Fig. 1: RNA ligase mechanism and identification of C12orf29 by chemical proteomics using modified Ap3A probe.
a Schematic display of the three-step mechanism of RNA ligation by a 5′–3′ RNA ligase. In step 1, the ligase is auto-AMPylated on the catalytic lysine using ATP as the co-substrate. In step 2, the AMP is transferred from the catalytic lysine to the 5′-PO4 end of RNA (pRNA), giving the RNA-adenylate intermediate (AppRNA). In step 3, the ligated RNA is obtained upon the attack of 3′-OH to the AppRNA in the presence of the ligase along with the liberation of AMP. PPi, pyrophosphate. b Structures of Ap3A analogues employed in this study. c Schematic display of the workflow for the identification of C12orf29. Cell lysates are incubated with C2-eAp3A or controls. AMPylated proteins are expected to bear ethynyl functionalities that enable selective modification with an affinity tag desthiobiotin (DB) via CuAAC. Labelled proteins are enriched and identified by ABPP, and further verified by immunoblotting. d Structure of the azide-bearing desthiobiotin as affinity tag. e Affinity enrichment of C12orf29 from two cell lysates verified by immunoblotting (representative images of n = 3). Source data are provided as a Source Data file.
