6 research outputs found
CGG repeat expansions in the FMR1 gene inhibit translation of Venus-ARC RNA in human fibroblasts.
<p>Panel A—Human fibroblasts from control individuals C0603 (control male, 31 repeats), GM00497 (control male, unknown repeat number), GM00498 (control male, unknown repeat number) and FMR1 premutation carriers FX08-2 (female, 31, 105 repeats), FX11-2 (female, 20, 79 repeats), FX13-2 (female, 33, 85 repeats), and WC26 (female, two premutation alleles, 60, 90 repeats) were microinjected with Venus-ARC RNA and after 2 hours Venus-ARC RNA and newly-synthesized Venus-ARC protein were imaged by dual channel confocal microscopy. Representative images are shown for untreated control and premutation cells and for premutation cells treated with TMPyP4. Scale bars indicate 5 micrometers. Panel B—Cumulative Kolmogorov Smirnov plots for specific translational activities (newly-synthesized Venus-ARC protein/microinjected Venus-ARC RNA) in individual cells for 3 control and 4 premutation fibroblast cell lines as described for Panel A, untreated (top) and treated with TMPyP4 (bottom). Panel C—Frequency distribution plots for specific translational activities (newly-synthesized Venus-ARC protein/microinjected Venus-ARC RNA) in cells for 3 control and 4 premutation fibroblast cell lines as described for Panel B, untreated (top) and treated with TMPyP4 (bottom). Panel D—PCR analysis of CGG repeat numbers in FX08-02, FX11-02, FX13-02 and C0603 fibroblasts with a 100 bp DNA ladder (left panel) and in WC26 and C0603 fibroblasts with a 1 kb ladder (right panel). In the panel on the left, in the FX08-2, FX11-2, and FX13-2 lanes, the band at the bottom of the gel represents female gender specific PCR product, the bands immediately above the gender specific band represent PCR products from CGG repeat alleles in the normal range and the bands above represent PCR products from expanded CGG repeat alleles. The fainter products near the top of the gel are of unknown origin. In the C0603 lane, the two bands near the bottom of the gel represent male and female gender specific PCR products, and the band above the gender specific bands represents PCR product from the normal CGG repeat allele. In the panel on right, the two bands in the WC26 lane both represent PCR products from expanded CGG repeat alleles and the single band in the C0603 lane represents PCR product from the CGG repeat allele in the normal range. Panel E—Table showing ID, gender, FMR1 alleles and CGG repeat numbers (based on panel D) for each cell line. Panel F—Western blotting of FMRP expression in full mutation (FX08-1, FX11-01, FX13-01) and premutation (FX08-2, FX11-02 and FX13-02) cell lines with actin loading controls.</p
CGG repeat expansions in the FMR1 gene disrupt regulation of calcium transients in human fibroblasts.
<p>Control (C0603) and premutation (FX08, 31,105 repeats) fibroblasts were loaded with fluorescent calcium indicator Fluo-4 and incubated with bradykinin to induce calcium transients. Cells were imaged by time-lapse confocal microscopy to visualize changes in intracellular calcium concentrations over time. Total fluorescence intensity over the entire cell was integrated in each time frame for 20 control and 20 premutation cells, in the absence or presence of TMPyP4. F/F<sub>0</sub> values for each cell are plotted, with initiation of primary calcium transients aligned at time = 0 s.</p
FCS photobleaching of ARC RNA and CGG 99 RNA molecules in individual granules in hippocampal neurons.
<p>Panel A—the FCS observation volume was positioned to encompass a single individual immobile granule containing differentially labeled fluorescent ARC RNA and CGG99 RNA molecules in a hippocampal neuron. Continuous illumination during the FCS measurement results in photobleaching of fluorescent RNA molecules of each type in the granule, which is recorded as count rate decay in each FCS channel. The numbers of fluorescent RNA molecules of each type in the granule are determined by dividing the total decay in counts during photobleaching by the counts per molecule for each RNA determined by FCS in solution. Panel B shows a scatter plot for numbers of ARC RNA and CGG99 RNA molecules in individual granules in hippocampal neurons in the absence of TMPyP4. Panel C shows a scatter plot for numbers of ARC RNA and CGG99 RNA molecules in individual granules in hippocampal neurons in the presence of TMPyP4. Panel D shows Kolmogorov-Smirnov plots of the ratios of CGG99 RNA molecules to ARC RNA molecules in individual granules in the absence (black symbols) and presence (red symbols) of TMPyP4.</p
CGG repeat RNA and ARC RNA are co-localized in granules and CGG repeat RNA inhibits translation of ARC RNA in hippocampal neurons.
