C9orf72 hexanucleotide repeat associated with amyotrophic lateral sclerosis and frontotemporal dementia forms RNA G-quadruplexes

Abstract

Large expansions of a non-coding GGGGCC-repeat in the first intron of the C9orf72 gene are a common cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). G-rich sequences have a propensity for forming highly stable quadruplex structures in both RNA and DNA termed G-quadruplexes. G-quadruplexes have been shown to be involved in a range of processes including telomere stability and RNA transcription, splicing, translation and transport. Here we show using NMR and CD spectroscopy that the C9orf72 hexanucleotide expansion can form a stable G-quadruplex, which has profound implications for disease mechanism in ALS and FTD.

Figure 1. Schematic representation of the parallel stranded GGGGCC RNA G-quadruplex.

(a) Tetrads are stabilized by eight hydrogen bonds (black lines), with metal ions sitting centrally (orange circle) and phosphate backbones laterally (blue squares). (b) Four stacked G-tetrads, anti-glycosidic torsion angles, with phosphate backbones connected through a propeller loop arrangement, comprised of the two cytosines, ensures a parallel topology.

Figure 2. The C9orf72 hexanucleotide repeat is predicted to form a G-quadruplex structure.

The G-quadruplex prediction tool GQRS mapper was used to identify potential G-quadruplex forming sequences in the entire C9orf72 genomic DNA sequence, which is shown with exons highlighted in red and the GGGGCC repeats in green. GQRS mapper provides a G-score (plotted in blue) which indicates the likelihood of G-quadruplex formation. (a) The highest G-score in the C9orf72 reference sequence from Ensembl GRCh37 corresponds to the three GGGGCC repeats and adjacent GGGGC which we have termed the minimal C9orf72 G-quadruplex repeat unit (C9Gru). (b) 800 GGGGCC repeats were added, which is representative of the disease-causing expansions, leading to an extended region of high G-score. Image is adapted from QGRS mapper (http://bioinformatics.ramapo.edu/QGRS/index.php). E1a = exon 1a; E1b = exon 1b.

Figure 3. NMR analysis of the C9orf72 GGGGCC RNA hexanucleotide repeat shows formation of G-quadruplexes.

The 1D proton spectrum of the C9Gru RNA oligonucleotide annealed in 10 mM K₂PO₄ 40 mM KCl buffer, pH 7.0, 298 K. Peaks in the imino proton region (arrow) between 10 and 11.5 ppm correspond to quadruplex formation.

Figure 4. CD analysis shows the GGGGCC RNA G-quadruplex structures are very stable, cation dependent and parallel oriented.

CD spectra of the C9Gru RNA oligonucleotide (4.6 μm) show a positive peak at 262 nm and a negative peak at 237 nm, which is characteristic of parallel-oriented G-quadruplex structures. (a) and (b) represent the temperature unfold and refold spectra respectively, with the RNA oligonucleotide in KCl 40 mM, K₂PO₄ 10 mM buffer. The peak at 262 nm only decreases at 85°C indicating a very stable structure. Temperature unfold (c) and refold (d) spectra of identical RNA in LiCl 50 mM, Na₂PO₄ 10 mM buffer. The characteristic cation dependence of G-quadruplex structures was confirmed by the observed reduced stability in the presence of Li⁺ ions.