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. 2014 Apr 17;55(4):2500-15.
doi: 10.1167/iovs.13-13574.

The Rpe65 rd12 Allele Exerts a Semidominant Negative Effect on Vision in Mice

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Free PMC article

The Rpe65 rd12 Allele Exerts a Semidominant Negative Effect on Vision in Mice

Charles B Wright et al. Invest Ophthalmol Vis Sci. .
Free PMC article

Abstract

Purpose: 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.

Keywords: RPE65; rd12; visual cycle.

Figures

Figure 1
Figure 1
The Rpe65 knockout and rd12 alleles did not complement. Knockout/rd12 offspring bred from KO/KO and rd12/rd12 parental mice did not have restoration of vision. OKT measurements in P120 +/+ (n = 11), KO/KO (n = 9), rd12/rd12 (n = 14), and KO/rd12 (n = 20) are shown. Knockout/KO, rd12/rd12, and KO/rd12 mice had significantly reduced visual acuity measures compared to +/+ mice. Knockout/rd12 and rd12/rd12 mice had significantly reduced responses compared to KO/KO mice but did not differ with respect to one another. **P < 0.001 compared with +/+. Data are presented as mean ± SD.
Figure 2
Figure 2
The rd12 allele caused visual acuity loss in a semidominant fashion. Mice harboring at least one copy of the rd12 allele lost visual function at earlier ages than mice that did not harbor the rd12 allele. Visual acuities of +/+ (solid line with diamond points; n = 8–13), KO/+ (dotted line with square points largely hidden behind the solid line; n = 8), rd12/+ (dotted and dashed line with circle points; n = 14–21), KO/KO (dashed line with square points; n = 6–9), rd12/rd12 (dotted line with asterisk points; n = 7–16), and KO/rd12 mice (dashed line with circle points; n = 20) from P30 to P210 are represented on the same graph. **P < 0.001 +/+ compared with rd12/+, ##P < 0.001 KO/KO compared with rd12/rd12, ooP < 0.001 KO/rd12 compared with rd12/rd12, significance determined through two-way repeated measures ANOVA with post hoc Student-Newman-Keuls testing. There was no difference between +/+ and KO/+ between P30 and P210, and visual acuity measures of the two strains overlap through the duration of the study. Data are presented as mean ± SD.
Figure 3
Figure 3
Mutant mice had reduced dark-adapted a- and b-wave amplitudes compared with +/+ mice from P30 to P90. Mice that were either homozygous or compound heterozygous for the rd12 allele trended toward a slightly faster progressive loss of ERG amplitudes than KO/KO mice. Responses from +/+ mice are shown to provide perspective to response losses observed in mutant mice (solid line to the left of mutant mouse responses). (A) Dark-adapted a-wave amplitudes from KO/KO (solid line; n = 8, 9, 9 at P30, P60, and P90, respectively), rd12/rd12 (long dashed line; n = 7, 7, 6 at P30, P60, and P90, respectively), and KO/rd12 (short dashed line; n = 16, 19, 19 at P30, P60, and P90, respectively) are shown at P30, P60, and P90. (B) Dark-adapted b-wave amplitudes from KO/KO (solid line), rd12/rd12 (long dashed line), and KO/rd12 (short dashed line) are shown at P30, P60, and P90. Based on ERG responses, mutant mice were approximately 103- to 104-fold less sensitive to light than wild-type mice. *P < 0.05, **P < 0.001 KO/KO compared with rd12/rd12; #P < 0.05, ##P < 0.001 KO/KO compared with KO/rd12; oP < 0.05, ooP < 0.001 KO/rd12 compared with rd12/rd12, significance determined through two-way repeated measures ANOVA with post hoc Student-Newman-Keuls testing. Data are represented as mean ± SD.
Figure 4
Figure 4
Knockout/KO and rd12/rd12 mice had a thinner retina than +/+ mice. Knockout/KO and rd12/rd12 mice had similar retinal morphology, with only slight retinal thinning compared with wild-type mice at P210. Bright-field images of retina cross sections stained with toluidine blue from +/+, KO/KO, and rd12/rd12 mice are shown at P60 (AC) and P210 (DF). Representative images from retina 1 mm superior of the optic nerve are shown. IS, inner segment; INL, inner nuclear layer; GCL, ganglion cell layer.
Figure 5
Figure 5
