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Human Molecular Genetics, 2003, Vol. 12, No. 23 3067-3074
DOI: 10.1093/hmg/ddg331
© 2003 Oxford University Press

The (CGG)n repeat element within the 5' untranslated region of the FMR1 message provides both positive and negative cis effects on in vivo translation of a downstream reporter

Li-Sheng Chen, Flora Tassone, Parminder Sahota and Paul J. Hagerman*

Department of Biological Chemistry, University of California, Davis School of Medicine, Davis, CA 95616, USA

Received July 11, 2003; Revised September 15, 2003; Accepted September 23, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The human fragile X mental retardation 1 (FMR1) gene contains a polymorphic (CGG) trinucleotide repeat element in its 5' untranslated region. Expansion of the (CGG)n element beyond 200 repeats (full mutation range) generally leads to transcriptional silencing; consequent loss of the FMR1 protein (FMRP) results in fragile X syndrome, the most frequent form of inherited mental impairment. For carriers of smaller expansions (55<=n<=200; premutation range), FMRP levels are gradually reduced with increasing repeat number, despite elevated FMR1 mRNA levels, suggesting that translation is impeded within the premutation range. To examine in more detail the influence of the CGG repeat on translation, CMV immediate–early promoter constructs, containing the FMR1 5'-UTR with various (CGG)n repeat lengths (0<=n<=99) and a downstream (luciferase) reporter, were transfected into two human cell lines, a neural cell-derived line (SK) and a fetal kidney cell-derived line (293). For both cell types, the CGG element exerts distinct effects on reporter expression, depending on the length of the repeat. For n>=30, luciferase expression decreases with increasing repeat length, consistent with earlier observations of decreased FMRP expression in peripheral blood leucocytes over the same repeat range, despite a slight increase in mRNA level for the larger repeats. Surprisingly, for smaller alleles (0<=n<=30), reporter expression actually increases by nearly two-fold with increasing repeat length in the absence of any change in mRNA level. These results suggest that the CGG repeat element can exert both positive (n<30) and negative (n>30) effects on translation. Interestingly, optimal translation appears to occur near the modal repeat number within the general human population.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Fragile X syndrome (MIM 3009550) is a trinucleotide repeat (CGG) expansion disorder and the most common heritable form of mental retardation. The syndrome generally results from silencing of the fragile X mental retardation 1 (FMR1) gene, with consequent absence of the FMR1 protein (FMRP), when the CGG element exceeds 200 repeats (full mutation) (1,2). Many carriers of premutation alleles (55–200 CGG repeats) or gray zone alleles (41–54 repeats) appear to be clinically uninvolved, although some do have subtle physical features suggestive of fragile X syndrome, with emotional or behavioral features occurring in ~25% of premutation carriers (3–5).

Two forms of clinical involvement, premature ovarian failure (POF) and fragile X-associated tremor/ataxia syndrome (FXTAS), appear to be unique to the premutation range. POF occurs in ~20% of female carriers of premutation alleles (6,7), with no increased incidence among full mutation females. FXTAS, a recently described neurological disorder involving intention tremor, gait ataxia, parkinsonism and dysautonomia, appears to affect more than one-quarter of carrier males over 50 years of age (8–12).

The polymorphic (CGG)n repeat, which lies in the 5'-untranslated region (5'-UTR) of the FMR1 mRNA, appears to exert a negative influence on translation in the premutation range, with a gradual reduction of FMRP levels occurring with increasing repeat number, despite increased levels of FMR1 mRNA (13,14). The reduced ratio of FMRP to mRNA suggests that premutation CGG expansions act, directly or indirectly, to block translation of the FMR1 message. Primerano and colleagues (15) recently provided direct evidence for the inhibitory role of premutation alleles in translation by analyzing the polysome profiles of lymphoblastoid cell lines derived from premutation carriers (97–195 CGG repeats); their results demonstrated that the translation efficiencies of premutation FMR1 mRNAs are progressively reduced with increasing CGG repeat expansions. However, the mechanism by which the CGG repeats inhibit translation is not known, nor is it known whether the effect of the CGG element is purely inhibitory at all repeat lengths within the normal and premutation ranges.

Trinucleotide repeat RNAs are known to form stable RNA hairpins (16), suggesting that the CGG repeat element might provide a direct structural impediment to translational initiation. However, to date, there are no reports of the influence of the CGG repeat on translation within the normal repeat range, particularly for smaller repeat lengths; although the structural model would predict a purely inhibitory role of the CGG element once the secondary structure, generated by the repeat, becomes sufficiently stable to affect initiation. To address this issue, we have analyzed the effect of increasing CGG repeat length on the in vivo translation of a reporter (luciferase) mRNA with a FMR1 5'-UTR, for a range of CGG elements encompassing the normal and low premutation ranges (0<=n<100 CGG repeats). This system has allowed us to control for any variations in RNA level. We observed that the CGG element leads to reduced translation of the reporter mRNA only for 5'-UTRs with greater than 30 CGG repeats, with the extent of inhibition being directly correlated with the number of CGG repeats (43<=n<=99). By contrast, translation increased by nearly 2-fold with increasing repeat length between 0 and 30 repeats. This latter observation, the first evidence of a positive role of the CGG repeat on translation, suggests that other factors, including trans-acting factors, may be important for translation of the FMR1 message.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Construction of reporter plasmids containing the FMR1 5'-UTR with polymorphic CGG elements
To evaluate the effect of the expanded CGG repeats within the FMR1 5'-UTR on the translation of downstream mRNA, a series of reporter plasmids, containing a chimeric FMR1 5'-UTR-firefly luciferase (FL) coding sequence under the control of the CMV immediate early (CMV IE) promoter, were constructed (Fig. 1A). pCMV-FMR(16)-FL was derived from pSP6-FMR1-FL (17) and was used as the starting point for the construction of the pCMV-FMR(n)-FL. The inserted FMR1 5'-UTR element extends from 135 bp upstream of the polymorphic CGG repeat element, and continues 69 bp downstream of the repeat to the start codon (Fig. 1B). The inserted FMR1 5'-UTRs in pCMV-FMR(0)-FL, pCMV-FMR(16)-FL and pCMV-FMR(30)-FL have been sequenced. The CGG element has been deleted in pCMV-FMR(0)-FL, which retains all other FMR1 5'-UTR sequences. pCMV-FMR(16)-FL contains 16 straight CGG repeats, whereas pCMV-FMR(30)-FL has two AGG interruptions at the 10th and 20th trinucleotide positions. The remaining inserts have been sized on polyacrylamide gels.



