CAG repeat expansions induce cytoplasmic RNA aggregation

Abstract

Expansions of ‘CAG’ trinucleotide repeats in the genome can cause over a dozen diseases, including Huntington disease and several spinocerebellar ataxias. Short tracts of these ‘CAG’ repeats are benign; however, mutant alleles that harbor an abnormally large number of consecutive ‘CAG’ motifs can result in disease. Mutant RNA molecules harboring an expanded repeat tract can promote pathology through at least two activities. First, these repeat-containing RNA molecules are prone to condensation within the nucleus. This results in the formation of liquid-like nuclear foci. These foci can sequester RNA-binding proteins, resulting in a loss of the RNA-binding proteins’ functions. Second, the expanded repeat tract in the RNA can undergo translation even if the repeat does not lie in a canonical coding sequence, a phenomenon termed repeat-associated non-AUG (RAN) translation. This can result in the production of toxic, repeat-containing proteins.

In this thesis, I present evidence that RNA molecules containing an expanded CAG repeat tract can condense in the cytoplasm to form gel-like aggregates. Unlike nuclear foci of CAG repeat RNAs, these cytoplasmic RNA aggregates are associated with RAN translation and with cytotoxicity. The repeat RNAs co-aggregate in the cytoplasm with their cognate repeat proteins. These aggregates sequester several typically-nuclear RNA-binding proteins, such as FUS and TDP-43. Inhibiting translation of the repeat RNAs prevents cytoplasmic RNA aggregation, cytoplasmic mislocalization of RNA-binding proteins, as well as toxicity.

In the second part of this thesis, I present evidence that the cytoplasmic repeat RNA aggregates also contain NEAT1, a lncRNA that typically localized to the nucleus. CAG repeat RNAs that do not undergo translation, and consequently do not form cytoplasmic RNA aggregates, do not cause NEAT1 mislocalization. Several proteins that bind NEAT1, including FUS, also mislocalize to the cytoplasmic RNA aggregates. Genetic knock-down of FUS is sufficient to induce cytoplasmic accumulation of NEAT1. These results suggest that depletion of FUS from the nucleus (e.g. through sequestration at cytoplasmic aggregates) increases the accumulation of NEAT1 in the cytoplasm.

Ultimately, this work opens new questions into the role of cytoplasmic RNA aggregates in CAG repeat expansion disorders and the role of FUS in nuclear retention of RNAs.