Postulated role of the 5′UTR structure in the pathogenesis of the -related CGG expansions diseases

<p><b>Copyright information:</b></p><p>Taken from "Facile FMR1 mRNA structure regulation by interruptions in CGG repeats"</p><p>Nucleic Acids Research 2005;33(2):451-463.</p><p>Published online 19 Jan 2005</p><p>PMCID:PMC548340.</p><p>© 2005, the authors © </p> Blue bars represent CGG tracts, while red rectangles symbolize AGG interruptions. The CGG region in the 5′UTR of mRNAs is shown in blue. The remaining portion of transcripts is shown in black. SDZ shows the RNA structure-dependent zone (see Discussion). Large CGG expansions (full mutations) in a vast majority of cases lead to the methylation-induced transcriptional silencing of the gene; however, translation abnormalities (bottom) were also reported ()

Structure analysis of 5′UTR fragments of the mRNA containing CGG repeats with single AGG interruption

<p><b>Copyright information:</b></p><p>Taken from "Facile FMR1 mRNA structure regulation by interruptions in CGG repeats"</p><p>Nucleic Acids Research 2005;33(2):451-463.</p><p>Published online 19 Jan 2005</p><p>PMCID:PMC548340.</p><p>© 2005, the authors © </p> () Native polyacrylamide gel electrophoresis of the 5′ labeled transcripts. Arrowheads indicate coexisting stable slow migrating (S) and fast migrating (F) conformers. The ds69 and ds107 represent dsRNA migration standards (see Materials and Methods). (–) Cleavage patterns obtained for 5′ end labeled conformers S and F of the fx4 transcript treated with: Pb(II), lanes 1 and 2 correspond to 0.25 and 1.0 mM lead acetate, respectively; S, lanes 1 and 2 correspond to 0.675 and 2.7 U/μl of S nuclease, respectively; T, lanes 1 and 2 correspond to 0.1 and 0.5 U/μl of T ribonuclease, respectively; U, lanes 1 and 2 correspond to 0.5 and 1 U/μl of U ribonuclease; lane C, incubation control; L, formamide ladder; Cl, cytosine-specific ladder; U, adenine-specific ladder. The first and the last cytosine of the CGG tract are indicated, as well as the adenine residue of the AGG interruption. The regions of the RNA molecules predicted to form single-stranded loops are shown. Electrophoresis was conducted in a 10% denaturing polyacrylamide gel. () Cleavage patterns obtained for 5′ end labeled conformers S and F of the fx5 transcript. The reaction conditions were the same as indicated in the panel B and C. () Results of the enzymatic probing of the 5′ end labeled fx6 transcript. The reaction conditions were the same as indicated in the panel B and C

Structure analysis of 5′UTR fragments of the mRNA containing CGG repeats with two AGG interruptions

<p><b>Copyright information:</b></p><p>Taken from "Facile FMR1 mRNA structure regulation by interruptions in CGG repeats"</p><p>Nucleic Acids Research 2005;33(2):451-463.</p><p>Published online 19 Jan 2005</p><p>PMCID:PMC548340.</p><p>© 2005, the authors © </p> () Cleavage patterns obtained for 5′ end labeled conformers S and F of the fx7 transcript harboring 28 CGG repeats with two AGG interruptions treated with Pb(II), S nuclease and T ribonuclease. The reaction conditions were the same as indicated in . The first and the last cytosines of the CGG tract, as well as both the adenine residues of the AGG interruptions are indicated. The regions corresponding to the loops , and are marked. Electrophoresis was conducted in a 10% denaturing polyacrylamide gel. () Cleavage patterns obtained for both the S and F conformers of the fx7 with: P, lanes 1–3 correspond to 5, 7.5 and 10 μg/ml of P nuclease, respectively; U, lanes 1 and 2 correspond to 0.5 and 1.0 U/μl of U ribonuclease, respectively. () Cleavage patterns obtained for conformer S of the fx7 transcript digested with V ribonuclease; lanes 1–3 correspond to 4.6, 9.1 and 18.2 μ/ml of V ribonuclease. () Results of the structure probing of the fx8 transcript (conformer S) with Pb(II), S nuclease and T ribonuclease. The reactions were carried out as described in the legend to the . () Proposed secondary structure models of the 5′UTR fragments containing 28 and 30 CGG repeats with two AGG interruptions. Models of the conformers S and F are shown separately for the transcript fx7. The only difference between the structure of the fx7 and fx8 transcripts is the length of the helical region 2 (h2). Thus, for clarity, only the helix h2 terminated with the loop is shown for the fx8 transcript conformer S. It is noted that the regions flanking the CGG tract in both the models, regardless of the number of repeats, adopted identical secondary structures. Specificity and intensity of the cleavages induced by S, T and U ribonuclease are indicated. Size of the symbols corresponds to the relative cleavage intensity. Shaded area represents V ribonuclease-sensitive regions. Terminal loops of the helical regions h2 (h2′), h3 (h3′) and h4 are designated as , and , respectively. Numbers indicating the trinucleotide repeats are italicized. Below, model for the temperature and low pH induced tertiary transition from the conformer S to the conformer F of the fx7 and fx8 transcripts (see Supplementary Figure 2)