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  		Figure 1. (A) Schematic of the pCMV-FMR(n)-FL constructs. The FMR1 5'UTR with n CGG repeats is designated FMR(n). In the control plasmids, the FMR1 5'-UTR is replaced by a multiple cloning site, as described in Materials and Methods. Luc, luciferase coding region; SV40 pA, SV40 late polyadenylation signal sequence. (B) Sequence of the FMR1 5'-UTR. The underlined A refers to a single nucleotide change introduced during cloning to match the FMR1 5'-UTR sequences. ATG, start of the luciferase coding sequence.

 
Initial attempts to clone 5'-UTRs with larger numbers of CGG repeats (premutation range), using commercially available high-copy number (pGL3; 500–700 copies/cell) plasmids, were unsuccessful, due to the highly unstable character of the CGG element in those vectors. Smears of partially or completely deleted insertions could be detected by PstI/NheI restriction digestion following attempts to clone inserts containing more than 50 CGG repeats (data not shown). Our experience is in agreement with several previous studies involving attempts to clone expanded FMR1 (CGG)n repeats (18,19).

Several factors appear to influence the stability of long-tract (CGG)n inserts in a recombinant plasmid, including repeat length, the presence of AGG interruptions, the orientation of the insert relative to the replication origin, the copy number of the host vector, and the E. coli host strain. Instability of GC-rich repeat sequences like the CGG expansions in FMR1 in bacterial hosts is sequence-specific and is presumably related to the formation of non-B DNA structures which block DNA polymerase progression (19–21).

To stably clone premutation CGG repeats in the premutation range, a low-copy number reporter construct was created, placing the CGG repeat in the (-) orientation relative to the direction of plasmid replication in E. coli to create pCMV-FMR(62)-FL and pCMV-FMR(99)-FL. This strategy was based on the observations of Hirst and White (19), who noted a strong orientation bias for repeat stability. The plasmids containing (CGG)62 and (CGG)99 propagated with reasonable stability (Fig. 2A) in host strain DH5{alpha}MCR when multiple 3 ml overnight cultures were initiated by single colony inoculate. To ensure the integrity of the CGG expansions, DNA isolates from each 3 ml culture were analyzed separately by restriction digest and Southern analysis. Rare deletion events were observed as generally large, single deletion events in a small number of the 3 ml overnight cultures. Finally, the doublets observed in lane 5 of Figure 2B were sized as 41 and 43 repeats, respectively; the presence of a doublet suggests that there are two discrete sizes of CGG expansion which are only two repeats apart and appeared as a single-size band in agarose gel electrophoresis (lane 5 in Fig. 2A). We designated this construct with the gray-zone insert, pCMV-FMR(42)-FL. The slight difference of repeat length in the particular construct may have arisen during replication in the host bacterial strain.



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  		Figure 2. Integrity of the (CGG)n elements in the pCMV-FMR(n)-FL constructs. The individual plasmid DNAs were digested with BlpI/NheI to release the fragments containing the CGG repeat. The resulting DNA fragments were subjected to agarose (2%) electrophoresis, and were visualized with ethidium bromide staining (A). A portion of each reaction mixture was subjected to Southern analysis using (CGG)10 as the probe (B). M: DNA size markers—100 bp ladder in (A) and MspI-digested pBR322 DNA in (B); lane 1, control; lane 2, 0 CGG; lane 3, 16 CGG; lane 4, 30 CGG; lane 5, 42 CGG; lane 6, 62 CGG and lane 7, 99 CGG.

 
The length of the CGG repeat element within the FMR1 5'-UTR affects expression of the luciferase reporter transcript
To investigate the influence of the length of the CGG repeat on translational efficiency, luciferase reporter activity was measured for both neurally-derived and non-neural cell lines (SK and 293, respectively) following a period of transient transfection by pCMV-FMR(n)-FL reporter constructs. The CGG repeat lengths (n) spanned the normal and low/mid premutation ranges (0<=n<=99 CGG repeats). Reporter gene expression was driven by a strong (CMV IE) promoter; the reporter coding sequence was followed by the SV40 late poly(A) signal (Fig. 1A). A control plasmid possessed a 90 bp multiple cloning site (MCS) between the CMV promoter and firefly luciferase coding sequence. The reporter gene constructs, together with the pCMV-RL (Renilla luciferase) internal control, were transiently transfected into both cell types, followed by incubation for 24 h. The transfected cells were subsequently assayed for the dual luciferase activities. For each sample, the firefly luciferase activity was measured first, followed by addition of Stop & Glo Regeant (Promega) to quench the firefly luciferase activity and concomitantly activate Renilla luciferase. The Renilla luciferase activity was assayed subsequently after a 1 s delay to allow complete activation. Firefly luciferase activities were first normalized to the activity of the Renilla luciferase (RL) reporter in each co-transfection experiment to correct for experimental variations in cell viability and in transfection efficiency. The corrected activity ratios were then normalized to the FL activity of the cells transfected with the control plasmid. Expression of the RL-corrected MCS/FL control was designated as 100% activity.

