CUG Repeats Present in Myotonin Kinase RNA Form Metastable “Slippery” Hairpins

Abstract

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...

Hairpin Structure-forming Propensity of the (CCTG·CAGG) Tetranucleotide Repeats Contributes to the Genetic Instability Associated with Myotonic Dystrophy Type 2

“…The purified and labeled oligonucleotides (4 -5 ϫ 10 5 cpm/reaction) in 10 mM Tris, 40 mM NaCl, and 10 mM MgCl 2 were denatured by heating at 80°C for 5 min followed by renaturation by gradually decreasing the temperature (2°C/min) to the indicated reaction temperature (61). The chemical and enzymatic probes were then added along with the carrier DNA (salmon sperm DNA, 1 g/ l) (Invitrogen).…”

“…5 and 6. The reactions were stopped by addition of a urea-EDTA dye solution (61) followed by quick freezing on dry ice.…”

Imperfect CAG Repeats Form Diverse Structures in SCA1 Transcripts

“…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)(18)(19)(20)(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).…”

Repetitive sequences that shape the human transcriptome

“…The characteristic feature of hairpins formed by the CNG repeats is that their structure rigidity increases with repeat length. For example, hairpin formed by the (CUG)49 has a stem structure which shows a melting temperature much higher than that of (CUG)21 and is completely resistant to the single strand specific probes [33]. Moreover, the long CUG and CAG hairpins behave as regular double-stranded RNA structures with typical RNA-A geometry [34].…”

“…We have shown that such repeats form stable hairpins, thus the sequestered proteins must be the dsCUG repeat binding proteins [33]. Such proteins were then isolated [45] and shown to co-localize with the expanded transcripts in nuclear foci [46][47][48].…”