Direct observation of RNA structure and dynamics in repeat-RNA assemblies using single molecule fluorescence microscopy.

Abstract

Huntington's disease (HD), a fatal neurodegeneration, results from somatic expansions of CAG repeats in exon-1 of the Huntingtin (HTT) gene. Genome-wide association studies (GWAS) implicate age of onset (AO) of HD to strongly depend on the length of uninterrupted CAG repeats with certain variations in flanking sequences acting as an AO enhancer or an AO delayer. Stable, GC-rich hairpins formed by expanded CAG repeats in the HTT-mRNAs are thought to trigger dysregulated protein sequestration and aberrant assembly of cytoplasmic and nuclear repeat-RNA-protein (RNP) aggregates. While aggregation is observed to be stochastic in cells, the mechanism of how repeat-RNA aggregate assembly is initiated remains unknown. Specifically, it remains unclear how the extended hairpin structures interact intermolecularly to aggregate and to what extents proteins contribute to RNA-RNA aggregation. Furthermore, it is unknown how sequence variations to the HTT-mRNA affects its aggregation propensities. To address these aspects of repeat-RNP aggregate assembly, we use in vitro transcribed HTT exon-1 RNA containing normal, expanded and interrupted CAG repeats to form repeat-RNA assemblies in vitro. Using a single molecule fluorescence microscopy method, we monitor, in real time, the dynamics and structural changes of single HTT RNA molecules bound to phase separated HTT RNA assemblies. Additionally, we probe how the RNA binding dynamics are affected by HTT-RNA flanking sequences.This study aims to provide mechanistic insights into how expanded repeat-RNP aggregate assembly is initiated in order to understand how physical interactions between triplet-repeat containing RNAs contributes to the assembly of macromolecular repeat-RNP structures implicated in neuronal toxicity. The outcome from the proposed study has potential to develop novel therapeutic target(s) for designing new approaches to prevent aberrant RNP foci formation.