Similar Regulation of Human Inducible Nitric-oxide Synthase Expression by Different Isoforms of the RNA-binding Protein AUF1*
- Andrea Pautz‡,
- Katrin Linker‡,
- Sebastian Altenhöfer‡,
- Sebastian Heil‡,
- Nadine Schmidt‡,
- Julia Art‡,
- Shirley Knauer§,
- Roland Stauber§,
- Navid Sadri¶,
- Adam Pont¶,
- Robert J. Schneider¶ and
- Hartmut Kleinert‡1
- ‡Department of Pharmacology, Johannes Gutenberg University, Obere Zahlbacher Strasse 67, D-55101 Mainz, Germany, §Molecular and Cellular Oncology, University Hospital of Mainz, Langenbeckstrasse 1, D-55101 Mainz, Germany, and ¶Department of Microbiology, New York University School of Medicine, New York, New York 10003
- 1 To whom correspondence should be addressed: Dept. of Pharmacology, Johannes Gutenberg University, Obere Zahlbacher Str. 67, 55101 Mainz, Germany. Tel.: 49-6131-393-3245; Fax: 49-6131-393-6611; E-mail: kleinert{at}mail.uni-mainz.de.
Abstract
The ARE/poly-(U) binding factor 1 (AUF1), a protein family consisting of four isoforms, is believed to mediate mRNA degradation by binding to AU-rich elements (ARE). However, evidence exists that individual AUF1 isoforms may stabilize ARE-containing mRNAs. The 3′-untranslated region of the human inducible nitric-oxide synthase (iNOS) contains five AREs, which promote RNA degradation. We have recently shown that the RNA-binding protein KSRP is critically involved in the decay of the iNOS mRNA. In this study we examined the effects of the individual AUF1 isoforms on iNOS expression. Overexpression of each AUF1 isoform reduces iNOS expression on mRNA and protein levels to the same extent by modulation of mRNA stability. Accordingly, knockdown of all or individual AUF1 isoforms by an RNA interference approach enhances iNOS expression. The AUF1 effect on iNOS expression is dependent on the iNOS 3′-untranslated region sequence, as demonstrated in transfection experiments with a reporter mRNA. Binding studies showed that all AUF1 isoforms interact with the same AU-rich region in the iNOS-3′-untranslated region. Cytokine stimulation altered intracellular AUF1 binding activities. These data demonstrate that AUF1 is an important factor that promotes iNOS mRNA degradation. Furthermore, all individual AUF1 isoforms act in a similar manner.
Footnotes
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↵2 The abbreviations used are: ARE, AU-rich element; UTR, untranslated region; AUF1, AU-rich element RNA-binding protein 1; CM, cytokine mixture; Dox, doxycycline; GFP, green fluorescence protein; EGFP, enhanced GFP; GST, glutathione S-transferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HuR, human antigen R; Luc, luciferase; KSRP, KH-type splicing regulatory protein; iNOS, inducible NO synthase; RT, reverse transcription; qRT, quantitative real time RT; BP, binding protein; PTB, polypyrimidine tract binding protein; siRNA, small interfering RNA; shRNA, short hairpin RNA; TTP, tristetraprolin; DRB, 6-dichloro-1-ribofuranosylbenzimidazole.
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↵* This work was supported by Grant 8312-38 62 61/322a and -b from the Innovation Foundation of the State of Rhineland-Palatinate, by the Collaborative Research Center SFB 553 (Project A7 (to H. K.), and by the Deutsche Forschungsgemeinschaft Grant LI 1759/1-1. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.
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- Received December 10, 2008.
- Revision received December 12, 2008.
- The American Society for Biochemistry and Molecular Biology, Inc.