<p>Panel A shows a schematic outline of the experimental protocol. Differentially labeled CGG repeat RNA and Venus-ARC RNA were co-injected into the cytoplasm of hippocampal neurons where they are localized in granules in dendrites. Venus-ARC RNA is translated in granules near dendritic spines. In some cases hippocampal neurons were incubated with membrane-permeant TMPyP4 before injecting RNA. Panel B shows the distribution of Venus-ARC RNA (red), CGG99 RNA (blue) and newly-synthesized Venus-ARC protein (green) in discrete granules in a three channel confocal images of a dendritic segment. Seven granules are visible in this dendritic segment, all of which contain Venus-ARC RNA, two of which also contain CGG99 RNA, and five of which lack detectable CGG99 RNA. Newly synthesized Venus-ARC protein is detected in the five granules lacking CGG99 RNA but not in the two granules containing CGG99 RNA. Scale bar indicates 1 micrometer. Panel C shows 3-dimensional graphs of fluorescence intensities for Venus-ARC RNA, Venus-ARC protein and CGG99 (right panel) or CGG0 (left panel) RNAs in a population of individual granules in hippocampal neurons. Panel D shows Kolmogorov-Smirnov cumulative frequency plots of specific translational activities (Venus-ARC protein/Venus-ARC RNA) for granules containing detectable (defined as integrated fluorescence intensity > 10 arbitrary units) CGG99 RNA (red), or in granules where CGG99 RNA was undetectable (defined as integrated fluorescence intensity < 10 arbitrary units) (black),. Specific translational activities were measured for granules in untreated (left panel) or in TMPyP4-treated (right panel) hippocampal neurons.</p
CGG repeat profiles and TMPyP4 binding for FMR1, CGG0, CGG30, CGG62 and CGG99 RNAs.
<p>Panels A-D—CGG repeat profiles for RNAs derived from the 5’UTR of FMR1 RNAs containing CGG 0, 30, 62, 99 were calculated using a sliding sequence algorithm (described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168204#sec002" target="_blank">Materials Methods</a>). Values of 1 in the profile indicate four consecutive CGG repeats in a row. Panel E—Binding of each CGG repeat RNA to serial dilutions of TMPyP4 was analyzed by SPR. Panel E shows representative SPR sensorgrams for binding of TMPyP4 to CGG 0, 30, 62 and 99 RNAs. Panel F—On-rates and off-rates determined by fitting SPR sensorgrams for each RNA to a heterogeneous ligand binding model. Apparent <i>K</i><sub><i>D</i></sub> values were calculated by dividing off rates by on rates NSp indicates non-specific binding, Sp indicates CGG-specific binding for each RNA.</p
CGG repeat profile and ribosome profile for FMR1 RNA.
<p>Panel A shows the CGG profile for exon 1 of FMR1 RNA, including the 5’UTR (blue) and the initial portion the ORF (red), calculated as described in Materials and Methods. There is a region of CGG repeats between positions 100–150, interrupted by a single AGG at position 131. Panel B shows the corresponding ribosome profile for exon 1 of FMR1 RNA. The number of sequence reads at each position reflects the probability of ribosomes located at that position. Increased ribosome probability downstream of position 200 presumably reflects ribosomes engaged in conventional translation of the FMRP ORF beginning at the AUG at position 230. Increased ribosome probability in upstream regions (1–100 and 150–200) may reflect ribosomes initiating translation at non-canonical AUG-like sites upstream of the CGG repeats or ribosomes engaged in RAN translation of 5’UTR CGG repeats, consistent with reports that translation of CGG repeats in FMR1 RNA may require non-canonical AUG-like sequences upstream of the CGG repeats [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168204#pone.0168204.ref011" target="_blank">11</a>]. The paucity of reads in the region between positions 100–150, which corresponds to the CGG repeat region, may reflect inefficient sequencing through long regions of CGG repeats [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168204#pone.0168204.ref024" target="_blank">24</a>]. Panel C shows the ribosome profile for the entire FMR1 gene (red) and the locations of individual exons (blue), with 5’ and 3’UTR regions indicated by thinner lines and ORF regions indicated by thicker lines. The exon 1 region shown in panels A and B is indicated.</p