Multiple Mechanisms Contribute to Leakiness of a Frameshift Mutation in Canine Cone-Rod Dystrophy

<div><h3></h3><p>Mutations in <em>RPGRIP1</em> are associated with early onset retinal degenerations in humans and dogs. Dogs homozygous for a 44 bp insertion including a polyA₂₉ tract potentially leading to premature truncation of the protein, show cone rod degeneration. This is rapid and blinding in a colony of dogs in which the mutation was characterised but in dogs with the same mutation in the pet population there is very variable disease severity and rate of progression.</p> <h3>Objective</h3><p>We hypothesized that this variability must be associated with leakiness of the <em>RPGRIP1</em> mutation, allowing continued RPGRIP1 production. The study was designed to discover mechanisms that might allow such leakiness.</p> <h3>Methods</h3><p>We analysed alternate start sites and splicing of <em>RPGRIP1</em> transcripts; variability of polyA_n length in the insertion and slippage at polyA_n during transcription/translation.</p> <h3>Results and Significance</h3><p>We observed a low rate of use of alternative start codons having potential to allow forms of transcript not including the insertion, with the possibility of encoding truncated functional RPGRIP1 protein isoforms. Complex alternative splicing was observed, but did not increase this potential. Variable polyA_n length was confirmed in DNA from different <em>RPGRIP1</em>^−/− dogs, yet polyA_n variability did not correspond with the clinical phenotypes and no individual was found that carried a polyA_n tract capable of encoding an in-frame variant. Remarkably though, in luciferase reporter gene assays, out-of-frame inserts still allowed downstream reporter gene expression at some 40% of the efficiency of in-frame controls. This indicates a major role of transcriptional or translational frameshifting in <em>RPGRIP1</em> expression. The known slippage of reverse transcriptases as well as RNA polymerases and thermostable DNA polymerases on oligoA homopolymers meant that we could not distinguish whether the majority of slippage was transcriptional or translational. This leakiness at the mutation site may allow escape from severe effects of the mutation for some dogs.</p> </div

Haplotypes spanning the 6.05 Mb flanking region of <i>RPGRIP1</i>.

<p> The ‘114’- major haplotype (yellow) corresponds to the haplotype predominant in <i>RPGRIP1</i>^−/− dogs, with the common PCR fragment pattern peaking at ‘114’ (A₂₉ insert) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051598#pone-0051598-g003" target="_blank">Figure 3</a>. Microsatellite marker alleles specific to the dogs with the ‘113’ pattern (A₂₈ insert) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051598#pone-0051598-g003" target="_blank">Figure 3</a> are indicated (blue). The polyA₂₈ allele was determined by cloning from genomic DNA (*) or PCR-fragment sizing (**).</p

Capillary electrophoresis of PCR products containing the polyA tract.

<p>PCR amplicon spanning the <i>RPGRIP1</i> polyA insertion was sized by capillary gel electrophoresis. The common electropherogram peak pattern from <i>RPGRIP1</i>^−/− MLHDs is represented by dogs MLD7 (b: late-onset affected, 9 y) and MLD4 (d: mid-onset affected, 5 y). In dogs MLD11 (a: clinically normal, 5 y) and MLD6 (c: clinically normal, 9 y), the highest peak in each electropherogram was shifted by 1 bp, to ‘113’, compared to the common PCR fragment peak pattern ‘114’. Note that the majority of the <i>RPGRIP1</i>^−/− dogs examined including both clinically affected and normal dogs showed the ‘114’ pattern. Direct cloning and haplotype analysis confirmed MLD6 as heterozygous for polyA_28/29 while the ‘114’ pattern corresponded to polyA_29/29.</p

p2 luc constructs used in dual-reporter luciferase assay.

<p>DNA sequences and the corresponding amino acids for plasmid constructs with polyA insertions (p2 luc/A_{28, 29 and 30}), and in-frame (p2 luc/F+) and <i>rluc</i>-only (p2 luc/F-, stop codon upstream of <i>fluc</i>). The polyA constructs shown indicate the three possible reading frames after the polyA sequence; only those with (3n-1) adenines, such as A₂₉, A₃₅, A₃₈ and A₄₁, lead to an in-frame <i>fluc</i>, unless the number of adenines is changed following transcription or the reading frame is altered during translation. (Note that this single base gain in the construct reading frame is specific to this reporter assay. In the cell, A₃₀, A₃₆, A₃₉ and A₄₂ would be in frame.) Blue and yellow highlights indicate <i>Renilla</i> and firefly gene sequences, respectively. The SalI and BamHI cloning sites are outlined. DNA sequence of the polyA tract is shown in red letters, while the flanking region (exon 3 of <i>RPGRIP1</i>) is in blue letters with the 15-bp duplication underlined.</p

Quantitation of cDNA fragments of different exonic regions of <i>RPGRIP1</i>.

<p>Retinal cDNA populations were analysed by quantitative RT-PCR in beagles of <i>RPGRIP1</i>^+/+ (blue) and <i>RPGRIP1</i>^−L/−L (red) genotypes. The absolute copy number of molecules in equal amounts of template cDNA is shown on a log scale. Each fragment was assayed in triplicate (technical replicates) and two replicate experiments and copy numbers of DNA molecules were calculated by comparison with control sequences cloned into plasmids.</p

Expression of <i>fluc</i> downstream of polyA tracts.