Replacing the control (MCS) 5'-UTR with the FMR(0) element did not significantly impede production of FL protein, a result that is somewhat surprising in view of the highly GC-rich character of the FMR1 5'-UTR (Fig. 1B). However, more surprising was the observation that the introduction of 16 or 30 CGG repeats resulted in a significant increase of FL expression in both SK cells (P=0.04, 16; P=0.02, 30) and 293 cells (P=0.001, 16; P<0.0001, 30) as shown in Figure 3. As the CGG repeat length is further increased from 30 CGG repeats to the gray zone (n=42 repeats) and low/mid premutation ranges (n=62, 99 repeats), there is a gradual decrease of FL reporter activity (Fig. 3), with FL levels for n=99 at around 60% of control levels and around 40% of peak (n=30 repeats) levels.



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  		Figure 3. Histograms of normalized firefly luciferase (FL) activity as a function of CGG repeat length, expressed as percentage of activity measured in SK and 293 cells (black and gray bars, respectively) transfected with control plasmid (without FMR1 5'-UTR). Results are the means±SE from three independent experiments, each consisting of four individual measurements. All values are also corrected for transfection efficiency by co-transfecting with a RL reporter with the control (MCS) 5'-UTR.

 
Differences in luciferase expression with increasing CGG repeat lengths appear to result from changes in both transcription and translation
All of the reporter constructs used in the current study possessed the identical CMV IE promoter elements; therefore, the levels of reporter (FL) mRNA produced by various constructs are expected to be the same, when corrected for variations in transfection efficiency (RL reporter activity). Nonetheless, FMR1 message levels have been shown to increase with increasing CGG repeat length in the premutation range (13,15,22). Moreover, there are reports that the CGG repeats in the context of either FMR1 or heterologous promoters affect the transcription of reporter gene (23). To determine whether the differences observed for FL expression with increasing CGG repeat length are in part due to differing levels of reporter RNA, the steady-state levels of both FL and RL (internal control) mRNAs were quantified using (fluorescence) RT–PCR. If CMV promoter-directed transcription of the FL reporter is not affected by the presence of CGG expansions within the FMR1 5'-UTR, the quantitative RT–PCR results are expected to show no significant dependence of FL mRNA levels on CGG repeat length. However, as shown in Figure 4, compared with the MCS control, transcription levels show a moderate increase in both cell lines for constructs possessing gray-zone or premutation alleles (n=42, 62 and 99). When the effects of the increased mRNA levels are taken into account, the decrease in FL translation with increasing CGG repeat length is more pronounced; the resulting FL/mRNA ratios are plotted in Figure 5.



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  		Figure 4. Histograms of relative luciferase mRNA levels in SK and 293 cells (black and gray bars, respectively), plotted as a function of CGG repeat length, expressed relative to Renilla mRNA levels, and normalized to FL mRNA levels for the control (MCS) reporter. Results are the means±SE from three independent experiments, each consisting of four individual measurements.

 


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  		Figure 5. FL reporter expression, corrected for relative FL mRNA levels, plotted as a function of CGG repeat length. Paired results of protein and mRNA levels from three independent experiments for both SK and 293 cells (black and gray bars, respectively) are expressed as ratios (protein/RNA). Results are the means±SE for three independent experiments.

 
We do not know at present why the luciferase mRNA levels are increased for CGG repeats in the premutation range. Such increases could be due to either increased transcriptional activity or an increase in mRNA stability. To address the latter possibility, we compared the rates of mRNA decay following actinomycin D treatment of SK cells transfected with reporter plasmids harboring either normal (30 CGG) or premutation (99 CGG) repeats. Transiently transfected cells were treated with actinomycin D 24 h after transfection, and levels of luciferase and glucoronidase (endogenous control) mRNAs were measured at various times within a 14 h period following addition of the drug. From the results of four separate trials, the half-life for firefly luciferase mRNA carrying 30 CGG repeats is 6.2±1.1 h, and 6.7±0.23 h for reporter gene carrying 99 CGG repeats; suggesting that the increase in mRNA levels in the premutation range are not due to increased message stability.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
To evaluate the influence of varying the CGG repeat length, in the context of the FMR1 5'-UTR, on the translation of a downstream reporter coding region, we placed a series of FMR1 5'-UTR elements (0<=n<=99 CGG repeats) in front of the coding region for firefly luciferase. The resulting set of FMR1 5'-UTR-luciferase reporter plasmids were then used to transfect both neurally-derived (SK) and non-neural (293) cell lines to quantify in vivo the influence of the CGG repeat on the efficiency of luciferase expression. To exclude the influence of varying CGG repeat length on transcription, reporter mRNA levels were determined using quantitative (fluorescence) RT–PCR following the period of transient transfection, and luciferase (protein) expression levels were normalized to the mRNA levels.

