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J Biol Chem, Vol. 273, Issue 23, 14588-14595, June 5, 1998
From the 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)230
than 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.
Friedreich ataxia
(FRDA)1 is the first
autosomal recessive neurodegenerative disease found to be caused by the
hyperexpansion of a triplet repeat sequence (TRS) (1), 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 (2-4). 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 (1, 5, 6). FRDA patients
carrying two expanded GAA·TTC repeats show very low levels of mature
frataxin transcript (1, 7, 8) and of frataxin (9), indicating
suppressed gene expression. Such a defect may be caused either by
reduced transcription or by abnormal post-transcriptional processing
(1, 9, 10). 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 (5). In addition, an inverse
correlation between the length of the smaller GAA·TTC repeat and the
residual amount of frataxin was observed in cultured cells from FRDA
patients (9).
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 (4, 11). Increasing the length of the
Pur·Pyr tract also promotes triplex formation (12). Ohshima et
al. (13) 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 (14-17) and
in vivo (18-20). However, GAA-containing RNA may also adopt
a secondary structure interfering with post-transcriptional processing
(21). 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 (11, 13).
We used cloned GAA·TTC repeats to investigate the possible effect of
this intronic sequence on gene expression in vivo. The cloned GAA·TTC tracts previously used by Ohshima et al.
(13) contain interruptions and no FRDA-derived flanking sequence.
Considering the effects of interruptions and of flanking sequences on
the biological properties of TRS (3, 4, 6, 22-28), 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 replication
in vivo.
Plasmid Construction--
Genomic DNA from a patient carrying
~700 GAA·TTC repeats was amplified by polymerase chain
reaction (1) using primers Not-Bam (5'-GGAGGGAACCGTCTGGGCAAAGG-3') and
2500-F (5'-CAATCCAGGACAGTCAGGGCTT-3') and then digested with
BamHI and BglII. The fragment containing (GAA·TTC)~700 along with flanking sequences (352 bp 5'
and 250 bp 3' to the TRS) was purified by a 1.2% agarose gel (13) and
ligated into the BamHI site of pUC19. The ligation mixture was transformed into Escherichia coli SURE
(Stratagene) by electroporation. The resultant recombinant
plasmid contained 110 GAA·TTC triplets in orientation II (Fig.
1A). The SacI-HindIII digest was
recloned into the SacI-HindIII site of
pUC18NotI and pUC19NotI (29) to give plasmids
containing (GAA·TTC)70 and (GAA·TTC)65 in
two different orientations, designating pRW3804 (orientation I) and
pRW3803 (orientation II), respectively. To generate longer repeats, the in vivo expansion method was performed as described
previously (13, 29). Briefly, after pRW3804 was grown in E. coli DH10B (Life Technologies) or SURE, the
SacI-HindIII digest was loaded on an agarose gel
and regions above 70 repeats were eluted and ligated into
pUC18NotI. The procedure was repeated several times, obtaining cloned GAA·TTC repeats ranging from 70 to 360 triplets.
Inhibitory Effects of Expanded GAA·TTC Triplet Repeats from
Intron I of the Friedreich Ataxia Gene on Transcription and Replication
in Vivo*
§,¶,
, and**§§
Centre de Recherche Louis-Charles Simard,
C.H.U.M., Campus Notre-Dame, 1560 rue Sherbrooke est, Montréal,
Québec H2L 4M1, Canada, the Institute of Biosciences and
Technology, Center for Genome Research, Department of Biochemistry and
Biophysics, Texas A&M University, Texas Medical Center, Houston, Texas
77030-3303, the ** Department of Medicine, Université de
Montréal, Montréal, Québec H2L 4M1, Canada, and the
Departments of Neurology and Neurosurgery,
McGill University, Montréal, Québec H3A 2B4, Canada
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
-Actin-125-Human
(Ambion), which contains a 127-bp cDNA fragment of the human
-actin gene (820-946), was cloned into the
EcoRI-KpnI site of pGEM-3Zf(
) (Promega) to
produce pMP125.
