Tandem RNA isolation reveals functional rearrangement of RNA-binding proteins on; CDKN1B/p27; Kip1; 3'UTRs in cisplatin treated cells

Post-transcriptional control of gene expression is mediated via RNA-binding proteins (RBPs) that interact with mRNAs in a combinatorial fashion. While recent global RNA interactome capture experiments expanded the repertoire of cellular RBPs quiet dramatically, little is known about the assembly of RBPs on particular mRNAs; and how these associations change and control the fate of the mRNA in drug-treatment conditions. Here we introduce a novel biochemical approach, termed tobramycin-based tandem RNA isolation procedure (tobTRIP), to quantify proteins associated with the 3'UTRs of cyclin-dependent kinase inhibitor 1B (; CDKN1B/p27; Kip1; ) mRNAs; in vivo; . P27; Kip1; plays an important role in mediating a cell's response to cisplatin (CP), a widely used chemotherapeutic cancer drug that induces DNA damage and cell cycle arrest. We found that; p27; Kip1; mRNA is stabilized upon CP treatment of HEK293 cells through elements in its 3'UTR. Applying tobTRIP, we further compared the associated proteins in CP and non-treated cells, and identified more than 50 interacting RBPs, many functionally related and evoking a coordinated response. Knock-downs of several of the identified RBPs in HEK293 cells confirmed their involvement in CP-induced; p27; mRNA regulation; while knock-down of the KH-type splicing regulatory protein (KHSRP) further enhanced the sensitivity of MCF7 adenocarcinoma cancer cells to CP treatment. Our results highlight the benefit of specific; in vivo; mRNA-protein interactome capture to reveal post-transcriptional regulatory networks implicated in cellular drug response and adaptation.