A crystal structure of a model of the repeating r(CGG) transcript found in fragile X syndrome.

Expanded repeats of r(CGG) in the 5’-untranslated region of the fragile X mental retardation protein mRNA cause fragile X and fragile X-associated tremor/ataxia syndromes. Expanded repeats fold into an RNA hairpin with repeating 5’-CGG/3’-GGC motifs. Herein, we report a structure of a model RNA duplex with three copies of the 5’-CGG/3’-GGC motif (PDB ID: 3JS2), refined to 1.36 . All three GG internal loops have N1-carbonyl, N7-amino pairs and are closed by standard Watson–Crick CG pairs. The results expand the available structures of triplet repeating transcripts and provide information to help understand how these RNAs bind small-molecule and protein ligands. Although RNA is an important target for small molecules, most functionally important RNAs have not been exploited in targeting endeavors. This is thought to be due generally to the lack of a fundamental understanding of RNA motifs that specifically bind small molecules and the small molecules that specifically bind RNA motifs. One important class of RNAs that can be exploited as a drug target by small molecules is that of triplet-repeating transcripts. A variety of diseases, including fragile X syndrome (FXS, r(CGG)), Friedreich’s ataxia (r(CGG)), the spinocerebellar ataxias (r(CAG) or r(CUG)), and myotonic dystrophy (r(CUG)) are caused by triplet-repeating RNAs. These expanded repeating transcripts fold into higher-order hairpin structures with regularly repeating 1 1 nucleotide internal loops separated by two GC base pairs, as determined by chemical probing of RNA structure (Figure 1A). Two different general mechanisms for how triplet-repeating transcripts cause disease have been established. In the first mechanism, which has been best established for Huntington’s disease, a repeating transcript of r(CAG) is present in an mRNA coding region. When that transcript is translated, polyQ proteins are synthesized and cause toxicity. In a second mechanism, which has been best established for myotonic dystrophy type 1 (DM1), the expanded repeat is present in a non-coding region in an mRNA, such as a 3’-untranslated region (UTR). The transcribed repeat sequesters RNA-binding proteins such as muscleblind-like protein 1 (MBNL1), and this controls premRNA splicing. Sequestration of MBNL1 by expanded r(CUG) repeats causes both translational defects of the mRNA with the expanded repeat and pre-mRNA splicing defects. Recent studies have shown that the pathology of fragile Xassociated tremor/ataxia syndrome (FXTAS) can be due to the latter disease mechanism and FXS is due to a well-established translational-defect mechanism. FXTAS is caused when 55– 200 r(CGG) repeats are present in the 5’-UTR of the fragile X mental retardation 1 mRNA (FMR1). A Drosophila model of FXTAS was developed by heterologous expression of 90 r(CGG) repeats that were not translated. The repeats alone caused a neurodegenerative phenotype associated with FXTAS. In patient-derived FXTAS cell lines, r(CGG) repeats form inclusion complexes that contain, for example, the Src-associated, 68 kDa (Sam68) protein in mitosis, heterogeneous nuclear ribonucleoprotein G (hnRNP-G) protein, and MBNL1. These studies also showed that r(CGG) repeats first bind a protein, which has yet to be determined, that first recruits Sam68, which further recruits hnRNP-G and MBNL1 to the r(CGG) repeats. Furthermore, pre-mRNA splicing of Sam68-controlled transcripts is affected in FXTAS-patient-derived cell lines. These mechanistic studies suggest a therapeutic strategy towards developing a treatment for FXTAS in which a small-molecule ligand binds expanded r(CGG) repeats and inhibits protein binding, thereby freeing the protein to perform its normal physiological roles. Such strategies have already been implemented for DM1. For example, oligonucleotides and a small molecule, pentamidine, target expanded r(CUG) repeats and correct DM1-associated splicing defects in mouse models. General strategies for targeting triplet-repeating transcripts with small molecules have been developed and are centered on a modular-assembly strategy. In order to develop an atomic understanding of the structure of r(CGG) repeats, we have refined diffraction data on a Figure 1. The secondary structure, refined structure, and crystal packing of the RNA construct. A) The secondary structure of the oligonucleotide r(CGG) repeat duplex model that allowed crystal growth. B) The global structure of the RNA including the electron density map at 1.0s. C) Side and D) top views of the crystal packing that was observed in the unit cell within 5 .