For the construction of pMP129, the HindIII-EcoRI
fragment of pIND/lacZ (Invitrogen) containing the
-galactosidase gene was filled in the overhangs with the Klenow
fragment of E. coli DNA polymerase I (New England Biolabs)
and dNTPs, and cloned into the MspI site of pSPL3.
pMP129-based plasmids containing (GAA·TTC)n were constructed
by digesting the pSPL3-based plasmids with either ApaI-PstI or HindIII and cloning the
released inserts into the ApaI-PstI or
HindIII sites of pMP129, respectively.
Luciferase gene fragments (661 and 1340 bp), obtained by digesting
pGL3-Control (Promega) with EcoNI or HindIII and
HincII followed by filling in the overhangs, were cloned
into the EcoRV site of pSPL3 in the antisense orientation to
give pMP175 and pMP177, respectively. pMP183 was constructed by
inserting the HindIII digest of pMP177 into the
HindIII site of pMP129.
For plasmid preparations, the recombinant plasmid DNA was transformed
into E. coli SURE by electroporation and the transformant was grown in 1 liter or 100 ml of LB with 75 µg/ml ampicillin at
37 °C until the end of logarithmic phase (OD600 = ~0.9). Plasmids were isolated by the alkali lysis method (30) and
purified by CsCl density gradient centrifugation or the QIAprep Plasmid
Kits (QIAGEN). Isolated plasmids were digested with appropriate
restriction enzymes. The digest was analyzed on an agarose gel, stained
with ethidium bromide, and photographed. The inserts in all of the recombinant plasmids were characterized by DNA sequencing on both strands to determine the repeat units. For plasmids containing more
than 59 GAA·TTC repeats, the triplet repeat units were estimated from
agarose gels and DNA sequencing to ±5 triplets.
Cell Culture and Transient Transfection for RNA Analyses-- COS-7 cells were grown in Dulbecco's modified Eagle's medium (ICN Pharmaceuticals) containing 50 units/ml penicillin-streptomycin and supplemented with 2 mM glutamine and 10% fetal bovine serum (ICN Pharmaceuticals). Cultures were maintained at 37 °C in a 5% CO2 atmosphere. COS-7 cells were plated on 60-mm diameter plastic dishes at a density of 4 × 105 cells/dish. After 24 h, the cells achieved 40-70% confluence and were transfected with 1 µg of pSPL3-based plasmid using cationic liposomes (Lipofectace, Life Technologies) (7 µg), according to the manufacturer's protocol. 48 h after transfection, total RNA was isolated by the TRIzol method (Life Technologies): 800 µl of TRIzol and 200 µl of chloroform were added to the cells and the nucleic acids were subsequently recovered by precipitation with 2/3 volume of isopropyl alcohol.
RNase Protection Assays--
pMP106 and pMP125 were used as a
template to make antisense RNA probes, R-MP106 and R-MP125, by in
vitro transcription from the T7 and SP6 promoters, respectively,
incorporating [
-32P]UTP (800 Ci/mmol, Amersham) with
the MAXIscript kit (Ambion). The reaction mixtures were loaded on a 5%
polyacrylamide electrophoresis gel containing 8 M urea, and
the radiolabeled RNA products were excised and eluted according to the
manufacturer's protocol. RNase protection assays were performed using
the RPAII kit (Ambion): 1 µg of total RNA was hybridized to 1.2 × 105 cpm and 2.2 × 104 cpm RNA probes
synthesized from pMP106 and pMP125, respectively, at 45 °C for
16 h in 20 µl of the hybridization buffer containing 80%
deionized formamide, 100 mM sodium citrate (pH 6.4), 300 mM sodium acetate (pH 6.4), and 1 mM EDTA. The
samples were digested with a mixture of RNase A (0.5 units) and RNase
T1 (20 units) in 200 µl of digestion buffer for 30 min at 37 °C.
RNA was precipitated with ethanol and dissolved in 8 µl of gel
loading buffer. Samples were fractionated on a 8% denaturing
polyacrylamide gel containing 8 M urea. After exposure of
the dried gel to x-ray film at 80 °C for 48 h, the amounts of
protected products was estimated using the AlphaImager version 3.0 (Alpha Innotech).
