CAG-Repeat RNA Hairpin Folding and Recruitment to Nuclear Speckles with a Pivotal Role of ATP as a Cosolute

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.

Automated summary

A hallmark of Huntington's disease is a prolonged polyglutamine sequence in the huntingtin protein and an expanded CAG triplet repeat region in the mRNA. The RNA foci and therapeutic approaches targeting the mRNA have attracted attention. Through experiments, it was found that CAG-RNA is destabilized in cells but remains folded in the cytoplasm and nucleus. ATP plays a crucial role in promoting unfolding and affecting CAG-RNA homeostasis.