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Originally published In Press as doi:10.1074/jbc.M405130200 on July 29, 2004

J. Biol. Chem., Vol. 279, Issue 40, 41563-41572, October 1, 2004
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Imperfect CAG Repeats Form Diverse Structures in SCA1 Transcripts*

Krzysztof Sobczak and Wlodzimierz J. Krzyzosiak{ddagger}

From the Laboratory of Cancer Genetics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland

Received for publication, May 10, 2004 , and in revised form, July 15, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The expanded CAG repeat in the coding sequence of the spinocerebellar ataxia type 1 (SCA1) gene is responsible for SCA1, one of the hereditary human neurodegenerative diseases. In the normal SCA1 alleles usually 1–3 CAT triplets break the continuity of the CAG repeat tracts. Here we show what is the structural role of the CAU interruptions in the SCA1 transcripts. Depending on their number and localization within the repeat tract the interruptions either enlarge the terminal loop of the hairpin formed by the repeats, nucleate the internal loops in its stem structure, or force the repeats to fold into two smaller hairpins. Thus, the interruptions destabilize the CAG repeat hairpin, which is likely to decrease its ability to participate in the putative RNA pathogenesis mechanism driven by the long CAG repeat hairpins.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The spinocerebellar ataxia type 1 (SCA1)1 belongs to a larger group of human hereditary neurological diseases that are caused by triplet repeat expansions in single affected genes (1–3). The best known examples of these diseases are: myotonic dystrophy (DM) caused by the expansion of CTG repeat in 3'-UTR of the DMPK gene; fragile X-associated tremor-ataxia syndrome (FXTAS), and mental retardation syndrome (FXS) resulting from, respectively, smaller or larger expansions of the CGG repeat in 5'-UTR of the FMR1 gene; as well as Huntington disease (HD) in which the CAG repeat expansion in the protein coding region of the IT-15 gene is implicated. One of the hallmarks of these diseases is genetic anticipation i.e. an earlier age of the onset and increased severity of clinical symptoms in subsequent generations, correlated with the increasing length of expanded alleles. The SCA1 usually appears in the fourth decade of life and death occurs between 10 and 15 years after the onset of symptoms. Typical clinical features of the SCA1 include ataxia, dysarthria and bulbar dysfunction. The neuropathology of the SCA1 is characterized by the progressive loss of neurons in the cerebellum, brain stem, and spinocerebellar tract.

The SCA1 gene that encodes nuclear protein ataxin-1 of a yet unknown function, harbors the expandable CAG repeat in its coding region (4–6). The repeat is polymorphic in length and usually contains CAT interruptions in a normal population. The SCA1 mutant alleles contain from 39 to about 80 uninterrupted CAG repeats (1–6) encoding the expanded polyglutamine (poly-Q) tracts in the aberrant ataxin-1. The SCA1 pathology correlates with the appearance of nuclear aggregates containing the mutated protein (7) but whether the aggregate formation is a symptom or cause of the disease is still a matter of debate (8–10). The aberrant ataxin-1 either in its soluble form or in the form of aggregates was shown to be toxic to neurons supporting the notion that the poly-Q tract itself may play a critical role in SCA1 pathogenesis (7–12). It may interfere with neuronal transcription by interacting with specific transcription factors in a poly-Q-dependent manner and induce apoptotic cell death (13). On the other hand, recent studies revealed the importance of the ataxin-1-specific sequence context for the protein function and showed that even the high level expression of an expanded poly-Q tract protein in the nucleus may be insufficient to cause disease (14). In this light the RNA-mediated mechanisms of pathogenesis, similar to those proposed for DM (15) and FXTAS (16–18) were also considered for polyglutamine diseases (17–21). According to this mechanism the expanded repeats in transcripts form long stable hairpins, which recruit double-stranded RNA-binding proteins in a repeat length-dependent manner, sequestering them from other transcripts and interfering with their normal functions (22).

The length of CAG repeat in the SCA1 gene ranges from 6 to 43 repeats and the number of CAT interruptions varies from 0 to 3 in a normal population (1–6, 23). Considering both repeat length and its interruption status about 40 different SCA1 alleles are known. The alleles containing two CAT interruptions strongly predominate in all analyzed populations (78–95%). They range in length from 23 to 43 repeats and a single CAG triplet always separates CAT interruptions. The alleles harboring one or three interruptions contribute to 0–11% and 0–4% of all alleles, and normal SCA1 variants containing uninterrupted repeat tracts of a maximum length of 21 repeats are also rare and contribute to 0–7% in different populations (23). In contrast to normal alleles, the great majority of pathologically expanded SCA1 variants do not contain repeat interruptions.

The postulated role of interruptions occurring within tandem repeats in genes is to prevent repeat expansion or contraction during DNA replication and repair. Their loss in the CAG repeat of the SCA1 gene generates much longer pure repeat tracts that are genetically less stable and more prone to expansion (23–27). There are several possible mechanisms by which the interruptions may enhance the stability of the repeat tracts (25–27). They may inhibit slippage between nascent and template strands by keeping them in register, or destabilize the hairpin structures aberrantly formed by slipped DNA strands.