Structure analysis of 5′UTR fragments of the mRNA containing uninterrupted CGG repeats

<p><b>Copyright information:</b></p><p>Taken from "Facile FMR1 mRNA structure regulation by interruptions in CGG repeats"</p><p>Nucleic Acids Research 2005;33(2):451-463.</p><p>Published online 19 Jan 2005</p><p>PMCID:PMC548340.</p><p>© 2005, the authors © </p> () Cleavage patterns obtained for 5′ end labeled fx2 transcript harboring 23 CGG repeats treated with: Pb(II), lanes 1–3 correspond to 0.25, 0.5 and 1.0 mM lead acetate, respectively; S, lanes 1–3 correspond to 0.675, 1.35 and 2.7 U/μl of S nuclease, respectively; T, lanes 1–3 correspond to 0.1, 0.3 and 0.5 U/μl of T ribonuclease, respectively; lane C, incubation control; L, formamide ladder; Cl, cytosine-specific ladder. The first and the last cytosine of the CGG tract are indicated. Electrophoresis was conducted in a 10% denaturing polyacrylamide gel. () Cleavage patterns obtained for 5′ end labeled fx3 transcript harboring 28 CGG repeats. The reaction conditions were the same as indicated in the panel A. () Cleavage patterns obtained for the terminal loops of the hairpin structures for fx1 transcript (odd number of CGG repeats) and fx3 transcript (even number of CGG repeats). Specificity and intensity of the cleavages induced by S and T nucleases are shown below. Size of the symbols corresponds to the relative cleavage intensity. () Proposed secondary structure model of the 5′UTR fragments containing 23 and 28 uninterrupted CGG repeats. It is noted that the regions flanking the CGG tract in both models, regardless of the number of repeats, adopted an identical secondary structure. The phosphodiester bonds susceptible to the lead-induced cleavages in the fx2 stem has only been shown for the 10th repeat of the hairpin for clarity; however, the same cleavage pattern was seen for all the remaining repeats of the CGG stem. The pattern of the lead ion cleavages observed in the terminal loop of fx3 is indicated. Phosphodiester bonds of the fx2 hairpin loop were very mildly cleaved by lead ions (repeats 13–15, not shown on the model). The sites of weakest cleavage induced by enzymatic probes in the CGG stem are not indicated. Numbers of the consecutive CGG repeats are italicized

Somatic instability of the expanded GAA repeats in Friedreich’s ataxia

<div><p>Friedreich’s ataxia (FRDA) is a genetic neurodegenerative disorder caused by transcriptional silencing of the <i>frataxin</i> gene (<i>FXN</i>) due to expansions of GAA repeats in intron 1. FRDA manifests with multiple symptoms, which may include ataxia, cardiomyopathy and diabetes mellitus. Expanded GAA tracts are genetically unstable, exhibiting both expansions and contractions. GAA length correlates with severity of FRDA symptoms and inversely with age of onset. Thus, tissue-specific somatic instability of long GAA repeats may be implicated in the development of symptoms and disease progression. Herein, we determined the extent of somatic instability of the GAA repeats in heart, cerebral cortex, spinal cord, cerebellar cortex, and pancreatic tissues from 15 FRDA patients. Results demonstrate differences in the lengths of the expanded GAAs among different tissues, with significantly longer GAA tracts detected in heart and pancreas than in other tissues. The expansion bias detected in heart and pancreas may contribute to disease onset and progression, making the mechanism of somatic instability an important target for therapy. Additionally, we detected significant differences in GAA tract lengths between lymphocytes and fibroblast pairs derived from 16 FRDA patients, with longer GAA tracts present in the lymphocytes. This result urges caution in direct comparisons of data obtained in these frequently used FRDA models. Furthermore, we conducted a longitudinal analysis of the GAA repeat length in lymphocytes collected over a span of 7–9 years and demonstrated progressive expansions of the GAAs with maximum gain of approximately 9 repeats per year. Continuous GAA expansions throughout the patient’s lifespan, as observed in FRDA lymphocytes, should be considered in clinical trial designs and data interpretation.</p></div

Instability analysis of the expanded GAAs in the <i>FXN</i> gene across different somatic tissues.

