Characterization of intermolecular base pairing using AMT crosslinking in mammalian cells during oxidative stress

Cellular stress induces global translational repression, leading to exposure of RNA sequences that could engage in intermolecular base pairing. However, characterization of these interactions in stressed cells remains limited. Here, we coupled RNA crosslinking with biotin pulldown to probe intermolecular base pairing in mammalian cells during oxidative stress. We found that oxidative stress downregulates intermolecular base pairing of a reporter mRNA engineered to enhance detection of such interactions. Consistently, crosslinking-dependent intermolecular base pairing was not readily detected among candidate mRNAs that accumulate in stress granules - RNA-rich condensates that form during stress. Furthermore, chemical probing of base accessibility revealed that while RNA regions within these transcripts remain structured during stress, they increase in structural diversity in a manner dependent on oxidative stress and the stress granule nucleating proteins G3BP1 and G3BP2. This enhanced structural heterogeneity may help reduce a sustained exposure of interaction-prone RNA sequences during stress such as those prone to crosslinking. We propose that the combined downregulation of intermolecular base pairing and increased RNA structural diversity provides a mechanism to preserve normal function of RNAs during stress, while simultaneously enabling the reversible assembly of stress granules.