The SCA1 alleles containing either the interrupted normal repeats or the expanded mutant repeats are transcribed, processed, transported to cytoplasm, and translated. The question of the possible differences in the efficiency of these processes between various normal and between the normal and mutant SCA1 transcripts has not been addressed so far. The physiological cellular function of the CAG repeats in transcripts, and the role of repeat interruptions is also completely unknown. To make the first step toward finding answers to these questions we describe here structures formed by the repeat regions in SCA1 transcripts representing different types of naturally occurring SCA1 alleles.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of DNA Templates and in Vitro Transcription—DNA templates for in vitro transcription were synthesized by PCR from gel-purified SCA1 amplification products containing different variants of the CAG repeat tracts as described earlier (23), using the forward primer (SCA1-FT7) containing a T7 RNA polymerase promoter 5'-TAATACGACTCACTATAGGGCAGTCTGAGCCA and reverse primer (SCA1-RT7) 5'-TCTGCTGGGCTGGTGG. The PCR was performed in a 50-µl reaction containing: a DNA template, 1 µM each primer SCA1-FT7 and SCA1-RT7, 10 mM Tris-HCl, pH 9.0, 1.5 mM MgCl2, 50 m M KCl, 0.1% Triton X-100, 200 µM of each of the dNTPs, and 1.5 units of TaqDNA polymerase (Promega) in the following conditions: 94 °C, 1 min; 35 cycles: 94 °C, 1 s, 62 °C, 1 s, 72 °C, 20 s.

A transcription reaction carried out in a 50-µl volume contained about 10 pmol of DNA template (10–20 µl of the PCR product described above), 400 units of T7 RNA polymerase (Epicenter), 3 mM guanosine, 0.5 mM each of ribo-NTPs, 10 mM dithiothreitol, 40 mM Tris-HCl (pH 7.9), 6 mM MgCl2, 2 mM spermidine, 10 mM NaCl, and 50 units of RNazin. Incubation was at 37 °C for 1 h. The RNA products were purified in denaturing 6% polyacrylamide gels, and 5'-end-labeled with 10 units of T4 polynucleotide kinase (Epicenter) and 50 µCi of [{gamma}-32P]ATP (4500 Ci/mmol; ICN) at 37 °C for 5 min.

Preparation of Human Fibroblast Whole Cell Extract—The whole cell extract was prepared from 6 x 107 human fibroblast cells growing in log phase according to modified standard procedure (28). In brief, the cells were pelleted and washed twice in phosphate-buffer saline and resuspended in a double volume of hypotonic swelling buffer (7 mM Tris-HCl, pH 7.5, 7 mM KCl, 1 mM MgCl2, 1 mM {beta}-mercaptoethanol) and after 10 min incubation on ice, they were homogenized 30x in Dounce homogenizer followed by the addition of one-tenth of the final volume of the neutralizing buffer (21 mM Tris-HCl, pH 7.5, 116 mM KCl, 3.6 mM MgCl2, 6 mM {beta}-mercaptoethanol). Finally homogenate was centrifuged at 21,000 x g for 5 min at 4 °C, and supernatant was stored at –70 °C with 40% glycerol.

Nuclease Digestions and Lead Cleavages in Water Solution and in Cellular Extract—Prior to structure probing reactions, the 32P-labeled RNAs were subjected to a denaturation and renaturation procedure in a solution containing 20 mM Tris-HCl (pH 7.2), 80 mM NaCl, 2 mM MgCl2, by heating the sample at 80 °C for 1 min and then slowly cooling to 37 °C. Limited RNA digestion was initiated by mixing 5 µl of the RNA sample (50,000 cpm) with 5 µl of a probe solution containing either lead ions or nucleases S1, Mung Bean or ribonucleases T1, T2 (final probe concentrations are specified in the figure legends). The structure probing experiments were performed either in 1 or 10 mM final concentration of MgCl2 (other reaction conditions were the same) and no significant differences in cleavage patterns were observed. RNase H digestion was performed by adding to the transcript solution, after denaturation and renaturation procedure (as above), one of the following three types of oligodeoxynucleotides: libraries of random 6-mers (100 pmol) or random 7-mers (100 pmol) or complementary to CAG repeats oligonucleotide with sequence 5'-TGCTGCT (5 pmol), and RNase H to the final concentration of 0.10 or 0.25 units/µl. All reactions were performed at 37 °C for 20 min and stopped by adding an equal volume of stop solution (7.5 M urea and 20 mM EDTA with dyes) and sample freezing.

The reactions in cell extracts were performed as follows. Before the nuclease treatment 1 µl of RNA sample in water solution (100,000 cpm) was preincubated at 37 °C for 5 min with 3 µl of cellular extract (protein concentration 2 mg/ml and the exogenously added Mg2+ and K+ ions were at about 0.6 mM and 15 mM concentration, respectively). Limited enzymatic digestion was initiated by adding to the RNA sample 2 µl of probe solution with different enzyme concentration: RNase T1 (0.05 and 0.1 units/µl), RNase T2 (0.02 and 0.05 units/µl), nuclease S1 (2.5 and 5.0 units/µl). All reactions were conducted at 37 °C for 5 min and stopped by phenol/chloroform extraction. After precipitation the RNA samples were diluted with the stop solution described above and loaded on the gel.

