Disulfide Tethering to Map Small Molecule Binding Sites Transcriptome-wide.

We report the development of Tether-seq, a transcriptome-wide screen to probe RNA-small molecule interactions using disulfide tethering. This technique uses s4U metabolic labeling to provide sites for reversible and covalent attachment of small molecule disulfides to the transcriptome. By screening under reducing conditions, we identify interactions that are stabilized by binding over those driven by the reactivity of the RNA sites. When applied to cellular RNA, Tether-seq with a disulfide analogue of risdiplam, an FDA-approved drug that targets RNA to treat spinal muscular atrophy (SMA), revealed a number of potential binding sites, most prominently at a site within the cytochrome C oxidase 1 (COX1) transcript. Structure probing by SHAPE-MaP revealed a structured motif and confirmed binding to the lead molecule. This work demonstrates that these screens have the power to identify binding sites throughout the transcriptome and provide invaluable insight into the thermodynamic properties that define small molecule binding.

Acetyl-methyllysine marks chromatin at active transcription start sites.

Lysine residues in histones and other proteins can be modified by post-translational modifications that encode regulatory information1. Lysine acetylation and methylation are especially important for regulating chromatin and gene expression2-4. Pathways involving these post-translational modifications are targets for clinically approved therapeutics to treat human diseases. Lysine methylation and acetylation are generally assumed to be mutually exclusive at the same residue. Here we report cellular lysine residues that are both methylated and acetylated on the same side chain to form Nε-acetyl-Nε-methyllysine (Kacme). We show that Kacme is found on histone H4 (H4Kacme) across a range of species and across mammalian tissues. Kacme is associated with marks of active chromatin, increased transcriptional initiation and is regulated in response to biological signals. H4Kacme can be installed by enzymatic acetylation of monomethyllysine peptides and is resistant to deacetylation by some HDACs in vitro. Kacme can be bound by chromatin proteins that recognize modified lysine residues, as we demonstrate with the crystal structure of acetyllysine-binding protein BRD2 bound to a histone H4Kacme peptide. These results establish Kacme as a cellular post-translational modification with the potential to encode information distinct from methylation and acetylation alone and demonstrate that Kacme has all the hallmarks of a post-translational modification with fundamental importance to chromatin biology.