<p>Genomic DNA was extracted from heart (H), cerebral cortex (Cc), spinal cord (Sc), cerebellar cortex (Cb) and pancreas (P) tissues and the GAA repeats in the <i>FXN</i> locus were amplified by PCR. The results from FRDA patients (<b>A</b>) F2 (<b>B</b>) F7 and (<b>C</b>) M6 shown as examples. (-) represents no-template control and (+) represents positive control for amplification of the expanded GAAs (genomic DNA isolated from fibroblasts obtained from an unrelated FRDA patient). (<b>D-F</b>) A GAA repeat tract at the 5q23 locus was amplified by PCR using the same genomic DNA templates used for reactions shown in (<b>A-C</b>). (<b>G-I</b>) A fragment spanning intron 1—exon 2 of the <i>FXN</i> gene, downstream of the GAA tract, was also amplified using the same templates to serve as a control for genomic DNA quality.</p

GAA repeat length in fibroblasts and lymphocytes of FRDA patients.

<p>GAA repeat length in fibroblasts and lymphocytes of FRDA patients.</p

Quantitative analysis of expanded GAA repeat instability in the <i>FXN</i> gene across different tissues.

<p>The expanded GAA repeats in the <i>FXN</i> gene were amplified from genomic DNA extracted from heart (H), cerebral cortex (Cc), spinal cord (Sc), cerebellar cortex (Cb) and pancreas (P) tissues isolated from FRDA patients. The band intensity of the PCR products and repeat size are shown for FRDA patients (<b>A</b>) F2, (<b>B</b>) F7, and (<b>C</b>) M6. Solid vertical lines represent the mean of all GAA repeat sizes detected across all five tissues for each patient. Gel lanes were manually outlined and gel bands were detected via the Image Lab 5.0’s band finder set to high sensitivity. Faint bands of PCR products not detected by the software were manually identified. Band boundaries, accounting for smearing, were automatically outlined by the program with final manual adjustments to include the entire spectrum of PCR products. Multiple PCR analyses with determinations of GAA lengths were performed to demonstrate reproducibility of PCR and reliability of measurements (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189990#pone.0189990.s004" target="_blank">S4 Fig</a>).</p

Determination of GAA repeat length in paired FRDA patient fibroblast (F) and lymphocyte (L) samples.

<p>(<b>A</b>) Agarose gel analysis of GAA repeat sizes in fibroblast/lymphocyte samples isolated from the same individual (FRDA patients FA1—FA16). (<b>B</b>) The mean sizes of the GAA1 and GAA2 alleles between all fibroblast and lymphocyte samples (n = 16) were calculated and compared. A p-value <0.05 denotes a significantly significant difference. (<b>C</b>, <b>D</b>) Correlation between the number of GAA repeats expanded in lymphocytes relative to size of the repeat tracts in fibroblasts and the number of GAA repeats in lymphocytes. The difference between the expanded GAA repeat lengths observed in lymphocyte and fibroblast samples (ΔGAA) was plotted against lymphocyte GAA sizes for each of the 16 FRDA paired samples. The analysis was performed for both alleles (<b>C</b>) GAA1 and (<b>D</b>) GAA2. The Pearson’s correlation coefficient (R) is indicated.</p

Assessing time-dependent changes of GAA tract length in FRDA lymphocytes.

<p>(<b>A</b>) The GAA repeat tract at the <i>FXN</i> locus was amplified using genomic DNA extracted from lymphocytes, which were isolated from 5 patients (F10, F11, F12, M7 and F13). Blood samples were taken at an initial timepoint (I) and a second time point 7–9 years after the initial sampling (II) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189990#pone.0189990.t003" target="_blank">Table 3</a>). The time-dependent changes in GAA repeat length were quantitated as (<b>B</b>) total GAA repeat gain/loss (ΔGAA) and (<b>C</b>) rate of the change (ΔGAA per year). (<b>D</b>) The Pearson’s correlation coefficient was calculated using the size of the GAA tract at time point I [GAA(I)] and the change of the number of GAAs between time points II and I [ΔGAA (II-I)]. Five pairs of samples (n = 10 alleles) were analyzed.</p