Northern Blot Analysis--
Antisense probes R-MP107 and R-MP125
were synthesized by in vitro transcription using T7 and SP6
RNA polymerases from pMP107 and pMP125, respectively, as described
above. 1 µg of total RNA was size-fractionated on a 1.0%
agarose-formaldehyde gel (Ambion) and transferred to
Hybond-N+ nylon membrane (Amersham) using the
NorthernMax kit (Ambion). After UV cross-linking, blots were hybridized
to either probe R-MP107 or R-MP125 at 65 °C for 16 h. The
membrane was exposed to x-ray film at 80 °C for 24 h and 7 days for probes R-MP125 and R-MP107, respectively.
Reporter Gene Expression Assays--
COS-7 cells were plated on
100-mm diameter plastic dishes at a density of 8 × 105 cells/dish. After 24 h, pMP129-based plasmid (3 µg) and a luciferase reporter plasmid pGL3-Control (Promega) (1 µg)
were introduced into COS-7 cells by cationic liposomes (LipofectAMINE,
Life Technologies) (30 µg), according to the manufacturer's
protocol. Transfected cells were harvested 48 h post-transfection
and lysed by 900 µl of reporter lysis buffer (Promega). The
-galactosidase and luciferase assays were performed using the
respective kits (Promega). Luciferase activity was measured using the
AutoLumat LB953 (EG&G Berthold).
Analysis of Replicated Plasmids in COS-7 Cells--
Plasmid DNA
was recovered from transfected COS-7 cells as follows: 3 µg of
pMP141, pMP180, pRW3823, pMP145, pMP165, pRW3827, pMP175, and pMP177
were separately introduced, along with 1 µg of pGL3-Control, into
COS-7 cells in 100-mm diameter plastic dishes using LipofectAMINE (30 µg) as described above. Transfected cells were washed twice with
ice-cold phosphate-buffered saline 48 h after transfection and
incubated with 900 µl of lysis solution containing 0.6% SDS and 10 mM EDTA for 20 min at room temperature. The lysate was
transferred into two 1.5-ml microcentrifuge tubes, mixed with 450 µl
of 2.5 M NaCl, and incubated at 4 °C for 16 h.
After centrifugation at 4 °C for 4 min at 14,000 × g, the supernatant was extracted with phenol twice and
chloroform once and precipitated with ethanol. The pellet was
resuspended in 250 µl of TE buffer (10 mM Tris·Cl, 1 mM EDTA, pH 8.0) and reprecipitated with 750 µl of
ethanol in the presence of 25 µl of 3 M sodium acetate
(pH 5.2). After centrifugation, plasmid DNA was isolated and
resuspended in H2O. 10 µg of plasmid DNA were digested
with the appropriate restriction enzymes in the presence of RNase A and
the digests were separated on agarose gels in 1 × TBE buffer (90 mM Tris borate, 2 mM EDTA, pH 8.3). DNA was
transferred onto nylon membranes (Hybond-N+, Amersham)
(30). After UV cross-linking, blots were hybridized to
32P-labeled probes indicated in the figures at 65 °C in
15 ml of hybridization solution (5 × SSC, 5 × Denhardt
solution, 1% SDS) with 100 µg/ml herring sperm DNA. Membranes were
washed with 2 × SSC, 0.1% SDS twice at 25 °C for 5 min and
0.2 × SSC, 0.1% SDS twice at 25 °C for 5 min, and then
exposed to x-ray film at 80 °C for 5-10 days. Band intensities on
autoradiograms were measured using the AlphaImager version 3.0 (Alpha
Innotech).
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RESULTS |
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Abstract
Introduction
Procedures
Results
Discussion
References
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Cloning of GAA·TTC Triplet Repeat Sequences in Plasmids-- Attempts were made to clone a polymerase chain reaction product from a FRDA patient containing (GAA·TTC)700 in pUC19. E. coli SURE transformants harbored plasmids with a family of repeat lengths containing 110 GAA·TTC triplets at most. TTC triplets were in the leading strand template of all these recombinant plasmids (Fig. 1A, orientation II). This first result indicated that GAA·TTC repeats cloned into pUC19 are unstable in E. coli, and the instability may be related to the direction of replication, as previously observed for CTG·CAG (29) and CCG·CGG (31). The insert containing (GAA·TTC)110 was then subcloned in both orientations into pUC18NotI (Fig. 1A, orientation I) and pUC19NotI (Fig. 1A, orientation II). The resulting recombinant plasmids, pRW3804 and pRW3803, contained 70 and 65 GAA·TTC triplets, respectively (Fig. 1A). These shorter GAA·TTC repeats were quite stable in both plasmids when grown in E. coli SURE, but pRW3804 was more stable than pRW3803 when grown in E. coli DH10B (data not shown). Hence, the stability of GAA·TTC repeats in E. coli is influenced by the direction of replication (see below, Fig. 1C).
