Mutant CAG repeats of Huntingtin transcript fold into hairpins, form nuclear foci and are targets for RNA interference

Preparation of RNA fragments of HTT and AR transcripts

The (CAG)₁₀(CCG)₁₀ and (CAG)₂₀ oligoribonucleotides were purchased from Metabion (Martinsried, Germany). Fragments of natural HTT and AR mRNAs were obtained by in vitro transcription of cDNA fragments obtained by PCR amplification. These HTT and AR fragments were selected based on structure prediction for RNAs differing in the length of the repeat flanking sequences using Mfold program (22,23). Results of the molecular modeling demonstrated that the structures formed by the repeats and their nearest flanks are autonomous and not influenced by long-range interactions. The template for HTT transcripts containing both the CAG and the CCG repeat tracts was amplified with Had1 T7 forward and Hd2 reverse primers, and the template for the AR transcripts was obtained with AR1 T7 forward and AR2 reverse primers (Table 1). Both forward primers contained the T7 RNA polymerase promoter in vitro transcription was carried out with T7 RNA polymerase (Epicenter) as described previously (24). RNA products were purified by denaturing PAGE and were 5′-end-labeled with [γ-³²P] ATP using T4 polynucleotide kinase (Epicenter).

RNA structure probing

Prior to structure probing reactions, gel purified 5′-end-labeled RNAs were subjected to denaturation and renaturation in a solution containing 20 mM Tris–HCl (pH 7.2), 80 mM NaCl and 2 mM MgCl₂ by heating at 90°C for 1 min and slowly cooling to 37°C. Incomplete RNA digestion was initiated by mixing 5 μl of RNA sample (20–60 nM; 50 000 cpm) and 5 μl of probe solution containing one of the four nucleases (S1, T1, T2 or V1). For the S1 nuclease reaction, 1 mM ZnCl₂ was present. In single-stranded RNA structures, T1 and T2 RNases exclusively cleave after G and A residues, respectively, whereas S1 nuclease cleaves after all residues (25,26) with some preference for Gs (27,28). In addition, ribonuclease V1 was used, which is specific for double-stranded regions of RNA (25) and exhibits a preference for U (28,29). All nuclease reactions were performed at 37°C for 10 min and were terminated by adding an equal volume of stopping solution (7.5 M urea and 20 mM EDTA with dyes) and freezing. Nuclease digestion products were separated by short (12% gel), medium (10%) and long (6%) denaturing electrophoresis, and these were run in parallel with products of alkaline hydrolysis (1-nt ladder) and limited T1 nuclease digestion (G-specific ladder) of the transcript to visualize RNA cleavage occurring in different sections of the studied molecules at high resolution. The denaturing gel contained 7.5 M urea, 90 mM Tris–borate buffer and 2 mM EDTA. Products of the RNA structure probing were visualized by autoradiography and Phosphorimaging (Typhoon, Molecular Dynamics). The intensity of cleavages induced by the different probes is proportional to the strength of radioactive signals from cleavage products which were measured and categorized into three groups: strong, medium and weak.

Cell culture and siRNA transfection

Human fibroblast cell lines from a normal control patient (GM07492) as well as heterozygous HD (GM04208–21/44 CAG, GM04281–17/68 CAG, GM03864–15/46 CAG and GM09197–21/151 CAG), DRPLA (GM13717–15/65 CAG), SCA3 (GM06153–18/69 CAG), SCA7 (GM03561–8/62 CAG) and DM1 patients (GM03987–13/500 CTG) were purchased from Coriell Cell Repositories (NJ, USA). Fibroblast cultures were maintained in MEM medium (Lonza) containing 10% FBS, 1× antibiotic antimycotic solution (Sigma) and 1× MEM non-essential amino acid solution (Sigma). For RNA fluorescence in situ hybridization (FISH) and immunofluorescence (IF), cells were grown on cover slips pre-coated with 0.05% gelatin in 1× PBS. Prior to transfection, siRNA duplexes were prepared as follows: 100 μM of single-stranded siRNAs (Table 1) (Metabion) were prepared at 20 μM in annealing buffer (Metabion). Then, siRNAs were denatured for 1 min at 90°C followed by slow cooling at room temperature for 40 min. The siRNAs were then diluted in serum and antibiotic-free Opti-MEM (Invitrogen) and mixed with the transfection reagent, Lipofectamine 2000 (Invitrogen). The mixture was added to the cells grown in a 6-well plate and incubated at 37°C for 4 h. Fluorescent siRNAs (BLOCK-iT Florescent Oligo, Invitrogen) were used to monitor transfection efficiency.

