomes.
Conclusions:
Loss of visual function in mice homozygous and heterozygous for the rd12 allele shows the allele acts in a dominant-negative manner to other Rpe65 mutant alleles, and that one copy of the rd12 allele is sufficient to drive this dominant-negative visual function loss. The rd12 allele does not produce a detectable protein, but does produce an mRNA that is appropriately transcribed, spliced, folded, and exported from the nucleus to the cytoplasm but does not traffic to polysomes, suggesting the mutant mRNA may induce harm to the visual system because of altered intracytoplasmic trafficking.
Keywords: 705 retinoids/retinoid binding proteins