Abstract 3741: Identification of RNA binding proteins influenced by ionizing radiation through RNA interactome capture

Abstract

Abstract Cells respond to ionizing radiation (IR) through the activities of constitutively expressed proteins and through changes in gene expression. Constitutively expressed proteins play well-defined roles in major components of the cellular radioresponse, such as DNA repair and cell cycle. However, the contribution of changes in gene expression to the radioresponse is less understood. We have previously demonstrated through polysome profiling and microarray analysis that translational control is a key component of radiation-induced changes in gene expression. In response to IR, specific transcripts are recruited to or away from polysomes without affecting the global polysome profile of the cells. One mechanistic explanation for this observation is that IR influences the activity of RNA binding proteins (RBPs), which regulate the inclusion or exclusion of transcripts from polysomes. Here, we utilized RNA interactome capture (RIC) to identify RBPs bound to transcripts in control and irradiated human cancer cells. Cells were either mock irradiated or treated with 2Gy. One hour post-IR, RNA and protein complexes were crosslinked with 254 nm UV light. mRNA was isolated from cell lysates with oligo(dT) magnetic beads followed by several rounds of highly stringent washes to remove non-specific interactors. RNA was removed from crosslinked proteins by RNase digestion. The proteins were separated on SDS-PAGE gels and subjected to in-gel trypsin digestion, and the subsequent peptides were analyzed by label-free quantification mass spectrometry using an Orbitrap Fusion mass spectrometer. Proteome Discoverer 2.2 was used to search the data against human proteins from the UniProt database using SequestHT with a 1% false discovery rate. We found that we were able to efficiently pull down RNA:protein complexes with a crosslinking dose of 150 mJ/cm2. As determined by silver staining, the protein pattern of crosslinked samples differed greatly from protein isolated from whole cell lysates. By western blot, this method highly enriched for known RBPs, such as PTBP1 and CUGBP1, while contamination from non-RBPs, such as actin and tubulin, was not detected. By mass spectrometry, we identified hundreds of RBPs in each replicate above the no crosslinking background control for both untreated and irradiated cells. However, more RBPs were consistently detected in the control cells. Examples of RBPs preferentially binding in control cells include SSB, a protein involved in a variety of RNA metabolic processes, and U2AF2, a protein involved in RNA splicing. Overall, these data demonstrate that RIC is a sensitive and rigorous method to identify RBPs and that RBPs may play a role in radiation-induced post-transcriptional gene regulation. Preferential binders identified by this method may provide further insight into pathways regulating tumor cell radiosensitivity. Citation Format: Stacey L. Lehman, Theresa Wechsler, Gaelyn C. Lyons, Lisa M. Jenkins, Kevin Camphausen, Philip J. Tofilon. Identification of RNA binding proteins influenced by ionizing radiation through RNA interactome capture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3741.