Analysis of the Reaction Products—To determine the cleavage sites, the products of lead-induced hydrolysis and nuclease digestion were separated in 8–15% polyacrylamide gels containing 7.5 M urea, 90 mM Tris borate buffer, and 2 mM EDTA, along with the products of alkaline hydrolysis and limited T1 ribonuclease digestion of the same RNA molecule. The alkaline hydrolysis ladder was generated by the incubation of the labeled RNA in formamide containing 0.5 mM MgCl2 at 100 °C for 10 min. The partial T1 ribonuclease digestion of the RNAs was performed under semi-denaturing conditions (10 mM sodium citrate, pH 5.0; 3.5 M urea) with 0.2 units/µl of the enzyme during incubation at 55 °C for 10 min. The cleavage sites characteristic for nucleases S1 and H were determined by comparison with the homologous S1 ladder (not shown). The RNA fragments generated by nucleases S1 and H terminate with 3'-hydroxyls and migrate slower than the corresponding formamide and T1 fragments. Electrophoresis was performed at 1500 V and was followed by autoradiography at –80 °C.

Electrophoresis in Nondenaturing Conditions—The RNA structure homogeneity was analyzed for each transcript by the electrophoresis of radiolabeled samples in 10% nondenaturing polyacrylamide gel 400/300/0.4 mm (acrylamide/bisacrylamide, 29/1) buffered with 45 mM Tris borate, at a fixed temperature of 37 °C. Prior to gel electrophoresis, the 32P-labeled transcripts were subjected to a denaturation and renaturation procedure in a solution containing 10 mM Tris-HCl (pH 7.2), 40 mM NaCl, 10 mM MgCl2, by heating the sample at 80 °C for 1 min. and slowly cooling it to 37 °C, and mixed with an equal volume of 7% sucrose with dyes. Electrophoresis was conducted at a constant power of 15 watts and was followed by phosphorimager analysis.

RNA Secondary Structure Modeling—RNA secondary structure prediction was performed using the mfold program version 3.1 (29). This program is designed to determine the optimal and suboptimal secondary structures of RNA calculated for 1 M NaCl solution at 37 °C, and to count free energy contributions for various secondary structure motifs.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Fifteen distinct SCA1 transcripts analyzed in this study differed in the number of CAG repeats from 27 to 54, while having the same specific flanking sequences 37 nt at the 5'-side and 59 nt at 3'-side of the repeat. Besides their length, the repeat regions varied also in the number and locations of the repeat interruptions (Table I). Accordingly, the transcripts could be divided into four groups. The first and the largest group (10 transcripts) included all those containing two CAU interruptions described by the formula (CAG)nCAUCAGCAU(CAG)m, in short n-1-1-1-m (interruptions are underlined). Within this group there were transcripts containing 27–36 repeats and either symmetrical or asymmetrical distribution of the interruptions. In the first subgroup (2 transcripts), the interruption region was flanked by 12 or 15 pure CAG repeats. In the larger second subgroup with the asymmetrical CAU distribution the longer tract of pure repeats was located either at the 5'-side n>m, or at 3'-side m>n of the interruption region. It is noteworthy, that within this group is a transcript from SCA1 allele from healthy individual, which contains as many as 43 repeats in the configuration 23-1-1-1-17. The genetic stability of such long alleles is secured by the presence of repeat interruptions (23, 30). In the second group there are two transcripts containing a single asymmetrically located CAU interruption in the tract of either 28 or 29 CAG repeats, and a transcript containing three interruptions in the 11-1-1-1-1-1-15 configuration is the only representative of the third group. Finally, the last group contains two transcripts harboring pure pathologically expanded repeat tracts of the 53 and 54 CAG repeats derived from SCA1 patients. Analyzed transcripts (Table I) represented all possible types of the interruption patterns described so far in world populations (23). In their selection the number and localization of CAU triplets as well as the total length of a triplet repeat region were taken into account.


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  		TABLE I
Characteristics of repeat interruption patterns in the analyzed SCA1 RNA fragments and the names of in vitro transcripts

 
All investigated transcripts were end-labeled and their solution structure was analyzed with a set of well-characterized chemical and enzymatic probes. Among the single-strand specific nucleases used were the nucleases S1 and Mung Bean showing little sequence specificity, RNase T2 cutting all internucleotide bonds but showing some preference for those after A-residues and G-specific RNase T1 (31, 32). The metal ion probes were represented by lead ions that cleave single-stranded RNA sections and relaxed parts of duplex structures (33–35). Also, the short oligodeoxynucleotide 5'-TGCTGCT (ctg-ODN), as well as the ODN libraries (6-mer and 7-mer) with a randomized sequence were used together with Escherichia coli RNase H, which executed RNA cleavages at the sites of RNA/DNA hybrids. Earlier studies have shown that RNA is cut at the 3'-side of the RNA/DNA hybrid and 5'-phosphate and 3'-OH groups are left at the cleavage site (36–38).

