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Novel role for molecular transporter importin 9 in posttranscriptional regulation of IFN-? expression.

ABSTRACT: IFN-? is a unique type I IFN whose constitutive expression in lung, brain, small intestine, and reproductive tissues is only partially understood. Our previous observation that posttranscriptional events participate in the regulation of IFN-? mRNA expression led us to investigate whether the 5' and/or 3' untranslated regions (UTR) have regulatory functions. Surprisingly, we found that full-length IFN-? 5'UTR markedly suppressed mRNA expression under basal conditions. Analysis of the secondary structure of this region predicted formation of two stable stem-loop structures, loops 1 and 2. Studies using luciferase constructs harboring various stretches of IFN-? 5'UTR and mutant constructs in which the conformation of loop structures was disrupted showed that loop 1 is essential for regulation of mRNA expression. Incubation of HeLa cell extracts with agarose-bound RNAs harboring IFN-? loop structures identified importin 9 (IPO9), a molecular transporter and chaperone, as a candidate that associates with these regions of the 5'UTR. IPO9 overexpression decreased, and IPO9 silencing increased basal IFN-? expression. Our studies uncover a previously undescribed function for IPO9 as a specific, and negative, posttranscriptional regulator of IFN-? expression, and they identify key roles for IFN-? stem-loop structure 1 in this process. IPO9-mediated effects on 5'UTRs appear to extend to additional mRNAs, including hypoxia-inducible factor-1?, that can form specific loop structures.

SUBMITTER: Matsumiya T

PROVIDER: S-EPMC3758363 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Novel role for molecular transporter importin 9 in posttranscriptional regulation of IFN-ε expression.

Matsumiya Tomoh T Xing Fei F Ebina Masayuki M Hayakari Ryo R Imaizumi Tadaatsu T Yoshida Hidemi H Kikuchi Hideaki H Topham Matthew K MK Satoh Kei K Stafforini Diana M DM

Journal of immunology (Baltimore, Md. : 1950) 20130712 4

IFN-ε is a unique type I IFN whose constitutive expression in lung, brain, small intestine, and reproductive tissues is only partially understood. Our previous observation that posttranscriptional events participate in the regulation of IFN-ε mRNA expression led us to investigate whether the 5' and/or 3' untranslated regions (UTR) have regulatory functions. Surprisingly, we found that full-length IFN-ε 5'UTR markedly suppressed mRNA expression under basal conditions. Analysis of the secondary st ...[more]

PMID: 23851686

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Project description:Calretinin (CALB2) is a diagnostic and prognostic marker in malignant pleural mesothelioma (MPM). We previously reported that calretinin expression is regulated at the mRNA level. The presence of a medium-sized (573 nucleotide) 3' untranslated region (3'UTR) predicted to contain binding sites for miR-30a/b/c/d/e and miR-9 as well as an adenine/uridine-rich element (ARE) in all three transcripts arising from the CALB2 gene, suggests that calretinin expression is regulated via posttranscriptional mechanisms. Our aim was to investigate the role of the CALB2-3'UTR in the posttranscriptional regulation of calretinin expression in MPM. CALB2-3'UTR was inserted downstream of the luciferase reporter gene using pmiRGLO vector and reporter expression was determined after transfection into MPM cells. Targeted mutagenesis was used to generate variants harboring mutated miR-30 family and ARE binding sites. Electrophoretic mobility shift assay was used to test for the presence of ARE binding proteins. CALB2-3'UTR significantly decreased luciferase activity in MPM cells. Analysis of mutation in the ARE site revealed a further destabilization of the reporter and human antigen R (HuR) binding to the ARE sequence was detected. The mutation of two miR-30 binding sites abolished CALB2-3'UTR destabilization effect; a transient delivery of miR-30e-5p mimics or anti-miR into MPM cells resulted in a significant decrease/increase of the luciferase reporter expression and calretinin protein, respectively. Moreover, overexpression of CALB2-3'UTR quenched the effect of miR-30e-5p mimics on calretinin protein levels, possibly by sequestering the mimics, thereby suggesting a competitive endogenous RNA network. Finally, by data mining we observed that expression of miR-30e-5p was negatively correlated with the calretinin expression in a cohort of MPM patient samples. Our data show the role of (1) adenine-uridine (AU)-binding proteins in calretinin stabilization and (2) miR-30e-5p in the posttranscriptional negative regulation of calretinin expression via interaction with its 3'UTR. Furthermore, our study demonstrates a possible physiological role of calretinin's alternatively spliced transcripts.

Dataset Information

Novel role for molecular transporter importin 9 in posttranscriptional regulation of IFN-? expression.

Publications

Novel role for molecular transporter importin 9 in posttranscriptional regulation of IFN-ε expression.

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