Knockout/KO and rd12/rd12 had similar ONL (A, B) and OS (C, D) thinning with age. Outer nuclear layer and OS thicknesses were not significantly different between KO/KO and rd12/rd12 at either P60 (A, C) or P210 (B, D). Quantitative measurements of ONL and OS thicknesses were taken at 500-μm intervals superior and inferior of the optic nerve at P60 and P210 in +/+ (n = 17 at P60, n = 12 at P210, solid line), KO/KO (n = 18 at P60, n = 9 at P210, dotted line), and rd12/rd12 (n = 7 at P60, n = 18 at P210, dashed line) mice. *P < 0.05, **P < 0.001 +/+ compared with rd12/rd12; #P < 0.05, ##P < 0.001 KO/KO compared with rd12/rd12, significance determined through two-way repeated measures ANOVA with post hoc Student-Newman-Keuls testing. Data are represented as mean ± SD.
Figure 6
Figure 6
Mice with Rpe65 mutations had large reductions in the number of cone nuclei. rd12/rd12 mice lost more cones on the superior portion of the retina than KO/KO mice at the same age. Cone nuclei were counted 250 μm, 1000 μm, and 2000 μm superior and inferior of the optic nerve in a 200-μm segment of the retina at P60 (A) and P210 (B). **P < 0.001 +/+ compared to rd12/rd12, #P < 0.05 KO/KO compared with rd12/rd12, significance determined through two-way repeated measures ANOVA with post hoc Student-Newman-Keuls testing. Data are presented as mean ± SD.
Figure 7
Figure 7
rd12/rd12 mice do not accumulate detectable amounts of RPE65 protein. rd12/rd12 mice did not have detectable amounts of a truncated RPE65 peptide fragment in soluble or insoluble fractions (detection limit defined in Supplementary Fig. S6). (A) Immunoblotting for RPE65 protein in the soluble RPE protein fraction; 1 μg of soluble +/+ RPE/choroid protein extract, 50 ng of the 44-amino acid antigen, 10 μg of soluble KO/KO RPE/choroid protein extract, and 10 μg of soluble rd12/rd12 RPE/choroid protein extract were loaded. Secondary-only controls are shown. (B) Immunoblotting for RPE65 protein in urea-soluble extracts with 8-day exposure to film; 20 μg protein were loaded in all lanes. Nonspecific binding of primary antibody to molecular weight ladder (Fig. 8A) was noted but was likely an artifact of nonspecific antibody being raised during the polyclonal antibody production.
Figure 8
Figure 8
rd12 mutant Rpe65 mRNA was exported to the cytoplasm. rd12/rd12 mice expressed appreciable Rpe65 mRNA that was exported to the cytoplasm. (A) +/+, KO/KO, and rd12/rd12 Rpe65 whole-cell mRNA levels. (B) Cells from RPE/choroid from P60 +/+ and rd12/rd12 mice were fractionated into their cytoplasmic and nuclear components and the RNA extracted for qRT-PCR. Rpe65 mRNA was present in similar levels in the nuclear fraction. *P < 0.05, **P < 0.001 significance tested through Student's unpaired t-test. Data are presented as mean ± SD, n = 10.
Figure 9
Figure 9
rd12 Rpe65 mRNA had a predicted secondary structure that closely resembled wild-type mRNA. Aside from an extra bubble (the size of a single base pairing) in the centroid structure (indicated by arrowhead), wild-type and rd12 mRNA structures were predicted to be identical. Predicted centroid structures of Rpe65 mRNA from wild-type, rd12, R91W, and tvrm148 alleles. Predicted mRNA secondary structures from wild-type and mutant mouse Rpe65 alleles predicted rd12 mRNA may adopt a structure that more closely resembled wild-type Rpe65 mRNA than other Rpe65 alleles.
Figure 10
Figure 10
Ribonucleic acid in situ hybridization showed similar localization of Rpe65 mRNA in both +/+ and rd12/rd12 RPE cells. There were no noticeable differences in the localization of rd12 mRNA with respect to wild-type mRNA. In situ hybridizations for Rpe65 (green), β-actin (red) mRNAs, and nuclei (blue; YO-PRO 1 iodide) from paraffin sections taken in +/+, KO/KO, and rd12/rd12 mice. (A) Knockout/KO mice had no detectable Rpe65 mRNA hybridization. (B) Rpe65 mRNA was found to localize throughout the cell in similar patterns in +/+ and rd12/rd12 mice, with much of the signal localized around the basal regions of the RPE. Signal was readily apparent in both the nucleus and cytoplasm of the RPE.
Figure 11
Figure 11