In the absence of any significant change in reporter mRNA from zero to normal range of CGG repeat lengths (P>0.1 in all cases), an unexpected finding of our experiments is that the efficiency of translation actually increases by nearly two-fold as the CGG repeat is increased from 0 to 30 CGG repeats. Were the CGG repeat to act by simply increasing the stability of the secondary/tertiary structure within the FMR1 5'-UTR, one would expect the translational efficiency of the native FMR1 message, or the FMR1 5'-UTR-luciferase chimeric message, to decrease with increasing CGG repeat length. Such a decrease may not be manifest until the CGG repeat length reaches a threshold value; however, an increase in translation is not expected. Secondary structures in many 5'-UTRs substantially block cap-dependent initiation when their calculated stabilization free energies reach -50 to -70 kcal/mol (24–28). However, for the FMR1 5'-UTR, the estimated energy of stabilization for the most frequent normal allele (30 CGG repeats) is already ~-50 to -60 kcal/mol (depending on the number of AGG interruptions) for the CGG repeat element alone, and exceeds (in magnitude) -160 kcal/mol for the entire 5'-UTR. These results suggest either that other mechanisms are at play in circumventing such stable structures in the FMR1 5'-UTR, or that the predicted secondary structures are not actually formed in vivo.

One possible mechanism for the observed increase in translation in the low normal range is that the CGG repeat element, in the context of flanking 5'-UTR sequences, recruits one or more transacting factor(s) to facilitate the translation of FMR1 mRNA, and that this is the normal function of the CGG element. Such a scenario has been reported in the case of HIV-1 TAR-containing RNA and its cellular binding protein TRBP (29). The TAR RNA structure is destabilized when the TAR–TRBP complex is formed (30); the unfolding of TAR structure thus increases its accessibility to the 43S translation initiation complex. We suggest that as-yet unidentified FMR1 5'-UTR-specific CGG-specific binding protein(s) may exist to carry out a similar function.

The gradual decline in the efficiency of translation, observed for the larger CGG repeats in the gray zone and premutation ranges, mirrors the decreases seen in FMRP levels in peripheral leucocytes (13,31) and lymphoblastoid cells (15). Primerano and coworkers (15) have demonstrated that translation of FMR1 mRNA is significantly impaired in lymphoblastoid lines from premutation carriers, as had been shown earlier for full mutation alleles (32). Moreover, it is noteworthy that there is an apparent decrease of translation efficiency between constructs with 30CGG and 43CGG (P<0.1 in SK cells and P<0.05 in 293 cells), which provides new evidence that a translational impediment may arise for alleles that are only 10–15 CGG repeats above the modal value of ~30 repeats in the general population. This gradual reduction in translational efficiency may indicate that the increasing thermodynamic stability of secondary structure within the 5'-UTR, due to the expanded CGG repeat, is beginning to influence the rate-limiting step(s) in translational initiation. Alternatively, such a decrease may reflect a limiting availability of one or more trans-acting factors, such that the molar concentration of CGG trinucleotides exceeds the availability of the hypothesized factors. This latter possibility is analogous to the mechanism underlying the RNA toxic gain-of-function model proposed for myotonic dystrophy (33–36). Finally, whereas it is possible that a decrease in apparent translational efficiency is the result of sequestration of message within the nucleus, we have not observed any significant nuclear sequestration of premutation mRNA in studies of lymphoblastoid lines (unpublished results). Furthermore, the studies of Primerano et al. (15) have provided a direct demonstration of reduced translational efficiency.

Using quantitative RT-PCR, we observed a modest but significant increase in reporter mRNA levels for (CGG)n elements in the premutation range (n=62, 99) relative to the mRNA levels in the normal range (n=16, 30). When comparing pooled replicates for the normal alleles (16 and 30) with those for the premutation alleles (62 and 99), significance was reached separately for both SK cells (P=0.019) and 293 cells (P=0.001), with a combined significance level (pooled data for both SK and 293 cells) of (P=3.3x10-5). Since the transcription of our FMR5'-UTR-luciferase reporter constructs is driven by the heterologous CMV immediate early promoter, the increase in mRNA levels with increasing CGG repeat length may reflect a general cis-enhancing effect of the (CGG)n element on transcription, i.e. one that does not require the FMR1 promoter for the enhancement effect.

The elevation of FMR1 mRNA among premutation carriers has been well-documented (13,14,22,37). Since the stability of FMR1 mRNA was shown to be similar between normal and premutation FMR1 transcripts, it was suggested that elevation of the FMR1 message is a response to lowered FMRP levels (13). Our mRNA decay data indicate that the elevated levels of luciferase mRNA in transfected cells harboring premutation CGG expansions is not due simply to increased mRNA stability, in accord with previous findings (13). The current results, albeit with a heterologous promoter, suggest that at least part of the mRNA elevation may be a direct (cis) effect of the CGG element, not a compensatory response to lowered FMRP. Additional experiments designed to resolve this issue using reporter constructs driven by the authentic FMR1 promoter are currently underway. There are clearly a number of possible means by which the CGG element could exert its cis effects, including a direct structural influence on pol II initiation, or an indirect effect via one or more transacting factors that may mediate the effects of the CGG repeat.