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DNA-BstEII digest and 100-bp ladder size markers,
respectively, with sizes in base pairs (bp) shown on the
left. The positions of the EcoRI-PstI
fragments containing (GAA·TTC)n are shown by
arrows on the right. C, stability of pSPL3-based
plasmids containing (GAA·TTC)n grown in E. coli
SURE. pSPL3-based plasmids containing (GAA·TTC)n in
orientations I and II, as defined in A, were digested with
EcoRI and NdeI and run on a 1.2% agarose gel.
The size and orientation of each repeat is shown on the top.
The bracket on the right indicates the positions
of migration of the EcoRI-NdeI fragments
containing (GAA·TTC)n. M1 and M2
indicate GAA·TTC Triplet Repeats Were More Stable in pSPL3 Than in pUC Vectors-- To investigate the effects of GAA·TTC triplet repeats on transcription and splicing (see below), we subcloned several such repeats from pUC vectors into the multicloning site of pSPL3 in both orientations (Fig. 2A). The subsequent in vivo expansion-deletion procedure generated GAA·TTC repeats ranging from 9 to 500 triplets (Fig. 1A). Surprisingly, GAA·TTC repeats were much more stable in pSPL3 than in pUC vectors (Fig. 1B). pSPL3-based plasmids containing up to 270 repeats were completely stable (Fig. 1B, lanes 2, 4, and 6), and pRW3824 containing 360 repeats showed only slight instability (lane 8). As expected, constructs containing 470 and 500 repeats showed an increasing frequency of deletions (data not shown). When comparing the stability of (GAA·TTC)360 in different orientations, the insert in orientation II (Fig. 1C, lane 9) was less stable than the one in orientation I (lane 8), as observed for pUC-based plasmids. To avoid contamination by deleted products, we only used pSPL3-based plasmids in which the cloned GAA·TTC repeat was completely stable for further experiments. These contained up to 230 triplets in orientation II, and up to 270 triplets in orientation I (Fig. 1C).
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Length and Orientation-dependent Reduction of Gene Expression by GAA·TTC in Transfected COS-7 Cells-- pSPL3 (Fig. 2A) harbors a reporter gene, derived from the HIV gp120 gene, composed by two exons (exons 1 and 2) separated by an intron derived from the HIV tat gene (33). This intron contains a multiple cloning site where (GAA·TTC)n repeats were inserted. Transcription is controlled by the SV40 early promoter. (GAA·TTC)n repeat-containing pSPL3-based plasmids were transfected into COS-7 cells and the reporter gene transcripts were detected by RNase protection analysis (Fig. 2B). We observed a reduction in the amount of the mature transcript containing both exons 1 and 2 (indicated as I in Fig. 2B) as the length of GAA·TTC repeats increased. Such reduction was much greater when (TTC)n was in the template strand, i.e. when GAA-containing RNA was synthesized (Fig. 2A, lower inset), than when (GAA)n was in the template strand, i.e. when UUC-containing RNA was formed (Fig. 2A, upper inset). Specifically, the mature transcript derived from (GAA)230-containing RNA was 6 times less abundant than the one derived from the (UUC)270-containing RNA (3% (Fig. 2B, lane 7) versus 17% (lane 4) of pSPL3 (lane 8)). The protected fragment indicated as II in Fig. 2B, whose size corresponds to exon 2 only, was also reduced as the repeat length increased, suggesting that accumulation of unspliced RNA was not occurring. The protected fragment indicated as III in Fig. 2B was also reduced in amount as the repeat length increased and is of uncertain nature, possibly resulting from an alternate splicing of exon 1 with part of exon 2. In the same figure, IV indicates a protected fragment corresponding to exon 1 only. Interestingly, its abundance remained stable in all samples, suggesting that the initiation of transcription was not affected by the length of the TRS.
