A passive protein environment offsets RNA folding energetics in cells

RNA structure can be predicted from its sequence in a dilute solution, but these predictions often don't hold true inside cells. Some RNA molecules retain their structure from in vitro conditions, while others become much less structured. To investigate the folding process of RNA within cells, the researchers used single-molecule FRET in conjunction with Xenopus oocyte extract.

This experimental setup allowed them to measure RNA folding and the formation of duplexes under conditions that closely resemble those found inside a living cell.

The proteins present in the extract acted as passive suppressors of base pairing. They appeared to slow down the rate at which RNA strands associated with each other, while leaving the process of duplex dissociation essentially unchanged. Importantly, this effect was not sequence-dependent and did not require the consumption of ATP.

By modeling this competitive binding process, the scientists were able to quantify its impact as an effective folding penalty of approximately 0.45 kcal/mol for every nucleotide in the RNA molecule.

This same effect was observed in lysates derived from HeLa cells, suggesting that it may be a common phenomenon across different cell types. When this finding was applied to chemical probing data obtained from inside cells, it was able to differentiate between the reactivity of around 42,000 stem-loops. In contrast, predictive models that were originally trained on RNA sequences in a dilute solution had classified nearly all of these stem-loops as being stably folded.

These results provide strong evidence that RNA folding within cells is influenced by a passive protein-mediated mechanism. This mechanism appears to play a key role in shifting the balance between paired and unpaired states in RNA molecules, without altering the fundamental rules of base pairing.