Chemical modifications on central biomacromolecules play a critical role in cellular physiology. Particularly, eukaryotic mRNA is extensively tuned post-transcriptionally with a diverse array of epitranscriptomic marks. Of these, the nucleobase N6-methyladenosine (m6A) can regulate mRNA stability, translation, splicing, and localization. Still, we lack a significant understanding of the exact mechanisms by which m6A governs these aspects of mRNA biology. This gap in our knowledge stems from the challenge in understanding the effect of these modifications on the protein-RNA interactome. Here, we present two complementary approaches to study m6A-regulated RNA-protein. First, we develop a chemical proteomics methodology relying upon photocrosslinking with synthetic diazirine-containing RNA probes and quantitative proteomics to profile ‘readers’ of m6A. We identify known m6A interactors, namely the YTH-domain-containing proteins and the m6A ‘eraser’ ALKBH5, and we also show that the disease-associated proteins FMR1 and LRPPRC can ‘read’ this mark. In addition, we find that stress granule proteins G3BP1/2, USP10, CAPRIN1, and RBM42 interact preferentially with unmethylated RNA. Second, we establish a high-throughput platform, relying upon in vitro selection with site-specifically modified random RNA library, to survey the binding specify of m6A reader proteins. We apply this scheme to characterize the m6A-dependent recognition motifs of ‘reader’ proteins YTHDF2 and YTHDC1 and find overlap with the canonical DR(m6A)CH motif, but overall distinct binding preferences for each protein. Taken together, our findings offer insights on the biochemical mechanisms underlying m6A function in biological settings and the sequence determinants necessary for recognition by its ‘readers,’ suggesting that the m6A interactome is more complex than previously appreciated. Ultimately, we envision that these approaches can be deployed together to investigate other interaction networks between protein and modified mRNA.

Chemical approaches to characterize N6-methyladenosine-regulated RNA-protein interactions

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

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