Pure Expanded CAG Repeats Form a Stable Hairpin Structure, Which Is Further Stabilized by Specific Flanking Sequences in the SCA1 Transcript—The normal SCA1 alleles containing pure uninterrupted CAG repeats are very rare and they were found in a few populations only. On the other hand, all mutant alleles contain pure repeat tracts. The structure probing results obtained for transcripts from two expanded SCA1 alleles are shown in Fig. 1. The most intense cuts generated by nucleases occur between the nucleotides A26 and A28 (i.e. between the adenyl residues of the CAG repeats 26 and 28) in the pure(CAG)53, and between A26 and A29 in pure(CAG)54. The rest of the repeat region is mildly cleaved by lead ions, which show some preference for cuts at the CpA bonds over the ApG, and cleavages at the internucleotide bonds C1pA1 and A1pG1 within the first CAG repeat are enhanced. Also, the enhanced RNase T2 and RNase H cuts can be detected in that region. What is noteworthy, is that none of the probes used cleaved short 6–7-nt sequences directly flanking the repeat region at both its sides. These results imply that the entire repeat region forms an autonomous structure, which includes the repeats themselves (hairpin H4) and the cleavage-resistant duplex formed by the six base pairs of its flanking sequences (H3) (Fig. 1B). This kind of structure explains why the different number of the reactive internucleotide bonds is observed in the terminal loop of the pure(CAG)53 and pure(CAG)54. In the former, the 4-nucleotide terminal loop occurs, whereas the 7-nucleotide loop region is present in the latter (Fig. 1B). These results are in accord with the observations made for shorter model transcripts (CNG)16 and (CNG)17 containing the artificial G-C clamp at the base of their hairpin stem (20). For comparison the model transcripts having not-clamped repeats were shown to have freedom of alignment and formed several alternative "slipped" structures each containing 4-nucleotide terminal loop (20). The lack of enhanced cuts at the 3'-terminal CAG repeats in pure(CAG)53 and pure(CAG)54 may serve as further argument for the existence of a natural clamp formed by a repeat flanking sequence in the SCA1 transcripts. This H3 duplex adds extra stability to the CAG repeat hairpin. Importantly, the same cleavage resistance of flanking sequences, which is shown for the two mutant transcripts is also observed in all other transcripts analyzed in this study. This means that the same type of structure (H3) is formed by flanking sequences in all these transcripts (Fig. 2). At the 3'-side of the repeat clamp another stable hairpin structure occurs (H5). It is composed of 11 uninterrupted base pairs and 7-nucleotide terminal loop, which is well mapped by the nucleases but not cleaved by RNase H in the presence of the 6-mer and 7-mer ODN libraries. The close proximity of the H3 and H5 helices, together with cleavage resistance of the region, which separates them, may suggest their involvement in the coaxial stacking, which could additionally stabilize the structure formed by the CAG repeat. According to the SCA1 mRNA structure modeling by the mfold algorithm, the helices H3 and H5 are also predicted to exist in the much longer mRNA fragments. Our experimental data show that the predicted single stranded regions that occur upstream of the H3 and downstream of the H5 are both efficiently cleaved by all the nucleases used and by the RNaseH/ODN library. The analysis of the transcripts containing much longer flanking regions confirmed the existence of the helices H3, H4, and H5 (data not shown).



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  		FIG. 1.
Long pure CAG repeat tracts of mutant SCA1 transcripts form clamped hairpin structures. A, cleavage patterns obtained for the 5'-end labeled pure(CAG)53 (upper electropherogram) and pure(CAG)54 transcript (lower electropherogram) denatured and renatured in buffer containing 1 mM concentration of Mg2+ ions and treated with S1 nuclease (0.5 units/µl, 1 units/µl and 2 units/µl; 1 mM ZnCl2 was present in each reaction); RNase T1 (0.5 units/µl, 1 units/µl, 1.5 units/µl); RNase T2 (0.1 units/µl, 0.2 units/µl, 0.4 units/liter); lane Ci, incubation control (no probe); lane F, formamide ladder; lane T, guanine specific ladder. Electrophoresis in an 8% polyacrylamide gel under denaturing conditions. Positions of selected G-residues of the CAG repeats and symbols of RNA structure modules present in this transcript are indicated. B, proposed secondary structure of the pure(CAG)54 transcript fragment containing CAG repeats as well as the terminal loop region of pure(CAG)53 shown in-frame. Cleavage sites and intensities are specified for each probe according to symbols shown in the legend. RNA structure modules present, their stability expressed in the free energy value (kcal/mol), and positions of selected G-residues from 1 to 54 of CAG repeats are also shown. The hairpin structure region that is repeated 3x and flanking sequences forming the H3 hairpin are shown by a gray background.

