Interrogating RNA Modifications Through Proximity Labeling and Platinum Molecular Probes

Abstract

Post-transcriptional modifications such as N6-methyladenosine (m6A) have been shown to regulate the stability, translation, and function of messenger RNA (mRNA) molecules. Agents, such as platinum therapeutics, are also known to introduce damage modifications to RNA transcripts that might influence their biological fates. Importantly, both classes of chemical modifications are believed to influence the localization of RNA, which is essential to faithfully executing biological processes in cells. Stress granules are transient, non-membrane bound organelles thought to be important subcellular destinations for mRNAs harboring such modifications. In this thesis, we developed biochemical strategies to uncover how chemical modifications impact RNA and its potential localization to stress granules. We developed an APEX proximity labeling approach that selectively labeled RNA in the mitochondria, cytosol, and stress granules. Our results revealed that labeled RNA could be enriched from living cells via our methodology for usage in mass spectrometry-based modification detection. To understand how platinum therapeutics modify RNA, we designed and employed clickable molecular probes reminiscent of Oxaliplatin. Although these molecular probes bound diverse RNA species in vitro and in vivo, we found that their primary mechanisms of action and eventual processing were still due to DNA binding. Our click chemistry approach uncovered that platinum molecular probes changed their cellular localization over time and, of note, induced stress granule formation. Taken together, our results suggest that converging these two lines of research could enable a powerful approach to unravel how chemical modifications influence RNA localization.