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...
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Published in | RNA biology Vol. 17; no. 1; pp. 33 - 46 |
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Main Authors | , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
Taylor & Francis
02.01.2020
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Subjects | |
Online Access | Get full text |
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Summary: | 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 3ʹUTRs 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 3ʹUTR. 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. |
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ISSN: | 1547-6286 1555-8584 |
DOI: | 10.1080/15476286.2019.1662268 |