Total RNA isolation and semi-quantitative RT–PCR

Total RNA was extracted from cells with TRIzol (Invitrogen). Equal amounts of RNA (1 μg) were reverse-transcribed with random primers (Promega) using SuperScript II Reverse Transcriptase (Invitrogen) according to the manufacturer’s protocol. Oligonucleotides and PCR conditions used for RT–PCR of human HTT, ATXN3, ATXN7 and ATN1 cDNA are shown in Tables 1 and 2, respectively. Each sample used was assayed in three independent RT reactions and normalized to GAPDH expression levels. PCR products were visualized by 1.3% agarose gel electrophoresis and quantified using Gel-Pro 3.1.

Subcellular fractionation and RNA extraction

Fibroblasts were harvested by trypsinization and collected in 15 ml tubes on ice, washed three times with cold PBS and pelleted at 500g for 3 min. Cell pellets were then split. One-fourth of the pellet was used for total RNA isolation with TRIzol (Invitrogen), whereas the remaining part was subjected to fractionation into the nuclear and cytoplasmic fractions with PARIS ^TM Kit (Ambion). RNA was extracted from the subcellular compartments according to the manufacturer’s guidelines. In general, 300–500 ng of RNA was used for cDNA preparation with SuperScript II Reverse Transcriptase (Invitrogen) and random hexamer primers (Promega). To control for genomic DNA contamination mock reactions were performed that lacked reverse transcriptase. Genomic DNA contamination was removed with DNase I treatment before RNA denaturation. The RNA samples were incubated with RNase-free DNase for 15 min at 25°C followed by DNase heat inactivation at 65°C for 10 min in the presence of 5 mM EDTA. Oligonucleotides used and PCR conditions for RT–PCR are shown in Tables 1 and 2, respectively. The depletion of nuclear RNA in cytoplasmic fraction was determined by RT–PCR with primers for HTT pre-mRNA and for 5′-external transcribed spacer (5′-ETS) of pre-rRNA. The 5′-ETS is cleaved prior to export of rRNA from the nucleus (30). PCR products were resolved in 1.3% agarose gel in TBE, stained with ethidium bromide and quantified with Gel-Pro 3.1. HTT mRNA levels in total RNA isolated from one-fourth of the cell pellet and in the cytoplasmic fraction were normalized relative to the levels of GAPDH mRNA whereas nuclear HTT mRNA level was normalized relative to the 5′-ETS.

RNA FISH, IF and microscopy

For RNA FISH and IF cells were fixed in 4% PFA/PBS at 4°C and washed three times in PBS, followed by permeabilization in 2% pre-chilled acetone/PBS for 5 min. Pre-hybridization was performed in 30% formamide and 2× SSC for 10 min, followed by hybridization in buffer containing 30% formamide, 2× SSC, 0.02% BSA, 66 µg/ml yeast tRNA, 10% dextran sulfate, 2 mM vanadyl ribonucleoside complex and 2 ng/µl DNA/LNA probes (CTG)₆-CA or (CAG)₆-CA. The probes were labeled at the 5′-end with Cy3 and modified at positions 2, 5, 8, 13, 16 and 19 with LNA. Post-hybridization washing was done in 30% formamide and 2× SSC at 45°C for 30 min followed by 1× SSC at 37°C for the next 30 min. Slides were either mounted in Vectashield medium (Vector Laboratories, INC. Burlingame, CA, USA) with DAPI or were used for subsequent IF. In control experiments, prior to RNA FISH cells were treated for 1.5 h at 37°C with 0.01 μg/ml RNase A (Sigma). For RNA FISH in combination with IF, the slides were treated as described above, but DAPI staining was omitted. The slides were incubated in blocking buffer (5% non-fat milk, 10% normal serum and 0.3% Triton X-100 in PBS) for 1 h at room temperature, and the reaction with rabbit anti-MBNL1 primary antibody (A2764) (a gift from Dr Thornton) was performed overnight at 4°C. After the reaction, the slides were washed in PBS three times for 5 min each and then incubated with a secondary anti-rabbit antibody conjugated to Cy2 (Jackson ImmunoResearch Laboratories, Inc.). Following PBS washes, the slides were mounted in Vectashield medium with DAPI. All FISH/IF images were acquired on a Zeiss Axiovert 200M microscope equipped with a cooled AxioCam HRc camera and processed with LSM 510 software.

