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* This work was supported in part by National Institutes of Health Grants NS34192 (to M. P.) and GM52982 (to R. D. W.) and grants from the Muscular Dystrophy Association, and Medical Research Council of Canada (to M. P.), and the Robert A. Welch Foundation (to R. D. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Recipient of a fellowship from the Human Frontier Science Program. ¶ Recipient of a fellowship from the Medical Research Council of Canada.
Friedreich ataxia (FRDA) is associated with the expansion of a GAA·TTC triplet repeat in the first intron of the frataxin gene, resulting in reduced levels of frataxin mRNA and protein. To investigate the mechanisms by which the intronic expansion produces its effect, GAA·TTC repeats of various lengths (9 to 270 triplets) were cloned in both orientations in the intron of a reporter gene. Plasmids containing these repeats were transiently transfected into COS-7 cells. A length- and orientation-dependent inhibition of reporter gene expression was observed. RNase protection and Northern blot analyses showed very low levels of mature mRNA when longer GAA repeats were transcribed, with no accumulation of primary transcript. Replication of plasmids carrying long GAA·TTC tracts (∼250 triplets) was greatly inhibited in COS-7 cells compared with plasmids carrying (GAA·TTC)9 and (GAA·TTC)90. Replication inhibition was five times greater for the plasmid whose transcript contains (GAA)230than for the plasmid whose transcript contains (UUC)270. Our in vivo investigation revealed that expanded GAA·TTC repeats from intron I of the FRDA gene inhibit transcription rather than post-transcriptional RNA processing and also interfere with replication. The molecular basis for these effects may be the formation of non-B DNA structures.
), a GAA·TTC repeat in the first intron of the frataxin gene. The GAA·TTC expansion accounts for about 98% of all FRDA chromosomes, with the remaining ones carrying frataxin point mutations. The recessive inheritance, nature, and intronic localization of the expanded sequence make FRDA an unique case in TRS-related diseases (
). However, the FRDA expanded GAA·TTC repeats show meiotic and mitotic instability as for other disease-associated TRS. In FRDA chromosomes, GAA·TTC repeat units vary from about 100 to more than 1,000 whereas less than 37 repeat units are found in normal chromosomes (
). Together with the identification of frataxin point mutations resulting in a defective or truncated protein, this finding defines FRDA as a frataxin deficiency disease, in accordance with its recessive inheritance. The sizes of the GAA·TTC repeats carried by each patient correlate with the age of onset and the severity of the disease, particularly for the smaller one (
The GAA·TTC tract is a polypurine·polypyrimidine (Pur·Pyr) sequence, which may form an intramolecular triple helix in vitro under appropriate conditions of pH, metal ions concentrations, and supercoiling (
) demonstrated that plasmids containing 38, 58, and 103 GAA·TTC triplets, but not 16 triplets, showed supercoil-induced relaxations when examined by two-dimensional-agarose gel electrophoresis, even at pH 8.3, suggesting that they may adopt a triple helical structure in vivo. Such structures inhibit gene expression by blocking the progression of RNA polymerase, as shown to occur for Pur·Pyr tracts both in vitro (
). Pur·Pyr sequences, including GAA·TTC tracts, can also interfere with DNA replication, since they have been shown to stall DNA polymerase in vitro, probably again as a consequence of intramolecular triplex formation (
), we constructed new recombinant plasmids containing from 9 to 500 GAA·TTC triplets along with some frataxin gene-derived flanking sequence. These data evidence that such TRS inhibit transcription and possibly DNA replicationin vivo.
The level of frataxin mRNA and protein is very low in tissue samples and cultured cells from FRDA individuals carrying intronic GAA·TTC expansions in both homologs of the frataxin gene (
). By using a simple in vivo model, we showed that an expanded intronic GAA·TTC TRS can suppress gene expression in a length- and orientation-dependent manner, consistent with the observations in FRDA. Inhibition of transcription seems to be the most likely mechanism. In addition, we obtained new information about factors affecting GAA·TTC repeat stability, along with evidence suggesting that this sequence may interfere with DNA replication.
To perform this study, we utilized cloned GAA·TTC repeats originally amplified from the first intron of the frataxin gene along with some flanking sequence. Previously cloned GAA·TTC TRS had been obtained using synthetic oligonucleotides (
). Contractions are much more common than expansions. The initial cloning of GAA·TTC repeats from polymerase chain reaction products also indicated that this TRS shows intrinsic instability in E. coli, which was exploited to clone repeat sequences ranging from 9 to 500 GAA·TTC triplets by using the in vivo expansion-contraction method (
), and this was shown to be the case, at least for the repeats amenable to sequence analysis (up to about 60 triplets).