 



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  		FIG. 2.
Specific flanking regions form identical structures in all SCA1 repeat variants. A, cleavage patterns of the 12-1-1-1-12 transcript treated with three nucleases, S1, T1, and T2, used at concentrations as specified in the legend to Fig. 1A. Electrophoresis in a 10% polyacrylamide gel in denaturing conditions. Positions of selected G-residues of CAG repeats and interruption positions marked by gray arrowheads are indicated. B, proposed secondary structure of two transcripts: the 12-1-1-1-12 and 15-1-1-1-15 with a symmetrically located interruption region. Position of U-residues of the interruptions are marked in gray ovals. Cleavage sites and intensities are specified for each probe used. The RNA secondary structure elements are designated as in the legend to Fig. 1B. C, melting of the 12-1-1-1-12 transcript structure as monitored by the S1 nuclease reaction at temperatures varying from 20 to 80 °C. The enzyme concentration was the same in all reactions (1 unit/µl with 0.25 mM ZnCl2 present) with different incubation times: 30, 20, 15, 10, 5, 2, and 1 min, respectively.

 
CAG Repeats Containing Either One or Two CAU Interruptions Usually Form a Single Hairpin Structure—Most of the normal SCA1 alleles contain two interruptions in their repeat tracts. The question we asked first was: what is the structural role of two CAU sequences located symmetrically within the CAG repeats in two SCA1 transcripts 12-1-1-1-12 and 15-1-1-1-15? The structure probing results (Fig. 2) show that the highest reactivity of all the nucleases used and RNase H/ODN is observed in the central region of the repeats in which also the interruptions are located. This reactive region is between the C13 and A16 in the 12-1-1-1-12 transcript (Fig. 2, A and B). In both transcripts the terminal loops are composed of 10 nucleotides and include both CAU triplets. The hairpin stem structure of the 12-1-1-1-12 transcript is stable as revealed by its temperature-induced melting followed by structure probing with the S1 nuclease (Fig. 2C). Only at the temperature of 70 °C and higher the structure formed by the helices H4 and H3 was melted and all internucleotide bonds of that region showed similar reactivity.

The structures formed by the repeats containing asymmetrically located interruptions are somewhat different (Fig. 3). In each case the most reactive regions are those in which the interruptions are located and also their closest neighborhood. In some transcripts with a small degree of asymmetry, in which the length difference between pure repeat tracts was one or two repeats ({Delta} = 1 or 2) the interruptions were located first in the asymmetric internal loop a and second in the 4-nucleotide ({Delta}= 1) or 7-nucleotide ({Delta}= 2) terminal loop b. The examples of such structures are presented in Fig. 3D. In transcripts in which one pure repeat tract is longer by more than three repeats than the other ({Delta} > 3) both interruptions occur in two asymmetric internal loops a and b whereas the terminal loop c is composed of 4 or 7 nucleotides of the centrally located CAG repeats (Fig. 3E). The short random ODNs bind more effectively to hairpin terminal loops than to the internal ones (Fig. 3B).



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  		FIG. 3.
Majority of the SCA1 repeat regions containing two interruptions form single hairpin structures. A, denaturing 15% polyacrylamide gel electrophoresis of the 12-1-1-1-14 and 12-1-1-1-15 transcripts treated with lead ions at concentrations: 0.25, 0.5, and 1 mM; as well as S1 and T1 nucleases at concentrations specified in the legend to Fig. 1A. B, product of the nuclease H cleavage generated after preincubation of the 13-1-1-1-14 transcript with short oligodeoxynucleotides. Lanes 7-mer, oligonucleotides with randomized 7-nt; lanes ctg, oligonucleotide of the 5'-TGCTGCT sequence complementary to CAG repeats. Other descriptions as in Figs. 1A and 2A. C, cleavages induced by S1 nuclease in the interruptions containing regions of three transcripts 12-1-1-1-14, 17-1-1-1-16 and 16-1-1-1-10 (probe concentrations as in the legend to Fig. 1A). D, proposed secondary structures of the SCA1 transcripts containing two interruptions between the pure CAG tracts differing by 1–2 repeats ({Delta}= 1 or 2). Positions of the U-residues of interruptions are marked in gray ovals, and positions of the selected G-residues of the CAG repeats are indicated. Cleavage sites and intensities are specified for each probe used as described in the Fig. 1B legend. Additionally, the putative binding sites of random 7-mers (light gray lines) and ctg-ODN (dark gray lines) are shown. Positions of selected G-residues of the CAG repeats and names of internal and terminal loops (a, b, c) are also indicated. E, as in C but for two transcripts containing two interruptions between pure CAG tracts differing by more than 3 repeats ({Delta} > 3).

 
Transcripts containing a single asymmetrically located interruption form hairpins with two loop regions. The internal loop a harbors the interruption and terminal loop b is the 4-nucleotide long in the 11-1-16 transcript and 7-nucleotide long in 12-1-16. Both types of loops are well recognized by all the structure probes used. It is also noteworthy that in the case of all of the above described transcripts the specific oligonucleotide ctg-ODN binds to the interrupted repeats with lower efficiency than to the pure repeats located in the single stranded regions. Notably, they also bind weakly to the repeats in the stem region but only at the 5'-side of the stem (Fig. 3B).