Selection of transcripts for structural analysis of HTT and AR triplet repeats

In the HTT locus, the polymorphic CCG tract is 12 bp downstream of the expansion-prone (CAG)_n. The AR locus is composed of (CTG)₃(CAG)_n with a monomorphic (CAG)₆ tract 18 bp downstream (Figure 1A and C). This study aimed to determine the structure of HTT and AR mRNA fragments within the triplet repeat regions and the few dozen neighboring nucleotides. For the RNA structure analysis we selected transcripts corresponding to the most frequent normal alleles of the HTT and AR genes and mutant allele’s characteristic of HD and Kennedy’s disease. The selection of normal triplet repeat variants was based on the results of our prior genotyping studies (31,32) and the data extracted for HTT and AR genes are shown in Figure 1B and D.

CAG and CCG repeats interact with each other in vitro in HTT transcripts

First, we aimed to determine whether the two neighboring repeat tracts in the HTT locus interact with each other and what kind of structure is formed by the selected sequence flanking the repeats. To do this, we first analyzed a transcript containing 10 CAG and 10 CCG repeats (HTT-10-10) in the context of its natural flanking sequence (Figure 2A, left and middle sections). The results of RNA structure probing experiments showed that repeated sequences of the transcript form an autonomous structure in which the CAG and CCG repeats interact with each other over nearly their entire length (Figure 2A, right section). Only a large 13-nt terminal loop (loop a) formed mainly by the specific sequence separating the two repeat tracts was well recognized by T1, T2 and S1 single strand-specific nucleases. The stem structure that was formed by the two different types of repeats, the CAG at its 5′ side and the CCG at 3′ side, was composed of repeated C-G and G-C base pairs and A:C mismatches.

To gain better insight into the structure and stability of hairpins composed of the interacting repeats in the HTT transcript, we prepared RNAs composed of CAG and CCG units only. Transcripts harboring (CAG)₁₀(CCG)₁₀ and (CAG)₂₀ were analyzed. Both RNAs were cleaved in the center by single strand-specific nucleases and the number of cleavages observed in that region was consistent with the coexistence of two slipped variants (Supplementary Figure S1A). To determine differences in the structural stability of the investigated transcripts, we performed UV-monitored structure melting experiments. The obtained results confirmed that the (CAG)₁₀(CCG)₁₀ transcript formed a significantly more stable structure than the (CAG)₂₀ transcript, with the folding free energy change ΔG^37°C=−15.38 ± 0.43 kcal/mol and ΔG^37°C=−5.8 ± 0.39 kcal/mol and melting temperatures of 77.5 and 62.4°C, respectively (Supplementary Figure S1B). This difference in stability is likely to be related to the nature of base mismatches occurring periodically in the stems of hairpins in both transcripts.