Northern blot analysis of total RNA from transfected cells (Fig. 2C) confirmed that GAA·TTC triplet repeats caused a length- and orientation-dependent reduction in the abundance of mature reporter gene RNA (arrow) (lanes 1-7). The most marked decrease was observed for the mature RNAs derived from (GAA)170 and (GAA)230 containing primary transcripts (lanes 6 and 7). Small amounts of unspliced transcripts (bracket) were detected for all templates, including the control plasmid (pSPL3 without insert, lane 8), possibly because of the high level of transcription generated by the SV40 promoter. The (GAA)170 and (GAA)230 containing primary transcripts (lanes 6 and 7) were reduced as much as the corresponding mature transcripts, and no partially spliced intermediates were revealed. This lack of accumulation of unspliced or partially spliced RNAs strongly suggests that long GAA·TTC triplet repeats interfere with transcription rather than splicing. We then investigated the effects of (GAA·TTC)n repeats on the expression of the protein product of the reporter gene. The lacZ gene open reading frame was inserted into exon 2 of the pSPL3-based plasmids containing (GAA·TTC)n (Fig. 3).-Galactosidase activity, resulting
from lacZ expression, decreased in transfected COS-7 cells
as the length of GAA·TTC repeats increased. However, this decrease
significantly exceeded that observed as a general consequence of
inserting longer sequences upstream of the lacZ open reading
frame only when GAA-containing RNA was synthesized. -Galactosidase
activity in cells transfected with pMP163, whose primary transcript
contains (GAA)230, was significantly lower than in cells
transfected with pMP183, in which a random sequence of similar length
was cloned instead of a GAA·TTC repeat, while lacZ
expression in cells transfected with pMP153, which synthesize RNA
containing (UUC)270, was not significantly lower than in
pMP183-transfected cells.
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Analysis of Replicated Plasmids Containing GAA·TTC Repeats in COS-7 Cells-- pSPL3-based plasmids containing between 9 and 270 GAA·TTC triplets in both orientations were transfected into COS-7 cells to study their replication properties. After 48 h, plasmid DNAs were isolated, digested with restriction enzymes to produce fragments containing (GAA·TTC)n, and analyzed by Southern blot (Fig. 4). No evidence of instability was found for GAA·TTC repeats containing up to 270 triplets in orientation A (lane 3) and 230 triplets in orientation B (lane 6). Additional analysis using different restriction enzymes of KpnI and PstI, which produced shorter fragments to give better separation between the shortest and longest repeats on a 1.0% agarose gel, also showed no apparent instability (data not shown). However, the hybridization signals for the longest GAA·TTC repeats were greatly reduced (lanes 3 and 6) compared with the shorter repeats. Relative replication efficiency in the pSPL3-based plasmids to a co-transfected pGL3-Control showed that the plasmids containing the longest GAA·TTC repeats were recovered in much smaller amounts than the plasmids containing (GAA·TTC)9 or (GAA·TTC)90 (pRW3827 (lane 6) and pRW3823 (lane 3) were 2 and 11% of pMP145 (lane 4), respectively). Two control plasmids, pMP175 and pMP177, in which a portion of the luciferase gene replaced the smallest and longest (GAA·TTC)n, respectively, were recovered in similar amounts, not significantly different from the recovered amounts of the shortest GAA·TTC-containing plasmids. This finding indicates a specific effect of GAA·TTC tracts rather than a generic lower efficiency of replication of longer plasmids. Interestingly, we recovered five times less pRW3827, which generates a (GAA)230 containing transcript (Fig. 2A, orientation B), than pRW3823, which generates a (UUC)270 containing transcript (Fig. 2A, orientation A). This finding suggests that the replication of a long GAA·TTC repeat may be influenced by transcriptional activity and possibly by interference of RNA molecules containing (GAA)n (see "Discussion").
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DISCUSSION |
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Abstract
Introduction
Procedures
Results
Discussion
References
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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 (1, 7-9). 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 (13). The longest repeat contained 103 triplets and was interrupted by AA·TT and AGG·CCT sequences. In FRDA, hyperexpanded GAA·TTC repeats are usually much longer and believed to be uninterrupted (6). Both repeat length and the presence of interruptions are known to influence TRS stability (3, 6, 23), and recent studies also suggested a role of flanking sequences (22-28). Therefore, we chose to work with repeats as close as possible to the naturally occurring sequence in the FRDA gene.