Long Repeat Tract with Asymmetric Interruptions Forms Two Stable Conformers—Unexpectedly, more complex cleavage patterns different from those generated in other transcripts were observed in the SCA1 transcript containing 43 repeats in the 23-1-1-1-17 configuration (Fig. 4A). This pattern of cleavages suggested the existence of at least two stable conformers under structure probing conditions. This prediction turned out to be correct as shown by the result of the transcript analysis by non-denaturing gel electrophoresis (Fig. 4B). Unlike other analyzed transcripts the 23-1-1-1-17 migrated as two distinct species. The question was what is the structure of each of these stable conformers? In answering this question the results of 16-1-1-1-10 transcript structure analysis turned out to be helpful (Fig. 3, C and E). This shorter transcript has a similarly localized interruption region, i.e. the length difference between the 5' and 3' pure CAG repeat tracts is identical ({Delta} = +6). Its cleavage pattern well corresponds to that observed at centrally located repeats in 23-1-1-1-17 (Fig. 4A). In both transcripts the most prominent T1 ribonuclease cuts (shown in black arrowhead in Fig. 4A) occur after G residues of the third CAG repeat counting from the interruption region (G14 and G21, respectively). Based on the presence of these cleavages we propose the single distorted hairpin structure for conformer I of 23-1-1-1-17 (Fig. 4C), which is similar to that determined for the corresponding 16-1-1-1-10 transcript (Fig. 3D). Additional cuts observed in 23-1-1-1-17 transcript occurred in the middle of the pure (CAG)23 repeats between the A11-A13, in the middle of the pure (CAG)17 repeats between A34 and A36 and in the interruption region at the repeats 24–26 (Fig. 4A). They fit well into the branched structure of conformer II composed of two smaller hairpins (H4a and H4b) each formed by pure repeat tracts at both sides of the interruption region. These hairpins are separated by the 9-nucleotide single-stranded spacer (loop a) that harbors both interruptions (Fig. 4C).



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  		FIG. 4.
Long transcript with two interruptions forms two different conformers. A, cleavage patterns obtained for similar transcripts ({Delta} = +6) 16-1-1-1-10 (left) and 23-1-1-1-17 (right) with use of ribonuclease T1 at concentration 1.0 units/µl and 2.0 units/µl (black arrowheads indicate the similar T1 cut positions in both transcripts as described in the text). Digestion sites of 23-1-1-1-17 differing in both conformers are marked at the right hand side of the electropherogram. Positions of selected G-residues of CAG repeats and interruption positions marked by gray arrowheads are indicated. B, non-denaturing 10% polyacrylamide gel electrophoresis of three SCA1 transcripts subjected to a denaturation/renaturation procedure before loading on the gel. Below, a comparison of the migration rates of the analyzed transcripts obtained from phosphorimaging. C, proposed secondary structures of co-existing conformer I and conformer II of the 23-1-1-1-17 transcript. The cleavages differing both conformers are shown. Gray ovals indicate the interruption positions. Other descriptions as in the legend to Fig. 1B.

 
Three Interruptions Rearrange the Structure of the Repeat Region—It appears from the analysis of the transcript in the 11-1-1-1-1-1-15 configuration (Fig. 5) that its structure differs from those of transcripts harboring two asymmetrically located interruptions (Fig. 3). The nucleases do not cut the internucleotide bonds belonging to the first repeats at the pure 3'-repeat tract. Such cleavages were observed for the similar transcript 12-1-1-1-17 in which a terminal loop was present at that site. Instead, the efficient cleavages occur in central parts of pure repeat tracts at A5-A7 and A23-A25 as well as in the region of interruptions A12-G17 (Fig. 5A). This cleavage pattern resembles that observed for conformer II of the 23-1-1-1-17 transcript (Fig. 4) and supports the four way junction structure within the repeat region. In this structure the helix H3, two hairpins composed of pure CAG repeats and lower stability hairpin containing the interruptions can be distinguished (Fig. 5B). The formation of a branched structure by the repeat region of this transcript is supported by its retarded migration during electrophoresis in non-denaturing polyacrylamide gels (Fig. 5C). It migrates slower than the transcript 17-1-1-1-16, which despite being 5 repeats longer has a more regular structure, and only slightly faster than transcript pure(CAG)54 that forms a single extended hairpin.



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  		FIG. 5.
The SCA1 transcript containing three interruptions forms branched hairpin structure. A, cleavages induced in the 11-1-1-1-1-1-15 transcript by S1 nuclease and T1 ribonuclease at concentrations indicated in the legend to Fig. 1A. Electrophoresis in a 10% polyacrylamideurea gel. Position of selected G-residues and regions of three terminal loops a–c are indicated. B, proposed secondary structure of the module H4 distinguishing the 11-1-1-1-1-1-15 transcript from transcripts containing two interruptions. Cleavage sites and intensities are specified for each of the probes used in this analysis as described in the Fig. 1B legend. Three terminal loops a–c are also indicated. C, electrophoretic mobility of six compared transcripts in the non-denaturing conditions. D, nuclease S1 cleavage patterns and the proposed secondary structure of the 11-1-16 transcript fragment with two looped-out regions a and b indicated. The interruption localization is marked by a gray oval.