Variations in the number of CAG repeats in HTT modulate RNA hairpin architecture

To determine the consequences of normal lengthening and pathogenic expansions of the CAG repeats on RNA structure, we analyzed normal HTT transcripts containing (CCG)₇ and 17 or 24 CAG repeats (Supplementary Figure S2) as well as the mutant transcript harboring 44 CAG repeats (Figure 2B). The overall structural architecture of the triplet repeat region in these four transcripts was very similar. In all analyzed RNAs, a large part of the stem structure was formed by the interacting (CAG)₇ and (CCG)₇ repeats, which is similar to that described above for the HTT-10-10 repeat (Figure 2A). The cleavage pattern detected in the terminal loop region and CAG/sep region of transcripts containing CAG repeat tracts longer than CCG repeat tracts is consistent with co-existence of two structural variants of this part of HTT hairpin (Figure 2B and Supplementary Figure S2). This conclusion is supported by the results of structural analysis of 3′-end labeled HTT transcripts (data not shown). The ability to form this kind of slipped structure is typical for trinucleotide repeats (24). The cleavage pattern generated by nucleases within the repeat flanking sequences of all compared HTT transcripts was almost identical (Figure 2A). Therefore, the structure formed by the two repeat tracts was indeed autonomous and was not influenced by the structure of the flanking sequence. The only significant difference between the repeat regions containing long normal repeats and the expanded mutant repeats was the length of the hairpin stem composed of pure CAG repeats.

The CAG repeat hairpin is stabilized by the neighboring short (CUG)₃ in the AR transcript

Next, we aimed to determine the influence on hairpin structure formation of two short repeated tracts, (CUG)₃ and (CAG)₆, located in close proximity to the polymorphic CAG repeats subject to expansion in the AR locus. For RNA structure probing experiments, we selected three normal AR gene variants containing 9, 21 and 22 CAG repeats (AR-9, AR-21 and AR-22) as well as one mutant transcript with an expanded (CAG)₄₆ tract (AR-46). Based on nuclease digestion, it appeared that in all transcripts, three CUG triplets interact with the 3′-end of the (CAG)_n sequence, forming a perfect 12-bp helical region together with a few nucleotides of repeat flanking sequence (Figure 3). The remaining polymorphic CAG repeats fold into an RNA hairpin structure, which contains either a 4- or a 7-nt terminal loop (Figure 3B, left and middle sections). Due to stabilizing interactions between CUG and CAG repeats in both normal and mutant AR transcripts, the slipped nature of the hairpin that is characteristic of triplet repeat sequences was not observed. The only difference between analyzed mRNA fragments with increasing numbers of CAG repeats was the length of the CAG hairpin structure (Figure 3B). In each AR transcript tested, the specific flanking sequence formed identical structures, and the short (CAG)₆ sequence located 18 nt downstream from the polymorphic CAG repeats folded into autonomous short hairpins (Figure 3A).

In summary, structural analysis of the complex triplet repeat regions of HTT and AR transcripts revealed that both CAG-harboring RNAs form in vitro hairpin structures that are stabilized by (CCG)_n or (CUG)₃ motifs adjacent to the polymorphic CAG. Due to the similarity in structural conformation and contribution of the neighboring repeats to the hairpin formation in HTT and AR transcripts, we selected human cells expressing an HTT mutant transcript for further in vivo studies. We utilized these cells to examine the colocalization of mutant CAG RNA with the dsRNA-binding protein MBNL1 and to test the susceptibility to RNA interference (RNAi) to characterize the in vivo structure.

Expanded CAG repeat RNAs are accumulated in HD nuclei and colocalize with MBNL1 protein

To determine whether mutant HTT transcripts are retained in the nuclei of cultured human HD cells, we performed FISH and tested for presence of CAG repeat aggregates. Fibroblast cells expressing expanded 68 and 151 CAG repeats from the HTT locus were used for this experiment. As a positive control for FISH we selected DM1 cells in which nuclear deposition of mutant CUG^exp transcripts has been previously shown (15). Figure 4 depicts that numerous nuclear RNA inclusions composed of CAG repeats were detected in the analyzed HD cells. We confirmed the presence of RNA in the nuclear inclusions with RNase A treatment (Figure 4B). Additionally, treatment with DNase did not affect the pattern of foci (data not shown). IF was performed to determine whether nuclear CAG aggregates in HD cells colocalize with the MBNL1 protein. Analysis of MBNL1 intracellular localization in HD cells revealed that the dsRNA-binding protein colocalized with nuclear CAG repeat inclusions as shown for CUG^exp foci in DM1 cells (Figure 4A). In contrast, in normal fibroblasts the protein was distributed throughout the cell.

