SUMMARY The inherited neurodegenerative disease Friedreich’s ataxia (FRDA) is caused by GAA•TTC triplet repeat hyper-expansions within the first intron of the FXN gene, encoding the mitochondrial protein frataxin. Long GAA•TTC repeats causes heterochromatin-mediated gene silencing and loss of frataxin in affected individuals. We report the derivation of induced pluripotent stem cells (iPSCs) from FRDA patient fibroblasts by transcription factor reprogramming. FXN gene repression is maintained in the iPSCs, as are the global gene expression signatures reflecting the human disease. GAA•TTC repeats uniquely in FXN in the iPSCs exhibit repeat instability similar to patient families, where they expand and/or contract with discrete changes in length between generations. The mismatch repair enzyme MSH2, implicated in repeat instability in other triplet repeat diseases, is highly expressed in pluripotent cells, occupies FXN intron 1, and shRNA silencing of MSH2 impedes repeat expansion, providing a possible molecular explanation for repeat expansion in FRDA.

The CAG repeat expansions that occur in translated regions of specific genes can cause human genetic disorders known as polyglutamine (poly-Q)-triggered diseases. Huntington’s disease and spinobulbar muscular atrophy (SBMA) are examples of these diseases in which underlying mutations are localized near other trinucleotide repeats in the huntingtin (HTT) and androgen receptor (AR) genes, respectively. Mutant proteins that contain expanded polyglutamine tracts are well-known triggers of pathogenesis in poly-Q diseases, but a toxic role for mutant transcripts has also been proposed. To gain insight into the structural features of complex triplet repeats of HTT and AR transcripts, we determined their structures in vitro and showed the contribution of neighboring repeats to CAG repeat hairpin formation. We also demonstrated that the expanded transcript is retained in the nucleus of human HD fibroblasts and is colocalized with the MBNL1 protein. This suggests that the CAG repeats in the HTT mRNA adopt ds-like RNA conformations in vivo. The intracellular structure of the CAG repeat region of mutant HTT transcripts was not sufficiently stable to be protected from cleavage by an siRNA targeting the repeats and the silencing efficiency was higher for the mutant transcript than for its normal counterpart.

Transcription stimulates the genetic instability of trinucleotide repeat sequences. However, the mechanisms leading to transcriptiondependent repeat length variation are unclear. We demonstrate, using biochemical and genetic approaches, that the formation of stable RNA · DNA hybrids enhances the instability of CTG · CAG repeat tracts. In vitro transcribed CG-rich repeating sequences, unlike AT-rich repeats and nonrepeating sequences, form stable, ribonuclease A-resistant structures. These RNA · DNA hybrids are eliminated by ribonuclease H treatment. Mutation in the rnhA1 gene that decreases the activity of ribonuclease HI stimulates the instability of CTG · CAG repeats in E. coli. Importantly, the effect of ribonuclease HI depletion on repeat instability requires active transcription. We also showed that transcription-dependent CTG · CAG repeat instability in human cells is stimulated by siRNA knockdown of RNase H1 and H2. In addition, we used bisulfite modification, which detects single-stranded DNA, to demonstrate that the nontemplate DNA strand at transcribed CTG · CAG repeats remains partially single-stranded in human genomic DNA, thus indicating that it is displaced by an RNA · DNA hybrid. These studies demonstrate that persistent hybrids between the nascent RNA transcript and the template DNA strand at CTG · CAG tracts promote instability of DNA trinucleotide repeats.RNA·DNA hybrids | transcription-induced instability | triplet repeats E xpansions of simple repeating sequences (microsatellites) are responsible for more than 20 human diseases (1). Moreover, instability of simple repeats (expansion as well as contraction) is observed throughout the genomes of all organisms studied, and it is considered an important source of genetic variation (2). The molecular basis of this unusual mutation mechanism has been studied extensively in the past 15 years using bacteria, yeast, flies, mice, and mammalian cells. Although these analyses uncovered several cis elements and trans-acting factors affecting repeat instability (3), a unifying, comprehensive model of the repeat expansion and contraction is lacking. Processes such as replication, recombination, and repair, which temporarily dissociate DNA complementary strands, strongly destabilize repeating sequences. Transient exposure of single-stranded DNA regions allows formation of non-B DNA structures in regions containing repeats. Virtually all current models of repeat instability incorporate non-B DNA structures as the proximate cause of instability (4, 5).Recently, transcription through tandem repeat sequences has emerged as an important factor promoting instability of repeat sequences (6-12). Although the synthesis of RNA does not change the length of a DNA template, it can lead to the formation of non-B DNA structures, as shown in E. coli, yeast, and higher organisms (7-10, 13). These secondary structures may interfere with the progress of RNA polymerase, calling into play various DNA repair processes, whose action leads to repeat expansion and contraction. In h...

