Structural insights into CUG repeats containing the ‘stretched U–U wobble’: implications for myotonic dystrophy

Abstract

Tracks containing CUG repeats are abundant in human gene transcripts. Their biological role includes modulation of pre-mRNA splicing, mRNA transport and regulation of translation. Expanded forms of CUG runs are associated with pathogenesis of several neurodegenerative diseases, including myotonic dystrophy type 1. We have analysed two crystal structures of RNA duplexes containing the CUG repeats: G(CUG)2C and (CUG)6. The first of the structures, analysed at 1.23 Å resolution, is of an oligomer designed by us. The second model was obtained after ‘detwinning’ the 1.58 Å X-ray data previously deposited in the PDB. The RNA duplexes are in the A-form in which all the C–G pairs form Watson–Crick interactions while all the uridine pairs can be described as U•U cis wobble having only one hydrogen bond between the bases. The residue, which accepts the H-bond, is inclined towards the minor groove. This previously unreported base pairing can be described as ‘stretched U–U wobble’. The regular hydrogen-bonding pattern of interactions with the solvent, the electrostatic charge distribution and surface features indicate the ligand binding potential of the CUG tracks.

Watson–Crick-like pairs in CCUG repeats: evidence for tautomeric shifts or protonation

“…A bending of the CCUG helix was also reported by Childs-Disney et al (2014). The bending/coiling effect was not observed in any of the tri-nucleotide repeats analyzed before (Kiliszek et al 2009(Kiliszek et al , 2010(Kiliszek et al , 2011(Kiliszek et al , 2012.…”

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.…”

“…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).…”

“…Nuclease Digestions and Lead Ion Cleavages-Prior to structure-probing reactions, 32 P-labeled RNAs were subjected to a denaturation and renaturation procedure in a buffer containing either 2 mM (buffer A) or 20 mM MgCl 2 (buffer B) and 80 mM NaCl, 20 mM Tris-HCl, pH 7.2, or in a buffer containing 2 mM MgCl 2 , 270 mM KCl, 30 mM NaCl, 20 mM Tris-HCl, pH 7.2 (buffer C) by heating the sample at 80°C for 1 min and then slowly cooling to the reaction temperature. Limited RNA digestion was initiated by mixing 5 l of RNA sample (20 -40 nM; 50,000 cpm) with 5 l of a probe solution containing lead ions, nuclease S1, mung bean, or ribonucleases T1, T2, Cl3, and V1.…”

Triplet repeats in transcripts: structural insights into RNA toxicity

“…In recent years, the crystal structures of short oligoribonucleotides composed of the CUG, CAG and CGG repeats have been determined (Mooers et al , 2005 ;Kiliszek et al , 2009Kiliszek et al , , 2010Kiliszek et al , , 2011Kumar et al , 2011 ). All these repeated sequences form duplex structures that stack onto each other and likely represent the stem portions of hairpins formed by long sequences of this type.…”