RNA molecules with trinucleotide repeat expansions are involved in the pathology of various neurodegenerative diseases, such as Huntington's disease. A central research objective is to understand how trinucleotide repeat RNAs fold and function in disease progression. Studies that investigate the sequence structure, stability and self‐assembly of such RNAs mainly focused on in vitro methods in dilute aqueous solution. Functional studies in cells reveal further aspects of transport, protein association or assembly in phase separated compartments. Our study aims to understand governing factors for folding stability of trinucleotide repeats on the cellular level by assessing the influence of key components of the cellular medium in vitro. We investigate (CAG)20 RNA in complex and cytomimetic media using Fast Relaxation Imaging. Using distinct cosolutes we analyze the different enthalpic and entropic contributions that determine folding stability. We find a remarkable destabilization and aggregation of the RNA in cellular lysate that can be attributed to specific and non‐specific interactions with the unfolded state.

A hallmark of Huntington's disease (HD) is a prolonged polyglutamine sequence in the huntingtin protein and, correspondingly, an expanded cytosine, adenine, and guanine (CAG) triplet repeat region in the mRNA. A majority of studies investigating disease pathology were concerned with toxic huntingtin protein, but the mRNA moved into focus due to its recruitment to RNA foci and emerging novel therapeutic approaches targeting the mRNA. A hallmark of CAG-RNA is that it forms a stable hairpin in vitro which seems to be crucial for specific protein interactions. Using in-cell folding experiments, we show that the CAG-RNA is largely destabilized in cells compared to dilute buffer solutions but remains folded in the cytoplasm and nucleus. Surprisingly, we found the same folding stability in the nucleoplasm and in nuclear speckles under physiological conditions suggesting that CAG-RNA does not undergo a conformational transition upon recruitment to the nuclear speckles. We found that the metabolite adenosine triphosphate (ATP) plays a crucial role in promoting unfolding, enabling its recruitment to nuclear speckles and preserving its mobility. Using in vitro experiments and molecular dynamics simulations, we found that the ATP effects can be attributed to a direct interaction of ATP with the nucleobases of the CAG-RNA rather than ATP acting as "a fuel" for helicase activity. ATP-driven changes in CAG-RNA homeostasis could be disease-relevant since mitochondrial function is affected in HD disease progression leading to a decline in cellular ATP levels.

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