One of the intriguing features of the CGG-repeat dependence of reporter expression is that its maximum occurs near the modal value (~30 CGG repeats) for the allele distribution in both human and non-human primate populations (38–41). Although additional experiments are required to clarify the functional relationship, if any, between the two distributions, it is interesting to speculate that the modal distribution reflects a combination of upward pressure (enhanced FMRP production with increasing CGG repeat size), and downward pressure from deleterious effects of alleles in the premutation range. With regard to the latter, premature ovarian failure (6,7) and FXTAS (6–8,10–12,42) are both disorders that may reflect the deleterious effects of premutation alleles. In this regard, all normal FMR1 alleles reported to date possess at least five copies of the CGG repeat (38–40). Furthermore, there is a trend toward an increase in the overall length of the FMR1 CGG repeat in the course of primate evolution (43). Thus, as our data suggest, the (CGG)n element should not be thought of simply as a structural impediment to translation; instead, it may play an important, positive role in FMRP expression within the normal population.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
PCR amplification of the FMR1 5'-UTR
PCR amplification was performed using, as templates, genomic DNA from selected males whose FMR1 CGG repeat lengths had been sized by Southern and/or PCR analysis. The primers (c: 5'-GCTCAGCTCCGTTTCGGTTTCACTTCCGGT; f: 5'-AGCCCCGCACTTCCACCACCAGCTCCTCCA) used to amplify the 30, 42 and 62 CGG repeat 5'-UTRs were the same as previously described (38). The PCR reactions were performed as described (44). For amplification of 62 CGG repeats, a 1 : 1 ratio of dGTP: deaza7GTP was substituted for 100% dGTP. For amplification of premutation allele with 99 CGG repeat, primer 723 (5'-TTCACTTCCGGTGGAGGGCCGCCTCTGAGC) replaced primer c as the forward primer. The latter PCR reactions were performed using the method of Hirst and White (19). The products obtained from genomic PCR amplification were secondarily amplified with primer f, and a forward primer containing a PstI restriction site (5'-AGCTTGGGCTGCAGGGCCTCAGTCAGGCG) to facilitate the subsequent cloning into reporter plasmids. All PCR products were gel-purified, double-digested with PstI and NheI restriction enzymes, and cleaned with PCR Purification Kit (Qiagen) before the ligation reactions. The amplified FMR1 5'-UTR region was sequenced after cloning into the reporter vector by dRhodamine terminator cycle sequencing on an ABI 310 genetic analyzer (PE Biosystems) using the GLprimer2 (Promega) located at the 5' end of firefly luciferase coding sequence.

Construction of FMR1 5'-UTR reporter plasmids
To construct pCMV-FMR5'-UTR(CGG)16-FL [abbreviated: pCMV-FMR(16)-FL], the pCMV-RL reporter plasmid (Promega) was digested with PstI and XbaI to release the Renilla luciferase (RL) coding region. This fragment was replaced by the PstI–XbaI fragment, which contains the firefly luciferase (FL) coding region and upstream FMR1 5'-UTR with 16 CGG repeats. The latter fragment was obtained from pSP6-FMR(16)-FL (17). The reporter constructs containing 30 and 42 CGG repeats in the FMR1 5'-UTR were generated by replacing the 260 bp PstI–NheI fragment of pCMV-FMR(16)-FL with restriction digests of PCR products as described above. pCMV-FMR(0)-FL was generated by replacing PstI–KpnI Renilla luciferase coding region with a short PstI–KpnI linker from pRL-FMR(0)-FL as described previously (17). To address the issue of instability associated with the extended CGG repeats, a low-copy-number vector was generated as follows: pBR322 (NEB) was digested with HindIII and PvuII to obtain the 2.3 kb fragment containing the low-copy-number (15–20 copies/cell) replication origin and ampicillin resistance gene. The linearized plasmid was ligated with an adaptor containing BglII and BamHI sites. The resulting plasmid was then digested with BglII and BamHI and ligated to the BglII–BamHI fragment from pCMV-FMR(30)-FL. The clone of (-) orientation was selected by differential BglII/BamHI restriction pattern to create the low-copy number version of pCMV-FMR(30)-FL. pCMV-FMR(62)-FL and pCMV-FMR(99)-FL were then derived from pCMV-FMR(30)-FL with the low-copy-number ori using the same cloning strategy described above. When these two constructs were propagated in DH5{alpha}MCR, the long CGG repeats could be stably amplified in 3 ml overnight cultures, with only rare deletion events. All plasmids were isolated by standard alkaline lysis methods followed by chromatographic purification using Plasmid Maxi/Mini-prep kits (Qiagen). Southern blot analysis was performed with DIG-end-labeled (CGG)10 oligonucleotides following protocols of the supplier (Roche).

Cell culture, transfection and dual luciferase assays
SK-N-MC cells (‘SK’; neuroepithelial origin; ATCC) and 293 cells (fetal kidney epithelium; ATCC) were maintained in Eagle's minimal essential medium supplemented with 2 mM l-glutamate, 0.1 mM non-essential amino acids, and 10% fetal calf serum. For transient transfection experiments, cells were plated in 12-well plates at a density of 3–5x105 cells/well and incubated overnight. Cells were fed with antibiotic-free medium 4 h before transfection. Mixtures of 2 µl of LipofectAMINE 2000 (Life Technologies Inc.), 1 µg of pCMV-FMR(n)-FL DNA and 0.1 µg pCMV-RL plasmid DNA in 100 µl Opti-MEM medium (LifeTechnologies, Inc.) were used in each transfection reaction.

Twenty-four hours after transfection, cells were lysed with passive lysis buffer (Promega), and dual-luciferase assays were performed according to the manufacturer's instructions (Dual-Luciferase Reporter Assay System, Promega). Briefly, 20 µl of cell lysate were mixed with 100 µl of Luciferase Assay Reagent II, and the FL activity of the mixture was measured for 10 s using an Lmax Microplate Luminometer (Molecular Devices) with dual injectors. After a 1 s delay, 100 µl of Stop and Glo reagent were added to quench the FL activity; the RL activity was subsequently measured for 10 s, followed by a 1 s delay. The FL activity was then normalized to RL activity for each sample. All the experiments were performed at least in triplicate.

