Dissecting the role of GGGGCC repeat RNA secondary structure in cellular toxicity associated with C9 FTD/ALS

Abstract

AbstractBackgroundAberrant expansions of simple nucleotide repeat sequences can cause more than fifty human diseases. For example, the most common genetic cause of Frontotemporal dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS) is the expansion of an intronic GGGGCC hexanucleotide repeat sequence in C9orf72 gene (C9 FTD/ALS). Transcribed GGGGCC repeat RNAs can form complex secondary structures, which facilitate unusual interactions with cellular RNA‐binding proteins (RBPs). Sequestration of crucial RBPs into nuclear foci can inhibit the normal functions of those proteins. In addition, expanded GGGGCC repeat RNAs can support the translation of highly toxic repetitive peptides through a non‐canonical process known as repeat associated non‐AUG translation (RAN translation), which may require some specialized factors. Here we sought to understand how RNA secondary structure influence GGGGCC RAN translation and repeat elicited toxicity in C9 FTD/ALS.MethodWe used chemical modification coupled with traditional high‐throughput sequencing and nano‐pore sequencing approaches to probe GGGGCC repeat RNA secondary structures inside cells. We further use in vitro and in vivo RAN translation assays to determine whether repeat RNA secondary structures influence RAN translation efficiency.ResultOur RNA structural studies revealed that GGGGCC repeat RNAs form primarily form hairpin structures in cells. With nanopore sequencing, we were also able to detect the possible presence of unfolded and/or g‐quadruplex folded GGGGCC repeat RNAs in cells. Folding GGGGCC RNAs into hairpins supports RAN translation whereas folding repeat RNAs into g‐quadruplex structures strongly inhibits RAN translation in both in vitro and in vivo assays. Our results suggest that RNA secondary structure can affect the RAN translation elongation efficiency and g‐quadruplex structures may lead to the termination of elongating ribosomes.ConclusionWe present a deep sequencing‐based chemical probing technique to evaluate the roles of repeat RNA secondary structures in RNA toxicity underlying dementia‐associated repeat expansion disorders. Repeat RNA structural information can be used to identify chemical compounds that can bind to the RNA and prevent cytotoxicity in cells.