Possible outcomes of polyribosome profiling. The goal of this experiment was to determine whether the mutant rd12 Rpe65 mRNA could be bound by ribosomes, and if so, how many ribosomes bind. Messenger ribonucleic acid–bound species were separated on a linear sucrose gradient, followed by isolation of RNA and qRT-PCR of Rpe65 mRNA from each fraction. (A) Sucrose gradient fractionation separated RNA binding protein (RBP)-bound RNAs into three possible species: mRNP-bound RNAs (green circles), monoribosome-bound RNAs (black lines with one blue oval), and polyribosome-bound RNAs (black lines with multiple blue ovals). The first three fractions (1–3) contained RNA species mostly bound by mRNPs; the fourth fraction (4) contained RNA species mostly bound by multiple ribosomes. The more ribosomes bound a single mRNA molecule, the further the species migrated down the gradient during centrifugation. (B) Because there were detectable amounts of full-size RPE65 protein from +/+ mice, it would be expected that most wild-type Rpe65 mRNA would be found in fractions 5 to 10 (this is in fact what was observed). There were four possible outcomes for the mutant mRNA in the cytoplasm: (1) active translation of a full-size peptide (which would require ribosomes to read through the PTC, require an IRES, or another sequence that would allow read through or suppression of termination in the mutant mRNA); (2) inefficient initiation of translation (the mRNA would be bound only by mRNPs, which we observed in rd12/rd12 mice); (3) stalled translation (a single ribosome would stall on the PTC during the pioneer round of translation); and (4) active translation of a truncated peptide (because of the short length of the read frame in the mutant mRNA with the PTC, only one or two ribosomes would bind to the mRNA at a single time). If there were active translation of full-size RPE65 peptides, most of the mRNA would be in fractions 5 to 10 (as was the case in +/+ mice). If there was no (or inefficient) initiation of translation, most mRNA would be found in fractions 1 to 3 (as was the case in rd12/rd12 mice). If there was active translation of a truncated RPE65 peptide in rd12/rd12 mice, most mRNA would be found in fractions 4 to 5.
Figure 12
Figure 12
rd12 Rpe65 mRNA was mostly found in ribosome-free mRNPs. Unlike wild-type mRNA, mutant rd12 mRNA was mostly contained in ribosome-free mRNP fractions. (A) Cytoplasmic extracts of RPE/choroid were separated by linear sucrose gradient centrifugation to yield mRNP fractions (1–3), monoribosome fractions (4), and polyribosome fractions (5–10). (B) Mono- and polyribosomal complexes disrupted by the addition of EDTA result in all Rpe65 mRNA being present in mRNP-containing fractions.
Figure 13
Figure 13
Rpe65 mRNA is mostly bound by mRNPs in rd12/rd12 mice. The area under the wild-type and rd12 curves in fractions 1 to 3 in the polyribosome profiles of Rpe65 mRNA were added together to generate the average amount of mRNA present in the mRNP fractions. Because three mRNA-bound species (mRNP-bound, monoribosome-bound, and polyribosome-bound mRNAs) are separated by linear sucrose gradient fractionation in this assay, the areas under the polyribosome curves were added together for each species. Mutant mRNA was much more enriched in mRNP-containing fractions and greatly reduced in actively translating polyribosome fractions in rd12 mice compared with wild-type. There were no significant differences between wild-type and rd12 mice in monoribosome-bound Rpe65 mRNAs, which could be due to either insufficient resolution in this experiment or because the mutant mRNA is still capable of binding monoribosomes. *P < 0.05, significance tested through Student's unpaired t-test. Data are presented as mean ± SD.
Figure 14
Figure 14
Proposed model for the foundation of the negative semidominant effects exerted by the rd12 allele on visual function. The rd12 allele is (a) transcribed, (b) spliced, (c) processed, and (d) exported from the nucleus to the cytoplasm like the wild-type allele. Once in the cytoplasm, the mutant mRNA is (e) trafficked to the ribosomes much like the wild-type mRNA. Unlike the wild-type mRNA, though, the mutant mRNA (f) becomes sequestered on mRNPs and are unable to bind to ribosomes for the first round (often called the pioneer round) of translation when nonsense RNA surveillance pathways could recognize the rd12 mRNA and degrade it. Consequently, (g) the mutant mRNA-mRNP complexes accumulate and form the foundation for currently unknown downstream mechanisms that cause visual dysfunction in a semidominantly inheriting fashion.

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