Quantitative RT–PCR for determination of relative mRNA levels
Total RNA was isolated from cells 24 hours after transfection using Trizol reagent (Invitrogen, Inc.). The isolated RNA was treated with a DNA-free kit (Ambion) to remove contaminating plasmid and genomic DNA. For each sample, 0.5 and 0.25 µg quantities of total RNA were reverse-transcribed separately with MMLV reverse transcriptase (Invitrogen Inc.) using random hexamer primers to ensure the linearity of RT–PCR response. After first-strand synthesis, the cDNA was quantified by TaqMan real-time PCR using gene-specific primers and the dual-labeled probes, 5'-FAM-deoxyoligonucleotide-TAMRA-3', which are complementary to the PCR amplicon. Fluorescence was detected with an ABI Prism 7700 sequence detection system (PE Biosystems). Amplification primers for FL were 5'-GCTCCAACACCCCAACATCT as the forward primer, and 5'-GTTCACCGGCGTCATCG as the reverse primer. Amplification primers for RL were 5'-GCTGTTATTTTTTTACATGGTAACGC as the forward primer, and 5'-CGCGCTACTGGCTCAATATG as the reverse primer. The TaqMan probe for FL was 5'-(FAM)-CGCAGGTGTCGCAGGTCTTCCC-(TAMRA)-3'; the TaqMan probe for RL was 5'-(FAM)-CCTCTTCTTATTTATGGCGACATG-(TAMRA)-3'. The amount of FL message in each RNA sample was quantified and normalized to RL content. Relative amounts of FL message were calculated by the comparative CT method and are expressed as the percentage of FL message measured in cells transfected with control plasmid. Details of the RT reactions, PCR cycling conditions, and means of analysis of the CT data were as described (13,22).

Reporter gene mRNA decay
293 and SK cells were transfected and incubated for 24 h for luciferase gene expression as described in the previous paragraph. Transcription was inhibited 24 h post-transfection by the addition of actinomycin D (10 µg/ml) to the culture media. Total RNA was isolated at various time intervals following actinomycin D treatment, and luciferase and (endogenous) beta-glucoronidase (GUS) mRNA levels were determined by (TaqMan) quantitative RT–PCR as described above.


   ACKNOWLEDGEMENTS
 
The authors wish to thank Dr John Hershey for helpful discussions concerning these investigations. This work was funded by a grant from the National Institute of Child Health and Development (HD 40661; P.J.H.), by support from the Boory and Cooper/Fishman/Kraff family funds, and by the UC Davis MIND Institute.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Department of Biological Chemistry, University of California, Davis, School of Medicine, One Shields Ave, Davis, CA 95616, USA. Tel: +1 5307547266; Fax: +1 5307547269; Email: pjhagerman{at}ucdavis.edu Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Verkerk, A.J., Pieretti, M., Sutcliffe, J.S., Fu, Y.H., Kuhl, D.P., Pizzuti, A., Reiner, O., Richards, S., Victoria, M.F., Zhang, F.P. et al. (1991) Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell, 65, 905–914.[CrossRef][Web of Science][Medline]

  2. Bardoni, B. and Mandel, J.L. (2002) Advances in understanding of fragile X pathogenesis and FMRP function, and in identification of X linked mental retardation genes. Curr. Opin. Genet. Dev., 12, 284–293.[CrossRef][Web of Science][Medline]

  3. Dorn, M.B., Mazzocco, M.M. and Hagerman, R.J. (1994) Behavioral and psychiatric disorders in adult male carriers of fragile X. J. Am. Acad. Child Adolesc. Psychiat., 33, 256–264.[Web of Science][Medline]

  4. Riddle, J.E., Cheema, A., Sobesky, W.E., Gardner, S.C., Taylor, A.K., Pennington, B.F. and Hagerman, R.J. (1998) Phenotypic involvement in females with the FMR1 gene mutation. Am. J. Ment. Retard., 102, 590–601.[CrossRef][Web of Science][Medline]

  5. Hagerman, R.J. and Hagerman, P.J. (2002) The fragile X premutation: into the phenotypic fold. Curr. Opin. Genet. Dev., 12, 278–283.[CrossRef][Web of Science][Medline]

  6. Allingham-Hawkins, D.J., Babul-Hirji, R., Chitayat, D., Holden, J.J., Yang, K.T., Lee, C., Hudson, R., Gorwill, H., Nolin, S.L., Glicksman, A. et al. (1999) Fragile X premutation is a significant risk factor for premature ovarian failure: the International Collaborative POF in Fragile X study–preliminary data. Am. J. Med. Genet., 83, 322–325.[CrossRef][Web of Science][Medline]

  7. Schwartz, C.E., Dean, J., Howard-Peebles, P.N., Bugge, M., Mikkelsen, M., Tommerup, N., Hull, C., Hagerman, R., Holden, J.J. and Stevenson, R.E. (1994) Obstetrical and gynecological complications in fragile X carriers: a multicenter study. Am. J. Med. Genet., 51, 400–402.[CrossRef][Web of Science][Medline]

  8. Hagerman, R.J., Leehey, M., Heinrichs, W., Tassone, F., Wilson, R., Hills, J., Grigsby, J., Gage, B. and Hagerman, P.J. (2001) Intention tremor, parkinsonism, and generalized brain atrophy in male carriers of fragile X. Neurology, 57, 127–130.[Abstract/Free Full Text]

