Global Discovery of Covalent Modulators of Ribonucleoprotein Granules.

Stress granules (SGs) and processing-bodies (PBs, P-bodies) are ubiquitous and widely studied ribonucleoprotein (RNP) granules involved in cellular stress response, viral infection, and the tumor microenvironment. While proteomic and transcriptomic investigations of SGs and PBs have provided insights into molecular composition, chemical tools to probe and modulate RNP granules remain lacking. Herein, we combine an immunofluorescence (IF)-based phenotypic screen with chemoproteomics to identify sulfonyl-triazoles (SuTEx) capable of preventing or inducing SG and PB formation through liganding of tyrosine (Tyr) and lysine (Lys) sites in stressed cells. Liganded sites were enriched for RNA-binding and protein-protein interaction (PPI) domains, including several sites found in RNP granule-forming proteins. Among these, we functionally validate G3BP1 Y40, located in the NTF2 dimerization domain, as a ligandable site that can disrupt arsenite-induced SG formation in cells. In summary, we present a chemical strategy for the systematic discovery of condensate-modulating covalent small molecules.

Mechanisms of epitranscriptomic gene regulation.

Chemical modifications on RNA can regulate fundamental biological processes. Recent efforts have illuminated the chemical diversity of posttranscriptional ("epitranscriptomic") modifications on eukaryotic messenger RNA and have begun to elucidate their biological roles. In this review, we discuss our current molecular understanding of epitranscriptomic RNA modifications and their effects on gene expression. In particular, we highlight the role of modifications in mediating RNA-protein interactions, RNA structure, and RNA-RNA base pairing, and how these macromolecular interactions control biological processes in the cell.

YTHDF2 Recognition of N1-Methyladenosine (m1A)-Modified RNA Is Associated with Transcript Destabilization.

Epitranscriptomic modifications play an important role in RNA function and can impact gene expression. Here, we apply a chemical proteomics approach to investigate readers of N1-methyladenosine (m1A), a poorly characterized modification on mammalian mRNA. We find that YTHDF proteins, known m6A readers, recognize m1A-modified sequences in a methylation-specific manner. We characterize binding of recombinant YTHDF1/2 proteins to m1A-modified oligonucleotides to demonstrate that these interactions can exhibit comparable affinity to m6A-recognition events and occur in diverse sequence contexts. Further, we demonstrate YTHDF2 interacts specifically with endogenously modified m1A transcripts. Finally, we deplete cellular YTHDF2 to show that the abundance of m1A-modified transcripts is increased in its absence. Similarly, increasing m1A levels through depletion of ALKBH3, an m1A eraser protein, destabilizes known m1A-containing RNAs. Our results shed light on the function of m1A on mRNA and provide a mechanistic framework to further evaluate the role of m1A in biological processes.