The structural basis of myotonic dystrophy from the crystal structure of CUG repeats

Abstract

Myotonic dystrophy (DM) type 1 is associated with an expansion of (>50) CTG repeats within the 3 untranslated region (UTR) of the dystrophin myotonin protein kinase gene (dmpk). In the corresponding mRNA transcript, the CUG repeats form an extended stem-loop structure. The double-stranded RNA of the stem sequesters RNA binding proteins away from their normal cellular targets resulting in aberrant transcription, alternative splicing patterns, or both, thereby leading to DM. To better understand the structural basis of DM type 1, we determined to 1.58-Å resolution the x-ray crystal structure of an 18-bp RNA containing six CUG repeats. The CUG repeats form antiparallel double-stranded helices that stack end-on-end in the crystal to form infinite, pseudocontinuous helices similar to the long CUG stem loops formed by the expanded CUG repeats in DM type 1. The CUG helix is very similar in structure to A-form RNA with the exception of the unique U-U mismatches. This structure provides a high-resolution view of a toxic, trinucleotide repeat RNA.toxic RNA ͉ U-U mismatches ͉ x-ray crystallography M yotonic dystrophy (DM) is the most common form of adult muscular dystrophy, affecting Ϸ1 in 8,000 individuals. There are two types of DM: type 1 (DM1) and type 2 (DM2). Both types are caused by a RNA gain-of-function mechanism (1, 2). DM1 is caused by a CTG repeat expansion within the 3Ј untranslated region (UTR) of the dystrophin myotonin protein kinase (dmpk) gene, whereas DM2 is caused by a CCTG repeat expansion within intron 1 of the Zn finger 9 (znf9) gene (3, 4). In both DM1 and DM2, normal individuals have Ͻ50 repeats, whereas individuals with DM1 and DM2 have hundreds or even thousands of repeats (5). Severity and age of onset of DM are correlated with repeat length. The clinical features of DM1 and DM2 are very similar and include muscle weakness, myotonia, cardiac arrhythmias, insulin resistance, cognitive impairment, and serological changes (6, 7). However, only DM1 has a severe congenital form.Dmpk and znf9 genes are on different chromosomes and encode very different proteins, yet both CUG and CCUG expansions cause DM, suggesting that the loss of function of DMPK and ZnF9 are not the primary causative agent of DM and that the repeat tracts themselves might be toxic. It is clear that the repeats cause DM because this difference is how affected individuals differ from normal individuals. In support of this model, the expression of an RNA containing Ϸ250 CUG repeats in mice causes characteristic features of DM1 (8). The CUG repeats form an extended stem-loop structure with U-U mismatches and G-C Watson-Crick base pairs (9, 10).The clinical features of DM appear to be caused by a ''toxic RNA gain-of-function'' mechanism in which the CUG repeat tracts bind and sequester specific RNA and DNA binding proteins. The CUG binding protein 1 appears to be up-regulated in the presence of extended CUG repeats and this increase might affect alternative splicing of genes relevant to the clinical features of . The muscleblind protein...

Structural Diversity of Triplet Repeat RNAs

“…CUG repeats are predicted to assume one of two different conformations depending on whether the uracil residue is inclined toward the minor groove of the CUG repeat RNA-A helix (32). The authors of the first reported crystal structure of CUG repeats (33) suggested that CCG repeats may form a similar structure as pyrimidine-pyrimidine mismatches could well accommodate within the RNA-A helix. They anticipated stronger structure distortions by purine-purine mismatches, which have higher hydrogen bond-forming potential.…”

“…The fact that different structures were often reported for the same repeat types by authors that used different methods reflects the atypical character of these sequences where the structures are highly sensitive to the length of the repeated sequence, RNA concentration, temperature, and ionic conditions. Nevertheless, the x-ray structures of short CUG repeat duplexes (32,33) may well represent the stem portions of hairpin structures formed by longer CUG repeats (5,16,26,29,39). Similarly, the NMR structure of short CGG repeats, which were forced to form hairpins (31), may well correspond to the stem structure of hairpins formed spontaneously by long CGG repeat tracts (5,25).…”

Microsatellite in the 3′ untranslated region of human fibroblast growth factor 9 (FGF9) gene exhibits pleiotropic effect on modulating FGF9 protein expression

“…On the other hand, the changed luciferase activities can also be caused by changed RNA stability in a post-transcriptional event. Other studies (Mooers et al, 2005;Ruggiero et al, 2003) have shown that microsatellite motifs in the 3'-UTR formed structural elements (stem-loops) and contributed to mRNA regulation. Because our prediction using the Mfold program showed two stem-loop structures formed by sequences containing the microsatellite motifs (Data not shown), we went on to examine mRNA stability in constructs with disease-associated (TG) 14 repeats and most-common (TG) 15 repeats.…”

Gene regulation by SINES and inosines: biological consequences of A-to-I editing of Alu element inverted repeats

“…In the corresponding mRNA transcript, the CUG repeats form an extended stem-loop structure. 76 The dsRNA of the stem sequesters RNA binding proteins such as muscleblind (MBNL) proteins family, and CUG binding protein 1 (CUG-BP1) away from their normal cellular targets, resulting in aberrant transcription, alternative splicing patterns, or both, thereby leading to DM. [77][78][79] Transcripts containing expanded CUG repeats form foci in the nuclei of DM1 cells, and these loci colocalize with MBNL protein family members.…”