  9. Brunberg, J.A., Jacquemont, S., Hagerman, R.J., Berry-Kravis, E.M., Grigsby, J., Leehey, M.A., Tassone, F., Brown, W.T., Greco, C.M. and Hagerman, P.J. (2002) Fragile X premutation carriers: characteristic MR imaging findings of adult male patients with progressive cerebellar and cognitive dysfunction. AJNR Am. J. Neuroradiol., 23, 1757–1766.[Abstract/Free Full Text]

  10. Greco, C.M., Hagerman, R.J., Tassone, F., Chudley, A.E., Del Bigio, M.R., Jacquemont, S., Leehey, M. and Hagerman, P.J. (2002) Neuronal intranuclear inclusions in a new cerebellar tremor/ataxia syndrome among fragile X carriers. Brain, 125, 1760–1771.[Abstract/Free Full Text]

  11. Jacquemont, S., Hagerman, R.J., Leehey, M., Grigsby, J., Zhang, L., Brunberg, J.A., Greco, C., Des Portes, V., Jardini, T., Levine, R. et al. (2003) Fragile X premutation tremor/ataxia syndrome: molecular, clinical, and neuroimaging correlates. Am. J. Hum. Genet., 72, 869–878.[CrossRef][Web of Science][Medline]

  12. Berry-Kravis, E., Lewin, F., Wuu, J., Leehey, M., Hagerman, R., Hagerman, P. and Goetz, C.G. (2003) Tremor and ataxia in fragile X premutation carriers: blinded videotape study. Ann. Neurol., 53, 616–623.[CrossRef][Web of Science][Medline]

  13. Tassone, F., Hagerman, R.J., Taylor, A.K., Gane, L.W., Godfrey, T.E. and Hagerman, P.J. (2000) Elevated levels of FMR1 mRNA in carrier males: a new mechanism of involvement in fragile X syndrome. Am. J. Hum. Genet., 66, 6–15.[CrossRef][Web of Science][Medline]

  14. Kenneson, A., Zhang, F., Hagedorn, C.H. and Warren, S.T. (2001) Reduced FMRP and increased FMR1 transcription is proportionally associated with CGG repeat number in intermediate-length and premutation carriers. Hum. Mol. Genet., 10, 1449–1454.[Abstract/Free Full Text]

  15. Primerano, B., Tassone, F., Hagerman, R.J., Hagerman, P., Amaldi, F. and Bagni, C. (2002) Reduced FMR1 mRNA translation efficiency in fragile X patients with premutations. RNA, 8, 1482–1488.[Abstract]

  16. Napierala, D. and Slomski, R. (1998) [Regulation of human DMD gene expression. An example of multiple utilization of genetic information]. Postepy Biochem., 44, 48–55.[Medline]

  17. Chiang, P.W., Carpenter, L.E. and Hagerman, P.J. (2001) The 5'-untranslated region of the FMR1 message facilitates translation by internal ribosome entry. J. Biol. Chem., 276, 37916–37921.[Abstract/Free Full Text]

  18. Shimizu, M., Gellibolian, R., Oostra, B.A. and Wells, R.D. (1996) Cloning, characterization and properties of plasmids containing CGG triplet repeats from the FMR-1 gene. J. Mol. Biol., 258, 614–626.[CrossRef][Web of Science][Medline]

  19. Hirst, M.C. and White, P.J. (1998) Cloned human FMR1 trinucleotide repeats exhibit a length- and orientation-dependent instability suggestive of in vivo lagging strand secondary structure. Nucl. Acids Res., 26, 2353–2358.[Abstract/Free Full Text]

  20. Trinh, T.Q. and Sinden, R.R. (1991) Preferential DNA secondary structure mutagenesis in the lagging strand of replication in E. coli. Nature, 352, 544–547.[CrossRef][Medline]

  21. Kang, S., Ohshima, K., Shimizu, M., Amirhaeri, S. and Wells, R.D. (1995) Pausing of DNA synthesis in vitro at specific loci in CTG and CGG triplet repeats from human hereditary disease genes. J. Biol. Chem., 270, 27014–27021.[Abstract/Free Full Text]

  22. Tassone, F., Hagerman, R.J., Loesch, D.Z., Lachiewicz, A., Taylor, A.K. and Hagerman, P.J. (2000) Fragile X males with unmethylated, full mutation trinucleotide repeat expansions have elevated levels of FMR1 messenger RNA. Am. J. Med. Genet., 94, 232–236.[CrossRef][Web of Science][Medline]

  23. Muller-Hartmann, H., Deissler, H., Naumann, F., Schmitz, B., Schroer, J. and Doerfler, W. (2000) The human 20-kDa 5'-(CGG)(n)-3'-binding protein is targeted to the nucleus and affects the activity of the FMR1 promoter. J. Biol. Chem., 275, 6447–6452.[Abstract/Free Full Text]

  24. Kozak, M. (1986) Influences of mRNA secondary structure on initiation by eukaryotic ribosomes. Proc. Natl Acad. Sci. USA, 83, 2850–2854.[Abstract/Free Full Text]

  25. Kozak, M. (1989) Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs. Mol. Cell. Biol., 9, 5134–5142.[Abstract/Free Full Text]

  26. Bouvet, P., Paris, J., Phillippe, M. and Osborne, H.B. (1991) Degradation of a developmentally regulated mRNA in Xenopus embryos is controlled by the 3' region and requires the translation of another maternal mRNA. Mol. Cell. Biol., 11, 3115–3124.[Abstract/Free Full Text]