Several factors are known to influence the stability of TRS, including length, presence of interruptions, characteristics of the vector, orientation relative to the unidirectional replication origin, and genetic backgrounds of host cells (
). The current study shows that the instability of GAA·TTC repeats was greater when GAA was the lagging strand template than when it was the leading strand template. According to the previously proposed model for CTG·CAG (
), this differential instability may be due to the ability of single-stranded GAA to adopt a more stable DNA secondary structure during replication than single-stranded TTC. The nature of such structure for the FRDA sequence remains undetermined and may differ from a hairpin, since the structures that single-stranded GAA and TTC can adopt were reported to be much less stable than those formed by single-stranded CTG, CAG, CCG, and CGG (
The stability of long GAA·TTC sequences in E. coli was also strongly influenced by the cloning vector. Long GAA·TTC repeats were much more stable when cloned into pSPL3 rather than into pUC vectors. Transcriptional activity may be involved, because CTG·CAG repeats have been shown to be more unstable when transcribed, both inE. coli (
). In pSPL3-based plasmids, the GAA·TTC repeats are located in the intronic region and were transcribed under the control of the SV40 early promoter. No transcription occurs when these plasmids are propagated in E. coli. Conversely, the GAA·TTC repeats are localized within a transcription unit in pUC vectors, possibly accounting for the observed greater instability.
We analyzed the effect of intronic GAA·TTC repeats on gene expression by transfecting COS-7 cells with constructs harboring GAA·TTC repeats of different lengths and orientations in an intron of a reporter gene. When (GAA)n was in the transcripts, as is the case in the frataxin gene, transcription and expression of the reporter gene were reduced proportionally to the repeat length. Repeats containing more than 33 triplets, close to the upper limit for normal alleles of the frataxin TRS (
), started to inhibit gene expression. No increase in unspliced or partially spliced transcript was observed, suggesting that a defect in RNA splicing caused by the expanded GAA·TTC repeat, proposed as a cause of reduced frataxin gene expression in FRDA (
), is unlikely. Along with the observation that transcription initiation is probably not affected, as suggested by RNase protection experiments, the occurrence of a transcriptional block at the repeat seems to be the most likely explanation for reduced gene expression. According to our observations, such a block is orientation-dependent, occurring only with transcription of GAA-containing RNA. Such purine-specific inhibition is in agreement with previous in vitro studies of Pur·Pyr sequences (
) proposed instead that purine-rich RNA may bind to the single pyrimidine-rich DNA strand generated by the formation of an intramolecular DNA triplex, resulting in its stabilization. According to this model, a wave of negative supercoiling following transcription (Fig. 5C) would trigger intramolecular DNA triplex formation. We propose that the GAA-rich transcript may bind the duplex DNA template, as in the previously proposed models (Fig. 5,B and C), interfering with RNA elongation and preventing further transcription.
Because TRS may be unstable when propagated in eukaryotic cells, including yeast (
), we considered the possibility that GAA·TTC repeats might contract or expand during replication in COS-7 cells. Transcriptional activity would then be affected by the resulting heterogeneity of repeat lengths. This does not appear to be the case, as GAA·TTC repeats recovered from transfected COS-7 cells were quite stable. However, TRS are known to interfere with DNA replication, and we indeed observed that plasmids containing long GAA·TTC repeats replicate at very low efficiency in COS-7 cells. Inhibition of in vitro DNA polymerization is known to occur for certain lengths of CTG·CAG, CCG·CGG, GTC·GAC, GAA·TTC, and GGA·TCC which is believed to be related to the formation of unusual DNA structures, including tetraplexes and triplexes (
). However, intramolecular DNA triplex formation does not explain why pRW3827, whose transcript contains (GAA)230, was five times less efficient in replication than pRW3823, whose transcript contains (UUC)270. To account for this effect, we speculate that binding of the GAA-rich transcript to the DNA template may further inhibit DNA polymerization and formation of such a complex structure may be influenced by repeat length. Although the replication efficiency of plasmids containing (GAA·TTC)90, in both orientations, was similar to plasmids containing (GAA·TTC)9, their transcription efficiency was reduced to about a half of (GAA·TTC)9. This differential effect of repeat length on transcriptionversus replication may be due to a different stability of the secondary structures in these processes. It should be noted that the initiation level of transcription was not correlated with the amount of DNA template. This may be due to saturation of transcription machinery in COS cells even with the lower level of DNA template, as previously suggested (
Our analysis of the effect of intronic GAA·TTC expansions on transcription and replication provides an initial understanding of the molecular mechanisms underlying the loss of function in FRDA. Some of these mechanisms may be common to other long tracts of intronic Pur·Pyr sequences, including GAAGGA·TCCTTC repeats in the human tumor necrosis factor receptor p75 gene (
We thank Drs. Albino Bacolla, Pawel Parniewski, and Margaret Labuda, and Sarn Jiralerspong for helpful suggestions. We also thank Dr. Jacques Drouin and Gino Poulin for use and assistance of the luminometer, and Dr. Pierre Chartrand for use of the electroporator.