44 research outputs found
MOESM1 of Ribosome profiling of the retrovirus murine leukemia virus
Additional file 1: Table S1. Library composition statistics. Figure S1. RPF distributions on host mRNAs. Figure S2. Length distributions of reads mapping to viral and host RNA. Figure S3. Alignment of the 5′ regions of gammaretroviral genomic sequences. Figure S4. Codon-based alignment of gammaretroviral pol genes downstream of the env splice acceptor
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 polyA<sub>29</sub> 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<sub>n</sub> length in the insertion and slippage at polyA<sub>n</sub> 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<sub>n</sub> length was confirmed in DNA from different <em>RPGRIP1</em><sup>−/−</sup> dogs, yet polyA<sub>n</sub> variability did not correspond with the clinical phenotypes and no individual was found that carried a polyA<sub>n</sub> 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
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><sup>+/+</sup> (blue) and <i>RPGRIP1</i><sup>−L/−L</sup> (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
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><sup>−/−</sup> dogs, with the common PCR fragment pattern peaking at ‘114’ (A<sub>29</sub> 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 (A<sub>28</sub> 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 polyA<sub>28</sub> allele was determined by cloning from genomic DNA (*) or PCR-fragment sizing (**).</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<sub>28, 29 and 30</sub>), 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 A<sub>29</sub>, A<sub>35</sub>, A<sub>38</sub> and A<sub>41</sub>, 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, A<sub>30</sub>, A<sub>36</sub>, A<sub>39</sub> and A<sub>42</sub> 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
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><sup>−/−</sup> 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><sup>−/−</sup> dogs examined including both clinically affected and normal dogs showed the ‘114’ pattern. Direct cloning and haplotype analysis confirmed MLD6 as heterozygous for polyA<sub>28/29</sub> while the ‘114’ pattern corresponded to polyA<sub>29/29</sub>.</p
Expression of <i>fluc</i> downstream of polyA tracts.
<p>The firefly to <i>Renilla</i> luciferase expression ratio in p2luc/A<sub>21, 25, 27–30, 35, 38–43</sub> 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<sub>3n+1, 3n−1 and 3n</sub> 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, polyA<sub>28</sub>) and from a <i>cord1</i>-affected MLHD from the original AHT colony (b: polyA<sub>29</sub>).</p
Minimal sequence requirements for MNV termination-reinitiation.
<p>A) Schematic of the p2luc-MNV reporter mRNA. The termination-reinitiation region (203 nt upstream and 52 nt downstream of the <b>UA<u>A</u></b><u>UG</u> motif) was cloned into the <i>SalI</i> and <i>BamHI</i> sites of the p2luc reporter plasmid. <i>HpaI</i> run-off transcripts for <i>in vitro</i> translation were generated using T7 RNA polymerase. The location of the T3 promoter present in the structure mapping construct p2luc-MNV-T3 is indicated. B) Deletion analysis of MNV termination-reinitiation. A series of p2luc-MNV variants were prepared with stepwise, in-frame deletions from the 5′ end of the inserted viral sequence. The wild-type (wt), premature stop (ps) and deletion mutant plasmids were linearised with <i>HpaI</i> and run-off transcripts translated in Flexi® RRL at a final RNA concentration of 50 µg/ml in the presence of [<sup>35</sup>S]-methionine and 140 mM added KCl. The products were resolved by 12% SDS-PAGE and visualised by autoradiography. The number of nucleotides of viral sequence remaining up to the AUG start codon of the MNV ORF is shown below the gel. The product of the full-length or truncated versions of the rlucVP1 ORF (predicted size of MNVwt is 42 kDa) is marked rluc, and the VP2fluc product (predicted size, 62 kDa) is marked fluc. The MNV ps rluc product is the shortest (predicted size, 33 kDa). RRF denotes the relative reinitiation frequency in comparison to MNVwt (set at 100). The figure in brackets represents the ratio of the intensity of the fluc and rluc products (adjusted for methionine content and expressed as a percentage) for the MNVwt mRNA.</p