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A single element in the 3'UTR of the apical sodium-dependent bile acid transporter controls both stabilization and destabilization of mRNA.

ABSTRACT: mRNA stability appears to play a key role in the ontogenic regulation of the apical sodium-dependent bile acid transporter (ASBT). The RNA-binding proteins Hu antigen R (HuR) and tristetraprolin (TTP) stabilize and destabilize ASBT mRNA, respectively. Potential HuR-binding sites were assessed by sequence analysis in the context of prior in vitro functional analyses of the rat ASBT 3'UTR. Wild-type and mutant-binding sites were investigated by gel-shift analysis using IEC-6 cell extracts. The functional consequences of binding site mutations were assessed using two different hybrid reporter constructs linking the 3'UTR element to either a luciferase or a β-globin coding mRNA sequence. A specific metastasis-associated gene 1 (MTA1) cis-element was identified in the ASBT 3'UTR that became associated with proteins in IEC-6 cell extracts and could be supershifted by anti-HuR or anti-TTP antibodies. Mutation of this cis-element abrogated the gel shift of IEC-6 proteins. Furthermore, hybrid constructs containing a mutant MTA1 element had reduced responses to modulation of HuR or TTP. For the first time, we have identified a single specific sequence element in the 3'UTR of the rat ASBT mRNA that mediates counter-regulatory changes in mRNA abundance in response to both HuR and TTP.

SUBMITTER: Soler DM

PROVIDER: S-EPMC5338459 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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A single element in the 3'UTR of the apical sodium-dependent bile acid transporter controls both stabilization and destabilization of mRNA.

Soler Dellys M DM Ghosh Ayantika A Chen Frank F Shneider Benjamin L BL

The Biochemical journal 20140901 3

mRNA stability appears to play a key role in the ontogenic regulation of the apical sodium-dependent bile acid transporter (ASBT). The RNA-binding proteins Hu antigen R (HuR) and tristetraprolin (TTP) stabilize and destabilize ASBT mRNA, respectively. Potential HuR-binding sites were assessed by sequence analysis in the context of prior in vitro functional analyses of the rat ASBT 3'UTR. Wild-type and mutant-binding sites were investigated by gel-shift analysis using IEC-6 cell extracts. The fun ...[more]

PMID: 24946903

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Project description:UnlabelledDeficiency of multidrug resistance 2 (mdr2), a canalicular phospholipid floppase, leads to excretion of low-phospholipid "toxic" bile causing progressive cholestasis. We hypothesize that pharmacological inhibition of the ileal, apical sodium-dependent bile acid transporter (ASBT), blocks progression of sclerosing cholangitis in mdr2(-/-) mice. Thirty-day-old, female mdr2(-/-) mice were fed high-fat chow containing 0.006% SC-435, a minimally absorbed, potent inhibitor of ASBT, providing, on average, 11 mg/kg/day of compound. Bile acids (BAs) and phospholipids were measured by mass spectrometry. Compared with untreated mdr2(-/-) mice, SC-435 treatment for 14 days increased fecal BA excretion by 8-fold, lowered total BA concentration in liver by 65%, reduced total BA and individual hydrophobic BA concentrations in serum by >98%, and decreased plasma alanine aminotransferase, total bilirubin, and serum alkaline phosphatase levels by 86%, 93%, and 55%, respectively. Liver histology of sclerosing cholangitis improved, and extent of fibrosis decreased concomitant with reduction of hepatic profibrogenic gene expression. Biliary BA concentrations significantly decreased and phospholipids remained low and unchanged with treatment. The phosphatidylcholine (PC)/BA ratio in treated mice corrected toward a ratio of 0.28 found in wild-type mice, indicating decreased bile toxicity. Hepatic RNA sequencing studies revealed up-regulation of putative anti-inflammatory and antifibrogenic genes, including Ppara and Igf1, and down-regulation of several proinflammatory genes, including Ccl2 and Lcn2, implicated in leukocyte recruitment. Flow cytometric analysis revealed significant reduction of frequencies of hepatic CD11b(+) F4/80(+) Kupffer cells and CD11b(+) Gr1(+) neutrophils, accompanied by expansion of anti-inflammatory Ly6C(-) monocytes in treated mdr2(-/-) mice.ConclusionInhibition of ASBT reduces BA pool size and retention of hydrophobic BA, favorably alters the biliary PC/BA ratio, profoundly changes the hepatic transcriptome, attenuates recruitment of leukocytes, and abrogates progression of murine sclerosing cholangitis.

Dataset Information

A single element in the 3'UTR of the apical sodium-dependent bile acid transporter controls both stabilization and destabilization of mRNA.

Publications

A single element in the 3'UTR of the apical sodium-dependent bile acid transporter controls both stabilization and destabilization of mRNA.

Similar Datasets

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