title: The Rpe65 rd12 allele exerts a semidominant negative effect on vision in mice.
abstract: OBJECTIVE
  The rd12 mouse was reported as a recessively inherited Rpe65 mutation. We asked if the rd12 mutation resides in Rpe65 and how the mutation manifests itself.
  
  
  METHODS
  A complementation test was performed by mating Rpe65(KO) (KO/KO) and rd12 mice together to determine if the rd12 mutation is in the Rpe65 gene. Visual function of wild-type (+/+), KO/+, rd12/+, KO/KO, rd12/rd12, and KO/rd12 mice was measured by optokinetic tracking (OKT) and ERG. Morphology was assessed by retinal cross section. qRT-PCR quantified Rpe65 mRNA levels. Immunoblotting measured the size and level of RPE65 protein. Rpe65 mRNA localization was visualized with RNA fluorescence in situ hybridization (FISH). Fractions of Rpe65 mRNA-bound proteins were separated by linear sucrose gradient fractionation.
  
  
  RESULTS
  The KO and rd12 alleles did not complement. The rd12 allele induced a negative semidominant effect on visual function; OKT responses became undetectable 120 days earlier in rd12/rd12 mice compared with KO/KO mice. rd12/+ mice lost approximately 21% visual acuity by P210. rd12/rd12 mice had fewer cone photoreceptor nuclei than KO/KO mice at P60. rd12/rd12 mice expressed 71% +/+ levels of Rpe65 mRNA, but protein was undetectable. Mutant mRNA was appropriately spliced, exported to the cytoplasm, trafficked, and contained no other coding mutation aside from the known nonsense mutation. Mutant mRNA was enriched on ribosome-free messenger ribonucleoproteins (mRNPs), whereas wild-type mRNA was enriched on actively translating polyribosomes.
  
  
  CONCLUSIONS
  The rd12 lesion is in Rpe65. The rd12 mutant phenotype inherits in a semidominant manner. The effects of the mutant mRNA on visual function may result from inefficient binding to ribosomes for translation.
authors.affiliation: Department of Ophthalmology, School of Medicine, Emory University, Atlanta, Georgia, United States.
authors.forename: Charles B
authors.initials: CB
authors.lastname: Wright
authors.forename: Micah A
authors.initials: MA
authors.lastname: Chrenek
authors.forename: Wei
authors.initials: W
authors.lastname: Feng
authors.forename: Shannon E
authors.initials: SE
authors.lastname: Getz
authors.forename: Todd
authors.initials: T
authors.lastname: Duncan
authors.forename: Machelle T
authors.initials: MT
authors.lastname: Pardue
authors.forename: Yue
authors.initials: Y
authors.lastname: Feng
authors.forename: T Michael
authors.initials: TM
authors.lastname: Redmond
authors.forename: Jeffrey H
authors.initials: JH
authors.lastname: Boatright
authors.forename: John M
authors.initials: JM
authors.lastname: Nickerson
mesh_terms: D000483:Alleles; D000818:Animals; D018389:Codon, Nonsense; D004195:Disease Models, Animal; D004596:Electroretinography; D005838:Genotype; D015151:Immunoblotting; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010641:Phenotype; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism*; D012313:RNA / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D012162:Retinal Degeneration / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology; D014792:Visual Acuity; D019745:cis-trans-Isomerases / Q000096:biosynthesis / Q000235:genetics*; D000099178:Retinoid Isomerohydrolase
chemical_list: D018389:Codon, Nonsense; D012313:RNA; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase
keywords: RPE65; rd12; visual cycle