  27. McMillan, J.P. and Singer, M.F. (1993) Translation of the human LINE-1 element, L1Hs. Proc. Natl Acad. Sci. USA, 90, 11533–11537.[Abstract/Free Full Text]

  28. Hess, M.A. and Duncan, R.F. (1996) Sequence and structure determinants of Drosophila Hsp70 mRNA translation: 5'UTR secondary structure specifically inhibits heat shock protein mRNA translation. Nucl. Acids Res., 24, 2441–2449.[Abstract/Free Full Text]

  29. Dorin, D., Bonnet, M.C., Bannwarth, S., Gatignol, A., Meurs, E.F. and Vaquero, C. (2003) The TAR RNA-binding protein, TRBP, stimulates the expression of TAR- containing RNAs in vitro and in vivo independently of its ability to inhibit the dsRNA-dependent kinase PKR. J. Biol. Chem., 278, 4440–4448.[Abstract/Free Full Text]

  30. Erard, M., Barker, D.G., Amalric, F., Jeang, K.T. and Gatignol, A. (1998) An Arg/Lys-rich core peptide mimics TRBP binding to the HIV-1 TAR RNA upper-stem/loop. J. Mol. Biol., 279, 1085–1099.[CrossRef][Web of Science][Medline]

  31. Tassone, F., Hagerman, R.J., Chamberlain, W.D. and Hagerman, P.J. (2000) Transcription of the FMR1 gene in individuals with fragile X syndrome. Am. J. Med. Genet., 97, 195–203.[CrossRef][Web of Science][Medline]

  32. Feng, Y., Zhang, F., Lokey, L.K., Chastain, J.L., Lakkis, L., Eberhart, D. and Warren, S.T. (1995) Translational suppression by trinucleotide repeat expansion at FMR1. Science, 268, 731–734.[Abstract/Free Full Text]

  33. Mankodi, A., Logigian, E., Callahan, L., McClain, C., White, R., Henderson, D., Krym, M. and Thornton, C.A. (2000) Myotonic dystrophy in transgenic mice expressing an expanded CUG repeat. Science, 289, 1769–1773.[Abstract/Free Full Text]

  34. Miller, J.W., Urbinati, C.R., Teng-Umnuay, P., Stenberg, M.G., Byrne, B.J., Thornton, C.A. and Swanson, M.S. (2000) Recruitment of human muscleblind proteins to (CUG)(n) expansions associated with myotonic dystrophy. EMBO J., 19, 4439–4448.[CrossRef][Web of Science][Medline]

  35. Finsterer, J. (2002) Myotonic dystrophy type 2. Eur. J. Neurol., 9, 441–447.[CrossRef][Web of Science][Medline]

  36. Mankodi, A. and Thornton, C.A. (2002) Myotonic syndromes. Curr. Opin. Neurol., 15, 545–552.[CrossRef][Web of Science][Medline]

  37. Salat, U., Bardoni, B., Wohrle, D. and Steinbach, P. (2000) Increase of FMRP expression, raised levels of FMR1 mRNA, and clonal selection in proliferating cells with unmethylated fragile X repeat expansions: a clue to the sex bias in the transmission of full mutations? J. Med. Genet., 37, 842–850.[Abstract/Free Full Text]

  38. Fu, Y.H., Kuhl, D.P., Pizzuti, A., Pieretti, M., Sutcliffe, J.S., Richards, S., Verkerk, A.J., Holden, J.J., Fenwick, R.G., Jr., Warren, S.T. et al. (1991) Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox. Cell, 67, 1047–1058.[CrossRef][Web of Science][Medline]

  39. Brown, W.T., Houck, G.E., Jr., Jeziorowska, A., Levinson, F.N., Ding, X., Dobkin, C., Zhong, N., Henderson, J., Brooks, S.S. and Jenkins, E.C. (1993) Rapid fragile X carrier screening and prenatal diagnosis using a nonradioactive PCR test. JAMA, 270, 1569–1575.[Abstract/Free Full Text]

  40. Zhong, N., Liu, X., Gou, S., Houck, G.E., Jr., Li, S., Dobkin, C. and Brown, W.T. (1994) Distribution of FMR-1 and associated microsatellite alleles in a normal Chinese population. Am. J. Med. Genet., 51, 417–422.[CrossRef][Web of Science][Medline]

  41. Garcia Arocena, D., Breece, K.E. and Hagerman, P.J. (2003) Distribution of CGG repeat sizes within the fragile X mental retardation 1 (FMR1) homologue in a non-human primate population. Hum. Genet., 7, 7.

  42. Leehey, M.A., Munhoz, R.P., Lang, A.E., Brunberg, J.A., Grigsby, J., Greco, C., Jacquemont, S., Tassone, F., Lozano, A.M., Hagerman, P.J. et al. (2003) The fragile X premutation presenting as essential tremor. Arch. Neurol., 60, 117–121.[Abstract/Free Full Text]

  43. Eichler, E.E., Kunst, C.B., Lugenbeel, K.A., Ryder, O.A., Davison, D., Warren, S.T. and Nelson, D.L. (1995) Evolution of the cryptic FMR1 CGG repeat. Nat. Genet., 11, 301–308.[CrossRef][Web of Science][Medline]

  44. Chong, S.S., Eichler, E.E., Nelson, D.L. and Hughes, M.R. (1994) Robust amplification and ethidium-visible detection of the fragile X syndrome CGG repeat using Pfu polymerase. Am. J. Med. Genet., 51, 522–526.[CrossRef][Web of Science][Medline]


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