<p>The firefly to <i>Renilla</i> luciferase expression ratio in p2luc/A_{21, 25, 27–30, 35, 38–43} constructs normalised to the in-frame control (p2luc/F+, 100%) in MDCK cells (solid bars) and COS-7 cells (shaded bars). The green, blue, and orange colours correspond to constructs with polyA_{3n+1, 3n−1 and 3n} inserts, respectively, where in this vector the blue bars represent the constructs that preserve the reading frame of the <i>fluc</i> gene. PolyA insert lengths not studied are printed in grey along the X axis. Error bars represent SD of three triplicate assays. p2luc/F- is a negative control for which no <i>fluc</i> expression is expected because of a termination codon upstream of the firefly gene.</p

Partial exon structure of canine <i>RPGRIP1</i>.

<p>Canine <i>RPGRIP1</i> structure identified by Kuznetsova et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051598#pone.0051598-Kuznetsova1" target="_blank">[12]</a> (a) and in this study by 5′ RACE (b) and RT-PCR (c). The red triangle points to the 44 bp insertion in exon 3 previously associated with <i>cord1</i> in a MLHD research colony <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051598#pone.0051598-Mellersh1" target="_blank">[8]</a>. The vertical double bar represents cDNA ends. Grey exon boxes indicate partially omitted sequences. Red exon boxes represent translation initiation site codon (ATG). (b) Blue and yellow underlines show location of primers used in RACE. †New transcript variants identified in the current study.</p

Sequences of the cloned polyA tract of the <i>RPGRIP1</i> insertion.

<p>Sequences were obtained from genomic DNA fragments cloned and amplified in <i>E coli</i>. Shown are electropherograms of single alleles isolated from MLD6 (a, polyA₂₈) and from a <i>cord1</i>-affected MLHD from the original AHT colony (b: polyA₂₉).</p

Complex cellular phenotype of V644del mutant channel.

<p><b>(A)</b> Cellular localization of YFP-tagged wild-type canine CNGA3 and CNGA3-V644del mutant in HEK tsA201 cells. Cells transfected with the wild-type construct showed specific fluorescence pattern of expression limited to the plasma membrane and Golgi-like organelles (arrow); an evident increase in intracellular aggregates was observed in cells transfected with V644del mutant construct consistent with abnormal trafficking and potential ER retention. Scale bar: 10μm. <b>(B)</b> cGMP- and cAMP-activated currents recorded from CNGA3-WT and a responsive patch expressing V644del mutant channels. Approximately 40% of V644del mutant patches had no cGMP-activated currents, in contrast to 100% responsive patches from the CNGA3-WT-transfected cells. This partial loss of channel activity might reflect incomplete subunit assembly associated with disruption of the coiled-coil structure as depicted in the simulation studies. The responsive patches showed cyclic nucleotide-activated currents with similar characteristics to WT channels. <b>(C)</b> Histograms of subcellular localization patterns monitored in HEK tsA201 cells co-transfected with V644del and CNGA3-WT cDNA constructs. Cells were transfected with either CNGA3-WT or CNGA3-V644del or both constructs at the indicated ratios. Each cell count represents >300 cells from at least 2 transfections (mean% ± SD).</p

R424W mutation disrupts salt bridge interaction and destabilizes the open state of pore in a homotetrameric CNGA3 model.

<p><b>(A)</b> Schematic representation of CNGA3 subunit consisting of six transmembrane (TM) spanning segments (S1-S6) and a pore domain between S5 and S6. The highlighted last residue of S6 (blue) is the site of canine CNGA3-R424W mutation; its predicted partner, glutamic acid E306, is the first residue of S4-S5 linker. <b>(B)</b> Amino acid sequence alignment of the S4-S5 linker and S6 segment of selected shaker K^<b>+</b> channel superfamily members. The TM regions of the CNG channel family were assigned using the crystal structure of the chimeric voltage-gated potassium channel Kv1.2/2.1 (PDB ID: 2R9R). Sequence alignments of S5 domain and pore region were omitted for clarity. The R424 residue is shown in blue and its interacting partner, E306 in red. The conserved salt bridges in the Kv channels show opposite charges at these positions. c = canine, b = bovine, h = human, r = rat, m = mouse. <b>(C)</b> Side view of the wild-type CNGA3 homotetramer model and the CNGA3-R424W mutant channel equilibrated in its environment. The voltage-sensing domain (S1-S4) is presented in green, the S4-S5 linker in purple and the pore-forming region (S5-S6) in grey. The residues E306 and R424 are shown as red and blue rods, respectively. The E306:R424 interaction (wild-type) or its loss (R424W mutant) is demonstrated on the higher magnification images. Carbon atoms are labeled in cyan, nitrogens in blue and oxygens in red. Other side chains were omitted for clarity. Note that R424 forms a salt bridge with the E306 molecule in three subunits out of four. <b>(D)</b> Bottom views of the wild-type CNGA3 and CNGA3-R424W mutant channels. S6 is represented as a grey solid surface highlighting the partial closure of the pore in the R424W mutant model.</p