CGG-repeat length threshold for FMR1 RNA pathogenesis in a cellular model for FXTAS - PubMed (original) (raw)

. 2011 Jun 1;20(11):2161-70.

doi: 10.1093/hmg/ddr101. Epub 2011 Mar 9.

Affiliations

CGG-repeat length threshold for FMR1 RNA pathogenesis in a cellular model for FXTAS

Gry Hoem et al. Hum Mol Genet. 2011.

Abstract

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder that affects carriers of premutation alleles (55-200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene. The presence of elevated levels of expanded mRNA found in premutation carriers is believed to be the basis for the pathogenesis in FXTAS, but the exact mechanisms by which the mRNA causes toxicity are not known. In particular, it is not clear whether there is a threshold for a CGG-repeat number below which no cellular dysregulation occurs, or whether toxicity depends on mRNA concentration. We have developed a doxycycline-inducible episomal system that allows us to study separately the effects of CGG-repeat number and mRNA concentration (at fixed CGG-repeat length) in neuroblastoma-derived SK cells. Our findings show that there is a CGG-repeat size threshold for toxicity that lies between 62 and 95 CGG repeats. Interestingly, for repeat sizes of 95 CGG and above, there is a clear negative correlation between mRNA concentration and cell viability. Taken together, our results provide evidence for an RNA-toxicity model with primary dependence on CGG-repeat size and secondary dependence on mRNA concentration, thus formally ruling out any simple titration model that operates in the absence of either protein-binding cooperativity or some form of length-dependent RNA structural transition.

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Figures

Figure 1.

Figure 1.

The episomal system used in the current study. (A) A set of episomal plasmids (pcep4-TreT-nCGG-eGFP) were constructed to express FMR1 5′ UTRs harboring various CGG repeats under the control of the

T

et-

R

esponsive promoter

E

lement (TRE). Downstream of the FMR1 5′ UTR is a chimeric intron and the eGFP gene. The episomes also contain the Epstein–Barr virus OriP, and express the EBNA1 gene to allow for stable maintenance and replication in mammalian cells. (B) The CGG repeats are stable in mammalian (SK) cells. The triplet repeats from each cell line were amplified by PCR (53) after 5–12 passages and were sized relative to a control (C) marker set (Bionexus Hi-Lo DNA Marker, Oakland, CA, USA).

Figure 2.

Figure 2.

Scatter plots showing the DOX-dependent increase in episomally transcribed (CGG)n-GFP mRNA levels for cells containing episomes with 30, 42, 54, 62 and 95 CGG repeats as indicated. The arrow on the y-axis indicates the endogenous level of FMR1 mRNA expressed by SK cells. In this and subsequent figures, error bars correspond to SEMs. Open symbols indicate leakage CGG-GFP mRNA expression in the absence of DOX; filled symbols indicate expression of CGG-GFP mRNA induced by DOX (range 0.003 to 0.3 μg/ml).

Figure 3.

Figure 3.

Increase in the percent of non-viable (PI-positive) cells as a function of the concentration of (nCGG)-eGFP reporter mRNA at 24 h post-DOX induction, and corresponding to DOX concentrations of 0, 0.003, 0.01, 0.03, 0.1 and 0.3 μg/ml. (A) Comparison of 95- (black circles) and 54-CGG-repeat (open circles) alleles. (B) Comparison of 30 (open circles), 42 (gray circles) and 62 (black circles) CGG-repeat alleles. Control reactions for cells possessing pcep4-TreT-D-eGFP mRNA, which does not contain the FMR1 sequence, were run in parallel as calibrators for relative mRNA quantification. eGFP/GUS mRNA ratios were calculated for all samples as a secondary fiducial as described by Tassone et al. (14). Relative RNA concentrations represent the ratio: (nCGG)–eGFP/D-eGFP RNAs.

Figure 4.

Figure 4.

Altered lamin A/C nuclear architecture in cells expressing an expanded CGG-repeat element. (A) Lamin A/C architecture in cells harboring 30 or 95 CGG repeats following 72 h of DOX treatment (DOX; 0.3 µg/ml) or in the absence of DOX (noDOX); in descending order: lamin A/C, 4′,6-diamidino-2-phenylindole (DAPI), merged image, respectively. (B) Example of a lamin A/C-positive intranuclear inclusion following 72 h induction of 95 CGGs in the presence of DOX (0.3 µg/ml). (C) Ratio of lamin A/C rings, DOX (0.3 µg/ml) versus no DOX as a function of the number of CGG repeats in SK cells. All staining experiments used rabbit anti-lamin A/C primary and Alexa 555 goat anti-rabbit (see Materials and Methods).

Figure 5.

Figure 5.

(A) The presence of γH2AX in the intranuclear inclusions in neurons of the cerebral cortex from post-mortem brain tissue of FXTAS patients. (B) Sequence coverage of H2AX by mass spectrometry (17). Amino acid sequences from peptides in isolated inclusions are underlined; asterisk indicates position of indeterminate amino acid. (C) Relative γH2AX levels by western blot analysis for SK cells expressing 95 (filled circles), 62 (gray circles) and 54 (open circles) CGG repeats 24 h post-DOX induction with varying concentrations of DOX, plotted as a function of the levels of eGFP reporter RNA (relative RNA levels calculated as in Fig. 3). All membranes were probed with β-tubulin antibody as a loading control, and the protein level is reported as the ratio of γH2AX to β-tubulin.

Figure 6.

Figure 6.

γH2AX activation is a late event relative to the rate of production of eGFP mRNA for the 95-CGG-repeat allele (filled squares) under conditions where there is no significant reduction in cell viability (0.0125 µg/ml); there is no significant increase in γH2AX with time for smaller CGG repeats (e.g. 30 CGG repeats; open squares). (A) Time dependence of increase in eGFP mRNA concentration (filled circles, 95 CGG repeats; open circles, 30 CGG repeats); (B) time dependence of γH2AX activation (filled squares; 95 CGG repeats, open squares; 30 CGG repeats).

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