Structural dynamics of double-helical RNAs composed of CUG/CUG- and CUG/CGG-repeats

Abstract

Human genetic trinucleotide repeat expansion diseases (TREDs) are characterized by triplet repeat expansions, most frequently found as CNG-tracts in genome. At RNA level, such expansions suggestively result in formation of double-helical hairpins that become a potential source for small RNAs involved in RNA interference (RNAi). Here, we present three crystal structures of RNA fragments composed of triplet repeats CUG and CGG/CUG, as well as two crystal structures of same triplets in a protein-bound state. We show that both 20mer pG(CUG)₆C and 19mer pGG(CGG)₃(CUG)₂CC form A-RNA duplexes, in which U·U or G·U mismatches are flanked/stabilized by two consecutive Watson–Crick G·C base pairs resulting in high-stacking GpC steps in every third position of the duplex. Despite interruption of this regularity in another 19mer, p(CGG)₃C(CUG)₃, the oligonucleotide still forms regular double-helical structure, characterized, however, by 12 bp (rather than 11 bp) per turn. Analysis of newly determined molecular structures reveals the dynamic aspects of U·U and G·U mismatching within CNG-repetitive A-RNA and in a protein-bound state, as well as identifies an additional mode of U·U pairing essential for its dynamics and sheds the light on possible role of regularity of trinucleotide repeats for double-helical RNA structure. Findings are important for understanding the structural behavior of CNG-repetitive RNA double helices implicated in TREDs.

Keywords:

• trinucleotide repeat expansion diseases
• RNA crystal structure
• protein-bound and protein-free U·U and G·U mismatches

Acknowledgments

This research was supported by Spanish Ministerio de Ciencia e Innovacion (MICINN, grant BFU2007-61715) and by ‘Acciones Especiales en la Comunidad Autónoma del País Vasco’ (Project AE-2008-1-9). We thank Dr Eugene Skripkin (Rib-X Pharmaceuticals, New Haven) for bringing our attention to the problem of the TNRs and for a fruitful collaboration during initial stage of this project.

Conflict of interest statement. None declared.

Protein Data Bank accession number

The atomic coordinates and structure factors have been deposited with the RCSB Protein Bank. See Table 1 for PDB accession codes.

Notes

aValues in parentheses correspond to the highest resolution shell.

bRefinement was carried out in the space group R3 (see text for detail).

cR-merge = Σ _hkl Σ _i |I(hkl) _i – <I(hkl)>|/Σ _hkl Σ _i <I(hkl) _i > where I(hkl) _i is the ith individual measurement of I(hkl) and <I(hkl)> is the mean value of intensities of all reflection with indices hkl; Σ _i is the sum over the individual measurements of a reflection with indices hkl; and Σ_hkl is the sum over all reflections.

aIn crystallization was used truncated fragment of p19 (residues 27–149).

bValues in parentheses correspond to the highest resolution shell.

cR-merge = Σ _hkl Σ _i |I(hkl) _i –<I(hkl)>|/Σ _hkl Σ _i <I(hkl) _i >where I(hkl)_i is the ith individual measurement of I(hkl) and <I(hkl)> is the mean value of intensities of all reflection with indices hkl; Σ _i is the sum over the individual measurements of a reflection with indices hkl; and Σ _hkl is the sum over all reflections.

aDistances, atoms, and water molecules are shown in Figures 1(B)–(D) and 9(B)–(E). Modes A, B, C of U·U pairing are shown in Figure 2(A)–(C).

bHydrogen bonds and repulsive contact are highlighted by bold and italic font, respectively.

cModes A′and B′ are named after modes A and B, to which they show a certain degree of resemblance.

dMolecule located on a crystallographic dyad; two halves are structurally equivalent.

aDistances, atoms, and water molecules are shown in Figures 3(B)–(C) and 9(A).

bHydrogen bonds and repulsive contact are highlighted by bold and italic font, respectively.