We show that CUG repeats form "slippery" hairpins in their natural sequence context of the myotonin kinase gene transcript. This novel type of RNA structure is characterized by strong S 1 and T 1 nuclease and lead cleavages in the terminal loop and by mild lead cleavages in the hairpin stem. The latter effect indicates a relaxed metastable structure of the stem. (CUG) 5 repeats do not form any detectable secondary structure, whereas hairpins of increasing stability are formed by (CUG) 11 , (CUG) 21 , and (CUG) 49 . The potential role of the RNA hairpin structure in the pathogenesis of myotonic dystrophy is discussed.Eleven human diseases associated with the expansion of trinucleotide repeats have been identified so far. The progress of research in this new area has been discussed in several recent reviews (1-5). One of these diseases is myotonic dystrophy (dystrophia myotonica (DM)), 1 the most prevalent form of muscular dystrophy in adults with a global incidence of 1 in 8000. The molecular basis of this multisystemic disease, with a complex clinical picture, is the expanded CTG repeat in the myotonic dystrophy protein kinase (DMPK) gene. The repeat expansions, which cause DM, range from 50 repeats in mildly affected patients to Ͼ2000 repeats in the most severe congenital cases (6 -8).Despite the fact that the nature of the underlying DM mutation has been known for the past 6 years, the molecular pathology of this disease is not understood. In particular, it has been difficult to find the molecular mechanism by which this specific mutation, located in the 3Ј-UTR of the DMPK gene, causes the dominantly inherited disease. One of several recent proposals links the inheritance pattern with the molecular effects observed at the RNA level. It takes advantage of the observation that dramatic decreases in both mutant and normal DMPK poly(A) ϩ RNAs, as compared with the primary transcripts, occur in the DM tissue (9). This suggests that the expanded CUG repeat may have a dominant effect either on the processing of both the normal and expanded transcripts or on their transport to the cytoplasm. Other authors observed only a decrease in the expanded poly(A) ϩ transcript (10). The specific (CUG) n -binding proteins, discovered recently (11-13), may be involved in normal DMPK RNA processing, transport, and/or translation. These RNA-protein interactions may be impaired by the expanded DMPK transcript.Understanding the molecular basis of the DM disease is hampered by the lack of knowledge of the RNA structure formed by the CUG repeats. To fill this gap and to provide a background for further studies on the role of RNA level effects in the pathogenesis of myotonic dystrophy, we have analyzed the structure of the repeat region of DMPK RNA. In this paper, we describe the properties of an unusual "slippery" RNA hairpin that contains a metastable stem. The stability of the hairpin increases with the repeat length, and we postulate that long stable hairpins are important factors in DM pathogenesis. MATERIALS AND METHODS DNA Templa...

RNA metabolism is a major contributor to the pathogenesis of clinical disorders associated with premutation size alleles of the fragile X mental retardation (FMR1) gene. Herein, we determined the structural properties of numerous FMR1 transcripts harboring different numbers of both CGG repeats and AGG interruptions. The stability of hairpins formed by uninterrupted repeat-containing transcripts increased with the lengthening of the repeat tract. Even a single AGG interruption in the repeated sequence dramatically changed the folding of the 5′UTR fragments, typically resulting in branched hairpin structures. Transcripts containing different lengths of CGG repeats, but sharing a common AGG pattern, adopted similar types of secondary structures. We postulate that interruption-dependent structure variants of the FMR1 mRNA contribute to the phenotype diversity, observed in premutation carriers.