CRAC datasets were collected for Eno1 and an untagged control (BY). Two replicates were collected for each.
UV crosslinking can be used to identify precise RNA targets for individual proteins, transcriptome-wide. We sought to develop a technique to generate reciprocal data, identifying precise sites of RNA-binding proteome-wide. The resulting technique, total RNA-associated protein purification (TRAPP), was applied to yeast (S. cerevisiae ) and bacteria (E. coli). In all analyses, SILAC labeling was used to quantify protein recovery in the presence and absence of irradiation. For S.cerevisiae, we also compared crosslinking using 254nm (UVC) irradiation (TRAPP) with 4-thiouracil (4SU) labeling combined with 350nm (UVA) irradiation (PAR-TRAPP). Recovery of proteins not anticipated to show RNA-binding activity was significantly higher in TRAPP compared to PAR-TRAPP. As an example of preferential TRAPP-crosslinking, we tested enolase (Eno1) and demonstrated its strong, but largely sequence independent, binding to RNA in vivo. We speculate that many protein-RNA interactions have biophysical effects on localization and/or accessibility, by opposing or promoting phase separation. Homologous metabolic enzymes showed RNA crosslinking in S. cerevisiae and E. coli, indicating conservation of this property. TRAPP allows alterations in RNA interactions to be followed and we initially analyzed the effects of weak acid stress. This revealed specific alterations in RNA-protein interactions; for example, during late 60S ribosome subunit maturation. Precise sites of crosslinking at the level of individual amino acids (iTRAPP) were identified following phospho-peptide enrichment combined with a bioinformatic pipeline (Xi). TRAPP is quick, simple and scalable, allowing rapid characterization of the RNA-bound proteome in many systems.
Shchepachev V, Bresson S, Spanos C, Petfalski E, Fischer L, Rappsilber J, Tollervey D (2019) Defining the RNA Interactome by Total RNA-Associated Protein Purification. Molecular Systems Biology