Label-Free Protein-RNA Interactome Analysis Identifies Khsrp Signaling Downstream of the p38/Mk2 Kinase Complex as a Critical Modulator of Cell Cycle Progression
Fig 1
Changes in protein-RNA interactions in response to etoposide treatment.
(A) Etoposide induces DNA double strand breaks after 6h of treatment as reported by the DSB marker γ-H2AX. (B) Schematics of the experimental procedure for the purification of RBPs show how UV-mediated crosslinking of RNA to interacting proteins was followed by poly-A selection to identify RBPs through LC/MS/MS. (C) Crosslinking followed by purification of mRNA-interacting proteins and nano LC/MS/MS identified 335 protein group hits of which 287 were known as RBPs. (D) Heat map of differentially abundant RBPs. (E) Vulcano plot representing changes in mRNA-protein interactions in response to etoposide treatment identifies Khsrp as the most significantly changed RBP in response to etoposide treatment. (F) Immunoblot analysis of proteins co-purified with poly-A-containing RNA validates protein-RNA interactome changes identified by label free LC/MS/MS (right panel). Whole cell lysates show no significant changes in protein levels of analyzed RBPs (left panel).