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Dihydrotanshinone-I interferes with the RNA-binding activity of HuR affecting its post-transcriptional function.

ABSTRACT: Post-transcriptional regulation is an essential determinant of gene expression programs in physiological and pathological conditions. HuR is a RNA-binding protein that orchestrates the stabilization and translation of mRNAs, critical in inflammation and tumor progression, including tumor necrosis factor-alpha (TNF). We identified the low molecular weight compound 15,16-dihydrotanshinone-I (DHTS), well known in traditional Chinese medicine practice, through a validated high throughput screening on a set of anti-inflammatory agents for its ability to prevent HuR:RNA complex formation. We found that DHTS interferes with the association step between HuR and the RNA with an equilibrium dissociation constant in the nanomolar range in vitro (Ki?=?3.74?±?1.63?nM). In breast cancer cell lines, short term exposure to DHTS influences mRNA stability and translational efficiency of TNF in a HuR-dependent manner and also other functional readouts of its post-transcriptional control, such as the stability of selected pre-mRNAs. Importantly, we show that migration and sensitivity of breast cancer cells to DHTS are modulated by HuR expression, indicating that HuR is among the preferential intracellular targets of DHTS. Here, we disclose a previously unrecognized molecular mechanism exerted by DHTS, opening new perspectives to therapeutically target the HuR mediated, post-transcriptional control in inflammation and cancer cells.

SUBMITTER: D'Agostino VG

PROVIDER: S-EPMC4639722 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Dihydrotanshinone-I interferes with the RNA-binding activity of HuR affecting its post-transcriptional function.

D'Agostino Vito Giuseppe VG Lal Preet P Mantelli Barbara B Tiedje Christopher C Zucal Chiara C Thongon Natthakan N Gaestel Matthias M Latorre Elisa E Marinelli Luciana L Seneci Pierfausto P Amadio Marialaura M Provenzani Alessandro A

Scientific reports 20151110

Post-transcriptional regulation is an essential determinant of gene expression programs in physiological and pathological conditions. HuR is a RNA-binding protein that orchestrates the stabilization and translation of mRNAs, critical in inflammation and tumor progression, including tumor necrosis factor-alpha (TNF). We identified the low molecular weight compound 15,16-dihydrotanshinone-I (DHTS), well known in traditional Chinese medicine practice, through a validated high throughput screening o ...[more]

PMID: 26553968

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Project description:Background & aimsPaneth cells secrete antimicrobial proteins including lysozyme via secretory autophagy as part of the mucosal protective response. The ELAV like RNA-binding protein 1 (ELAVL1, also called HuR) regulates stability and translation of messenger RNAs (mRNAs) and many aspects of mucosal physiology. We studied the posttranscriptional mechanisms by which HuR regulates Paneth cell function.MethodsIntestinal mucosal tissues were collected from mice with intestinal epithelium (IE)-specific disruption of HuR (IE-HuR-/-), HuRfl/fl-Cre- mice (controls), and patients with inflammatory bowel diseases and analyzed by histology and immunohistochemistry. Paneth cell functions were determined by lysozyme-immunostaining assays. We isolated primary enterocytes from IE-HuR-/- and control mice and derived intestinal organoids. HuR and the chaperone CNPY3 were overexpressed from transgenes in intestinal epithelial cells (IECs) or knocked down with small interfering RNAs. We performed RNA pulldown assays to investigate interactions between HuR and its target mRNAs.ResultsIntestinal tissues from IE-HuR-/- mice had reduced numbers of Paneth cells, and Paneth cells had fewer lysozyme granules per cell, compared with tissues from control mice, but there were no effects on Goblet cells or enterocytes. Intestinal mucosa from patients with inflammatory bowel diseases had reduced levels of HuR and fewer Paneth cells. IE-HuR-/- mice did not have the apical distribution of TLR2 in the intestinal mucosa as observed in control mice. IECs from IE-HuR-/- mice expressed lower levels of CNPY3. Intestinal organoids from IE-HuR-/- mice were smaller and contained fewer buds compared with those generated from controls, and had fewer lysozyme-positive cells. In IECs, knockdown of HuR decreased levels of the autophagy proteins LC3-I and LC3-II, compared with control cells, and prevented rapamycin-induced autophagy. We found HuR to interact directly with the Cnpy3 mRNA coding region and increase levels of CNPY3 by increasing the stability and translation of Cnpy3 mRNA. CNPY3 bound TLR2, and cells with knockdown of CNPY3 or HuR lost membrane localization of TLR2, but increased cytoplasmic levels of TLR2.ConclusionsIn studies of mice, IECs, and human tissues, we found HuR to increase expression of CNPY3 at the posttranscriptional level. CNPY3 is required for membrane localization of TLR2 and Paneth cell function.

Dataset Information

Dihydrotanshinone-I interferes with the RNA-binding activity of HuR affecting its post-transcriptional function.

Publications

Dihydrotanshinone-I interferes with the RNA-binding activity of HuR affecting its post-transcriptional function.

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