Under certain conditions, TRS are unstable during replication in recombinant plasmids in E. coli (29, 31, 34-37) and Saccharomyces cerevisiae (38-40). 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 (23). These expanded clones would be expected to contain uninterrupted TRS, since expansions occur by triplet multiplication within the repeats (29), 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 (29, 31, 35-37). The instability of the GAA·TTC repeats was clearly dependent on the direction of replication, as previously observed for CTG·CAG (29) and CCG·CGG (31). Single-stranded CTG, CAG, CCG, and CGG repeats are known to form hairpin structures of variable stability (4, 41, 42). The molecular basis of expansions versus deletions of CTG·CAG (29) and CGG·CCG (31) was explained on the basis of preferential stabilization of transient loop structures during replication (29). 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 (29), 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 (41).
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 in E. coli (34) and in transgenic mice (22). 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 (1, 5, 6), 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 (10), 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 (14-17), which indicated that under physiological conditions Pur·Pur·Pyr triplex structures are preferentially formed and in vitro transcription of purine-rich RNA is specifically reduced. Griffin et al. (14-16) suggested that the underlying molecular mechanism is the formation of an intermolecular RNA·DNA hybrid triplex structure (Fig. 5B). Grabczyk and Fishman (17) 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.
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Because TRS may be unstable when propagated in eukaryotic cells, including yeast (38-40) and transgenic mice (22, 43-45), 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 (13, 46-48). In vivo, replication of long (CCG·CGG)n in the FMR1 gene is slowed down in cultured cells (49), and stalling of replication of long CTG·CAG and CCG·CGG repeats was observed in E. coli (50). The slowed replication of long GAA·TTC tracts that we observed may possibly be due to stalling of DNA polymerase caused by a Pur·Pur·Pyr intramolecular triplex (11). 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 transcription versus 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 (51).
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 (52), and GAA·TTC and
GAG·CTC triplets in the cardiac -myosin heavy chain (MYH6) gene
(53), whose biological roles are currently unknown.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
|---|
* 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.
§§ To whom correspondence should be addressed. Tel.: 514-281-6000 (ext. 8928); Fax: 514-896-4762; E-mail: pandolm{at}magellan.umontreal.ca.
1 The abbreviations used are: FRDA, Friedreich ataxia; TRS, triplet repeat sequences; Pur·Pyr, polypurine·polypyrimidine; bp, base pair(s).
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REFERENCES |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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G. Raca, E. Yu. Siyanova, C. T. McMurray, and S. M. Mirkin Expansion of the (CTG)n repeat in the 5'-UTR of a reporter gene impedes translation Nucleic Acids Res., October 15, 2000; 28(20): 3943 - 3949. [Abstract] [Full Text] [PDF]
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M. Ristow, M. F. Pfister, A. J. Yee, M. Schubert, L. Michael, C.-Y. Zhang, K. Ueki, M. D. Michael II, B. B. Lowell, and C. R. Kahn Frataxin activates mitochondrial energy conversion and oxidative phosphorylation PNAS, October 12, 2000; (2000) 220403797. [Abstract] [Full Text]
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 R. G. Jaffrey, S. C. Pritchard, C. Clark, G. I. Murray, J. Cassidy, K. M. Kerr, M. C. Nicolson, and H. L. McLeod Genomic Instability at the BUB1 Locus in Colorectal Cancer, but not in Non-Small Cell Lung Cancer1 Cancer Res., August 1, 2000; 60(16): 4349 - 4352. [Abstract] [Full Text]
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E. Grabczyk and K. Usdin The GAATTC triplet repeat expanded in Friedreich's ataxia impedes transcription elongation by T7 RNA polymerase in a length and supercoil dependent manner Nucleic Acids Res., July 15, 2000; 28(14): 2815 - 2822. [Abstract] [Full Text] [PDF]