 
Structures of the SCA1 Transcripts Are the Same in Cellular Extracts—The question often asked is whether the structures determined in vitro are also present in their natural cellular environment? In an attempt to answering this question we performed structure probing experiments in the whole cell extract from the human fibroblast cell line. It turned out from the analysis of two transcripts: 12-1-1-1-17 (Fig. 6) and 12-1-1-1-14 that the cuts generated by nucleases S1, T2, and T1 closely resembled those obtained in buffered water solution in the presence of Na1+ and Mg2+ ions only, and the observed differences were rather quantitative than qualitative supporting the notion that the structures which are most stable in vitro are likely to exist also in cells (39–42).



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  		FIG. 6.
The CAG repeat region of the SCA1 transcript forms the same structure in vitro and in cellular extract. Cleavage patterns of the 12-1-1-1-17 transcript treated with three nucleases, S1, T2, and T1 in two different reaction conditions: in water solution (left electropherogram) and in human fibroblast cellular extract (right electropherogram). The probe concentrations used were as specified in the legend to Fig. 1A and described under "Experimental Procedures." Electrophoresis was performed in a 10% polyacrylamide gel in denaturing conditions. Positions of selected G-residues of the CAG repeats and interruption positions marked by gray arrowheads are indicated.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we have determined the structures of numerous variants of the CAG repeat region in the SCA1 transcript. These variants are representative for the alleles most commonly occurring in a normal population and for those present in SCA1 patients. It turned out from this analysis, that the uninterrupted repeat tracts in two mutant SCA1 transcripts: pure(CAG)53 and pure(CAG)54, which are typical repeat lengths in SCA1 patients, form undistorted hairpin structures. They contain a stem composed of the periodically repeated two G-C and C-G pairs followed by A-A mismatch, and terminal loops of different sizes depending on the repeat number. This is so because a strong natural clamp is formed by the specific flanking sequences at the base of the repeat hairpin stem. This clamp forces the repeats to a single alignment, and the 7-nucleotide loop is formed in the transcript containing an even number of repeats whereas the 4-nucleotide loop is present in those having an odd repeat number. This pattern of the stem structure and loop sizes follows those observed for shorter (CAG)16 and (CAG)17 repeat tracts containing an artificial G-C clamp (20). Thus, it may be concluded from the above that both the uninterrupted normal repeats as well as expanded repeats present in mutation carriers form the same type of regular hairpin structure (Fig. 7). The only difference between transcripts from normal and mutant alleles is the length and stability of the repeat hairpin stem.



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  		FIG. 7.
The CAU interruptions shape the structure of CAG repeat regions in SCA1 transcripts. Left panel, schematic structures of repeat regions in mutant transcripts representing both typical and the shortest mutant alleles. Right panel, RNA structures of repeat regions from representative types of normal alleles either uninterrupted or containing CAU interruptions at various locations. Continuous horizontal gray line (threshold stem size) separates the normal from the putative pathogenic RNAs as described in the text. The length limits of hairpin stems composed of pure CAG repeats are indicated by the corresponding numbers for each transcript shown. Note that the maximum length of the hairpin stem, which is expressed in the number of CAG repeats, is double that of the shorter pure CAG repeat tract for single distorted hairpins. In the case of branched structures the corresponding value equals the length of the longer pure CAG repeat tract. These length limits were calculated taking into consideration all known SCA1 repeat configurations (23).

 
The main focus of this study was on the role of the CAU sequences interrupting the CAG repeat tracts in the SCA1 transcript. In this discussion we will consider them as the single G -> U base substitutions occurring at either one, two, or three closely located sites within the CAG repeat. In the regular CAG repeat hairpin stem such substitutions would result in the U-C or C-U base oppositions if they were located within the 5'- or 3'-part of the repeated sequence, respectively. Often the interruptions occur centrally within the repeat tract and the question was: whether they accommodate themselves in the repeat hairpin stem or reside in the terminal loop? The answer is that the C-U oppositions are reluctant to stay in the stem and they enlarge the loop structure.

The next question was: how do the U bases behave when their locations are more remote from the terminal loop? Will they be forced by their putative stem neighbors to stay within the stem or not? The answer to this question is also negative. They either form the local internal loops, which are well recognized by the single-strand specific probes or they redesign the structure of the repeat region entirely by splitting the repeated sequence into two separate hairpin structures (Fig. 7).

