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

Abstract

Intermolecular base pairing is essential for normal gene regulation but can also cause neurodegeneration when dysregulated. Despite its importance, intermolecular base pairing remains poorly characterized in cells. We coupled AMT crosslinking with RNA pulldown to probe intermolecular base pairing in mammalian cells under oxidative stress, a state that drives G3BP1/2-dependent stress granule assembly, translational repression, and increased exposure of RNA sequences available for intermolecular interactions. AMT captured intermolecular base pairing interactions among reporter RNAs in vitro and the known interaction between the U3 snoRNA and pre-18S rRNA in cellulo. Although detection sensitivity was limited for endogenous mRNAs, we found that base pairing among inducibly expressed reporter mRNAs is sensitive to RNA sequence, oxidative stress, and G3BP1/2. Furthermore, chemical probing revealed that base accessibility to DMS within non-translated regions of candidate RNAs remains similar between stressed and unstressed cells, and those lacking G3BP1/2, indicating that RNAs remained state under these conditions. Our findings establish an approach for studying RNA base pairing in cells and suggest that intermolecular base pairing may act together with RNA structure to preserve RNAs during oxidative stress.