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M. Cossee, H. Puccio, A. Gansmuller, H. Koutnikova, A. Dierich, M. LeMeur, K. Fischbeck, P. Dolle, and M. Koenig Inactivation of the Friedreich ataxia mouse gene leads to early embryonic lethality without iron accumulation Hum. Mol. Genet., May 1, 2000; 9(8): 1219 - 1226. [Abstract] [Full Text] [PDF]
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H. Puccio Recent advances in the molecular pathogenesis of Friedreich ataxia Hum. Mol. Genet., April 1, 2000; 9(6): 887 - 892. [Abstract] [Full Text] [PDF]
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C. J. Cummings and H. Y. Zoghbi Fourteen and counting: unraveling trinucleotide repeat diseases Hum. Mol. Genet., April 1, 2000; 9(6): 909 - 916. [Abstract] [Full Text] [PDF]
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 S. I. Bidichandani, C. A. Garcia, P. I. Patel, and M. M. Dimachkie Very Late-Onset Friedreich Ataxia Despite Large GAA Triplet Repeat Expansions Arch Neurol, February 1, 2000; 57(2): 246 - 251. [Abstract] [Full Text] [PDF]
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M. B Delatycki, R. Williamson, and S. M Forrest Friedreich ataxia: an overview J. Med. Genet., January 1, 2000; 37(1): 1 - 8. [Abstract] [Full Text]
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 K. Ohshima, N. Sakamoto, M. Labuda, J. Poirier, M. L. Moseley, L. Montermini, L. P. W. Ranum, R. D. Wells, and M. Pandolfo A nonpathogenic GAAGGA repeat in the Friedreich gene: Implications for pathogenesis Neurology, November 1, 1999; 53(8): 1854 - 1854. [Abstract] [Full Text] [PDF]
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M. Pandolfo Molecular Pathogenesis of Friedreich Ataxia Arch Neurol, October 1, 1999; 56(10): 1201 - 1208. [Abstract] [Full Text] [PDF]
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J. P. Jakupciak and R. D. Wells Genetic Instabilities in (CTG{middle dot}CAG) Repeats Occur by Recombination J. Biol. Chem., August 13, 1999; 274(33): 23468 - 23479. [Abstract] [Full Text] [PDF]
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R. R. Iyer and R. D. Wells Expansion and Deletion of Triplet Repeat Sequences in Escherichia coli Occur on the Leading Strand of DNA Replication J. Biol. Chem., February 5, 1999; 274(6): 3865 - 3877. [Abstract] [Full Text] [PDF]
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R. D. Wells, P. Parniewski, A. Pluciennik, A. Bacolla, R. Gellibolian, and A. Jaworski Small Slipped Register Genetic Instabilities in Escherichia coli in Triplet Repeat Sequences Associated with Hereditary Neurological Diseases J. Biol. Chem., July 31, 1998; 273(31): 19532 - 19541. [Abstract] [Full Text] [PDF]
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A. Pluciennik, R. R. Iyer, P. Parniewski, and R. D. Wells Tandem Duplication. A NOVEL TYPE OF TRIPLET REPEAT INSTABILITY J. Biol. Chem., September 8, 2000; 275(37): 28386 - 28397. [Abstract] [Full Text] [PDF]
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N. Sakamoto, J. E. Larson, R. R. Iyer, L. Montermini, M. Pandolfo, and R. D. Wells GGA{middle dot}TCC-interrupted Triplets in Long GAA{middle dot}TTC Repeats Inhibit the Formation of Triplex and Sticky DNA Structures, Alleviate Transcription Inhibition, and Reduce Genetic Instabilities J. Biol. Chem., July 13, 2001; 276(29): 27178 - 27187. [Abstract] [Full Text] [PDF]
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N. Sakamoto, K. Ohshima, L. Montermini, M. Pandolfo, and R. D. Wells Sticky DNA, a Self-associated Complex Formed at Long GAA{middle dot}TTC Repeats in Intron 1 of the Frataxin Gene, Inhibits Transcription J. Biol. Chem., July 13, 2001; 276(29): 27171 - 27177. [Abstract] [Full Text] [PDF]
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M. Ristow, M. F. Pfister, A. J. Yee, M. Schubert, L. Michael, C.-Y. Zhang, K. Ueki, M. D. Michael II, B. B. Lowell, and C. R. Kahn Frataxin activates mitochondrial energy conversion and oxidative phosphorylation PNAS, October 24, 2000; 97(22): 12239 - 12243. [Abstract] [Full Text] [PDF]
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