7308–7315 Nucleic Acids Research, 2011, Vol. 39, No. 16 doi:10.1093/nar/gkr368 Published online 19 May 2011 Crystal structures of CGG RNA repeats with implications for fragile X-associated tremor ataxia syndrome Agnieszka Kiliszek, Ryszard Kierzek, Wlodzimierz J. Krzyzosiak and Wojciech Rypniewski* Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland Received March 15, 2011; Revised and Accepted April 27, 2011 ABSTRACT The CGG repeats are present in the 50-untranslated region (50-UTR) of the fragile X mental retardation gene FMR1 and are associated with two diseases: fragile X-associated tremor ataxia syndrome (FXTAS) and fragile X syndrome (FXS). FXTAS occurs when the number of repeats is 55–200 and FXS develops when the number exceeds 200. FXTAS is an RNA-mediated disease in which the expanded CGG tracts form stable structures and sequester important RNA binding proteins. We obtained and analysed three crystal structures of double-helical CGG repeats involving unmodified and 8-Br modified guanosine residues. Despite the presence of the non-canonical base pairs, the helices retain an A-form. In the G–G pairs one guanosine is always in the syn conformation, the other is anti. There are two hydrogen bonds between the Watson–Crick edge of G(anti) and the Hoogsteen edge of G(syn): O6·N1H and N7·N2H. The G(syn)-G(anti) pair shows affinity for binding ions in the major groove. G(syn) causes local unwinding of the helix, compensated elsewhere along the duplex. CGG helical structures appear relatively stable compared with CAG and CUG tracts. This could be an important factor in the RNA’s ligand binding affinity and specificity. INTRODUCTION Tandem repeats of the CGG trinucleotide motif are abundant in human genome and occur in numerous genes and transcripts (1). The repeat tracts are often poly- morphic in length in human population and may play a regulatory role in gene expression. The CGG repeats present in the 50-untranslated region (50-UTR) of the fragile X mental retardation gene FMR1 are associated with several distinct phenotypes (2). In normal population the number of CGG repeats varies in the range 5–54 (2,3); 45–54 repeats fall into a subclass named ‘the grey zone’ associated with an increased likelihood of inter- generational pathogenic expansions (2,4). Tracts of 55–200 CGGs are premutations that can cause progressive neurodegenerative disorder fragile X-associated tremor ataxia syndrome (FXTAS) in elderly males (5,6). Female premutation carriers are at risk of developing premature ovarian failure (7). More than 200 CGG repeats are full mutations resulting in fragile X syndrome (FXS), the most common inherited mental retardation syndrome in man (8). FXTAS is an RNA-mediated disease in which the level of FMR1 mRNA is signiﬁcantly elevated (9,10). The expanded tracts form stable structures and sequester im- portant RNA binding proteins which are normally required for splicing and other cellular processes (11,12). The protein sequestration results in the formation of intranuclear inclusions in neurons and astrocytes (12,13) and triggers a dynamic formation of aggregates and de- regulation of alternative splicing of a number of genes in model cellular systems (11). In addition, the presence of stable CGG structures inhibits FMR1 translation at the initiation step, resulting in a deﬁciency of the encoded FMRP protein (9,14,15). FMRP is an important RNA binding protein involved in mRNA trafﬁcking between the cell nucleus and the cytoplasm and in regulating trans- lation at the synapse (16). The structure of the CGG repeats in FMR1 transcripts is a hairpin whose stem is formed by alternating C–G, G–C and the non-canonical G–G base pairs (17). Isolated (CGG)20 repeats are thermodynamically the most stable hairpins of all the (CNG)20 (N stands for C or G or A or U) repeats (18,19) which means that G–G pairs are the strongest of all homobasic interactions. According to an NMR study (20), the opposing G–G bases are highly dynamic in CGG repeat hairpin, having one G residue in anti and the other in syn conformation. *To whom correspondence should be addressed. Tel: +48-61-8528503; Fax: +48-61-8520532; Email: wojtekr@ibch.poznan.pl The Author(s) 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.