What is specific about the U-C and C-U oppositions that they have been selected to destabilize and reorganize hairpin structures formed by the CAG repeats? Why are they so rare in natural RNAs compared with other non-WC base pairs? Which of the postulated two geometries of the U-C pairs occurs in the RNAs: the U-C+ (with the neighboring carbonyl and imino groups involved in the formation of hydrogen bonds) or the neutral U-C pair (with N3-imino proton of U forming a water-mediated hydrogen bond to the N3 of C)? Some answers to these questions come from the crystal structures of RNA duplexes containing the U-C pairs (43–45). It appears that the RNA duplex containing two tandem U-C mismatches in its central positions is very unstable compared with a similar duplex with the U-U mismatches (43). The presence of eight water molecules, four per each U-C pair, bound to stabilize these pairs may be the factor that causes a severe nearly 2-fold widening of the major groove as compared with A-RNA, resulting in duplex destabilization (43). It is tempting to speculate that a similar effect may be the driving force for the CAG repeat duplex destabilization in the SCA1 transcripts. The periodic A-A mismatches that make the stem structure more relaxed as compared with the regular duplex may facilitate both the local structure changes and its global rearrangements.

Thus, the number and localization of U-residues within the CAG repeat tract in the SCA1 transcript are the factors determining structure of the repeat region. We postulate that the role of the CAG repeat interruptions is either the weakening or effective shortening of hairpins formed by pure repeats (Fig. 7), most likely to diminish their ability to interact with double-stranded RNA-binding proteins. Transcripts containing the uninterrupted double-stranded CAG repeats were shown earlier to activate the RNA-dependent protein kinase PKR (46, 47), and form a complex with the specific 63 kDa CAG repeatbinding protein (48). Such interactions resulting in protein activation or sequestration were considered as effects potentially triggering the mechanism of RNA-mediated pathogenesis also in a group of neurodegenerative diseases caused by the CAG repeat expansions (17–21,49). The prominent role of RNA is well documented for myoblast pathogenesis in two forms of myotonic dystrophy (15), and RNA toxicity is becoming recognized as an underlying cause of the more recently described FXTAS (16–18). In the case of DM1 and DM2 the repeat length-dependent sequestration of the muscleblind family proteins (22) is postulated, whereas the specific proteins that bind to the CGG repeat in FXTAS patients have not been characterized yet. It will be intriguing to see the extent to which the RNA-mediated pathogenesis shapes the polyglutamine toxicity in the cerebellum neurons of SCA1 patients. In Fig. 7 we presented in a simplified way the different types of structures formed by the repeat regions in the investigated SCA1 transcripts in order to see how they fit into the model of a putative RNA role in the SCA1 pathogenesis. The shortest CAG repeat tract found in SCA1 gene of spinocerebellar ataxia type 1 patients contains 39 pure CAG repeats. Both structures shown in Fig. 7 (left panel) have reached the pathogenic threshold, which is defined by the repeat hairpin stem composed of at least 38 CAG repeats. On the contrary, none of the transcripts from the normal SCA1 alleles shown in Fig. 7 (right panel) reaches this threshold. However, there are some unusual SCA1 alleles e.g. (CAG)45-CAT-CAG-CAT-(CAG)10 for which their total length (58 repeats) suggested an early onset of the disease at 22 years of age, whereas the actual age at SCA1 onset was 50 years (30). In addition, one of the SCA1 variants analyzed in this study, which belonged to an unaffected carrier, had 43 repeats in the 23-1-1-1-17 configuration (Fig. 7) i.e. did not reach the pathogenic threshold. In a 45-1-1-1-10 transcript similar type of structure to that of 23-1-1-1-17 is expected. The longer arm of this branched structure passes the pathogenic threshold reaching 45 CAG repeats. Thus, differences in the structures of transcripts from different SCA1 alleles may well explain their variable effects on phenotype.

Finally, the SCA1 gene is one of three genes implicated in triplet repeat expansion diseases that are equipped in the elaborated system of interruptions in repeat tracts. The SCA1 is, however, unique in the sense that its interruption system, which was likely developed to prevent DNA repeat expansions, has the potential to operate also on the levels of RNA and protein. The destabilization of longer potentially pathogenic CAG hairpins in RNA, and the insertion of histidines into the polyglutamine tracts in ataxin-1 may be considered the possible second and third lines of defense against SCA1 pathogenesis.


   FOOTNOTES
 
* This work was supported by the State Committee for Scientific Research, Grants No 2P05A-08826, PBZ-KBN-040/P04/2001, and Foundation for Polish Science, Grant 8/2000. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Laboratory of Cancer Genetics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14 St., 61-704 Poznan, Poland. Tel.: 48-61-8528503; Fax: 48-61-8520532; E-mail: wlodkrzy{at}ibch.poznan.pl.

1 The abbreviations used are: SCA1, spinocerebellar ataxia type 1; UTR, untranslated region; ODN, oligodeoxynucleotide; nt, nucleotide. Back



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K. Sobczak and W. J. Krzyzosiak
CAG Repeats Containing CAA Interruptions Form Branched Hairpin Structures in Spinocerebellar Ataxia Type 2 Transcripts
J. Biol. Chem., February 4, 2005; 280(5): 3898 - 3910.
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