Abstract
Growing evidence suggests a key role for RNA binding proteins (RBPs) in genome stability programs. Additionally, recent developments in RNA sequencing technologies, as well as mass-spectrometry techniques, have greatly expanded our knowledge on protein-RNA interactions. We here use full transcriptome sequencing and label-free LC/MS/MS to identify global changes in protein-RNA interactions in response to etoposide-induced genotoxic stress. We show that RBPs have distinct binding patterns in response to genotoxic stress and that inactivation of the RBP regulator module, p38/MK2, can affect the entire spectrum of protein-RNA interactions that take place in response to stress. In addition to validating the role of known RBPs like Srsf1, Srsf2, Elavl1 in the genotoxic stress response, we add a new collection of RBPs to the DNA damage response. We identify Khsrp as a highly regulated RBP in response to genotoxic stress and further validate its role as a driver of the G1/S transition through the suppression of Cdkn1aP21 transcripts. Finally, we identify KHSRP as an indicator of overall survival, as well as disease free survival in glioblastoma multiforme.
Highlights
In response to genotoxic stress, cells activate a complex, kinase-based signaling network, which is commonly referred to as the DNA damage response (DDR) [1, 2]
We show how label-free LC/MS/MS can be used for the profiling of functional RBPmRNA interactome changes in response to genotoxic stress induced by etoposide
Protein levels in whole cell lysates showed no change in response to etoposide treatment, while the etoposide-induced changes that were observed in the RNA-bound fractions could be reproduced by immunoblotting. These data suggest that the interaction between the RNA binding proteins (RBPs) under investigation and their client mRNAs is altered after genotoxic stress (Fig 1F, left panel)
Summary
Mouse embryonic fibroblasts (MEFs) were isolated as described previously [29], and cultured in high-glucose DMEM supplemented with 10% heat-inactivated FBS, 1% HEPES, 100 U/mL penicillin, and 100 μg/mL treptomycin (Gibco). Cells were treated with either etoposide (Sigma, E1383) or DMSO (vehicle / mock control; Carl Roth, A994.2). Mk2/3 knock out animals were previously published and were a kind gift from Matthias Gaestel at the Hannover Medical School [30]. Khsrp-/- MEFs were previously published and were a kind gift from Ching-Yi Chen at the University of Alabama at Birmingham [31]. Colony formation assays were performed as previously described [11]. Animal keeping was authorized by the “Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen” with the license 87– 51.04.2010.A006
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