Targeting CAG repeats by RNA interference in human HD cells

To determine whether the structure of the repeat region in the HTT transcript is susceptible to RNA interference, we utilized several siRNA duplexes (Table 1). Three patient derived HD fibroblast cell lines containing 15/46, 21/44 and 17/68 CAG repeats in the HTT gene were transfected with siRNAs, and the silencing effect was assessed for each HTT allele 24 h post-transfection by RT–PCR amplification of the repeat region (Figure 5A). Two siRNAs (siHD sep and siCUG7/CAG7) targeting the hairpin and two (siHD 5′ and siHD 3′) targeting flanking sequences (Figure 5B) were highly effective in silencing HTT transcripts. However, only the siCUG7/CAG7 siRNA showed some allele discrimination, cleaving the mutant transcripts more efficiently. This result implies that the structure formed by the CAG repeats in the analyzed HD cells was not stable enough to form a barrier for RNA-induced silencing complex (RISC) activity. In contrast to the four active siRNAs, the CCG repeat targeting RNA duplex did not trigger any considerable silencing effect, which may be explained by its high stability and inability to activate RISC (data not shown).

Next, to determine the CAG repeat tract cleavage sites by the repeat targeting siRNA, we performed 5′-RACE analysis. As controls for this experiment, we used siRNAs targeting non-repeating regions of the HTT transcript (Supplementary Figure S3). As expected, the HTT-specific siRNAs gave rise to single discrete amplification products with lengths corresponding to sites of AGO2-mediated cleavage. In contrast, multiple cleavage sites were observed in the CAG repeat region for HD cells treated with the CUG7/CAG7 siRNA, indicating that the repeated sequence was recognized by RISC at numerous positions (Supplementary Figure S3).

In addition to HD cells, we analyzed SCA3, SCA7 and DRPLA fibroblast cell lines to determine whether similar allele discrimination by the CAG repeat targeting duplex siCUG7/CAG7 could be achieved in cellular models of other polyglutamine diseases (Figure 6). In all CAG expansion-containing cell lines, the siRNA duplex used at two different concentrations was effective in silencing targeted genes. The efficiency of silencing was higher for mutant than for normal alleles. This result may be a consequence of the presence of more potential RISC binding sites in mutant alleles.

Downregulation of huntingtin transcript by siCUG7/CAG7 duplex predominates in the cytoplasm

In order to determine whether the observed siRNA silencing of the HTT mRNA took place only in the cytoplasm or also in the nucleus of HD cells, we used fractionated samples of RNA. Fibroblasts transfected with 20 nM siCUG7/CAG7 duplex were partitioned into the nuclear and cytoplasmic fractions and each of them was individually analyzed for the RNAi effect. The depletion of nuclear RNA from the cytoplasmic fraction was demonstrated by minute amounts of the HTT pre-mRNA and the 5′-ETS fragment of pre-rRNA in the cytoplasmic fraction (Figure 7). Mature HTT transcripts were detected in both the nuclear and cytoplasmic RNA isolated from HD cells expressing either 21/44 or 17/68 CAG repeats. Upon siCUG7/CAG7 treatment, we observed a decreased level of transcripts from both HTT alleles and the silencing was significantly more pronounced in the cytoplasm than in the nucleus. In cells expressing 21/44 CAG repeat RNAs the relative HTT mRNA levels decreased to 0.16 ± 0.005 in the cytoplasm and to 0.85 ± 0.03 in the nuclear fraction. Similar correlation was found in fibroblasts containing 17/68 CAG repeats in HTT locus where the relative HTT mRNA levels in the cytoplasm diminished to 0.4 ± 0.06 while in the nucleus only to 0.88 ± 0.08. Analysis of HTT pre-mRNA expression confirmed the low level of nuclear silencing with siCUG7/CAG7 in both the analyzed cell lines (Figure 7). Therefore, utilization of siCUG7/CAG7 duplex targeting HTT transcript caused its efficient silencing which was significantly more pronounced in the cytoplasmic fraction of RNA. It is also possible that the minor HTT silencing which we observed in the nuclear fraction resulted from contamination of the RNA with traces of the cytoplasmic fraction rather than being an effect of the nuclear RNAi.

Supplementary Data

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