BPS2026 – Untangling the folding and aggregation of triplet repeat RNA

In Huntington’s disease (HD) and related disorders, expansion of CAG trinucleotide repeats in the huntingtin gene produces a toxic gain of function that leads to the death of striatal neurons. Huntingtin mRNA containing CAG repeats aggregates and sequesters essential RNA binding proteins, dysregulating pre-mRNA splicing and translation. Although the aggregation of repeat-containing RNA correlates with neurotoxicity, the physical basis for this correlation is not understood. To disentangle the physical mechanism of RNA aggregation, we used single-molecule force spectroscopy to probe the folding and unfolding pathways of huntingtin mRNA fragments containing different numbers of CAG repeats. Whereas short, non-pathogenic CAG RNAs form single hairpins that unfold reversibly and cooperatively, expanded CAG RNA forms many structures that unravel non-cooperatively. Slippage of CAG base pairs is punctuated by unfolding of stable base pairs with adjacent CCG codons, creating partially folded structures that readily bind other RNA molecules. The rate of interstrand interactions correlates with the propensity of RNA sequences to phase separate in vitro and aggregate in cells. We show how the combination of CAG and CCG trinucleotides creates a stick-slip behavior that explains the aggregation propensity of expanded repeat mRNAs.