Repeat RNA structure and protein interactions influence RAN translation and neurodegenerative phenotypes in models of FTD and ALS

Abstract Background The most common genetic cause of Frontotemporal dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS) is an intronic GGGGCC hexanucleotide repeat expansion in C9orf72 (C9FTD/ALS). Transcribed GGGGCC repeat RNAs sequester RNA‐binding proteins (RBPs) into nuclear and cytoplasmic foci, which inhibits their normal functions. GGGGCC repeat RNAs also support translation of toxic repeat peptides through a process known as repeat associated non‐AUG (RAN) translation. Here we sought to understand how repeat RNA secondary structure and RBP engagements that occur during RNA transcription, processing, and trafficking influence GGGGCC RAN translation and repeat elicited toxicity, with a goal of identifying novel therapeutic targets. Method We developed a cellular RNA‐capture methodology coupled to mass spectrometry to identify GGGGCC repeat RNA‐interacting proteins within specific cellular compartments. In parallel, we used chemical approaches to probe GGGGCC repeat RNA secondary structures inside cells and RAN translation assays to determine whether repeat RNA secondary structures and/or interacting RBPs influence RAN translation efficiency. We employed Drosophila and neuronal models of repeat expansions to evaluate potential RBPs as disease modifiers. Result Our RNA structural studies revealed that GGGGCC repeat RNAs form primarily hairpin structures, rather than G‐quadruplex structures, in cells. Folding repat RNAs into such hairpins supports RAN translation whereas folding repeat RNAs into g‐quadruplex structures strongly inhibits their translation. We identified multiple RBPs enriching in the nuclear fraction of GGGGCC RNA‐interactome, including hnRNPH1 and SR (serine/arginine‐rich domain) proteins, which are known components of the nuclear foci found in C9FTD/ALS patient cells. Our comparative analysis found that lowering SR protein expression or modifying its phosphorylation modified toxicity in Drosophila models of multiple repeat expansion disorders. Currently we are investigating how repeat RNA structures influence RBP interactions and whether structure dependent RBP interactions selectively modulate RAN translation. Conclusion We present a comprehensive approach to evaluate the roles of repeat RNA secondary structures and critical RBPs in RNA toxicity underlying dementia‐associated repeat expansion disorders.