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AUF1 and HuR Proteins Stabilize Interleukin-8 mRNA in Human Saliva
V. Palanisamy1,
N.J. Park1,
J. Wang1 and
D.T. Wong1,2,3,4,5,*
1 School of Dentistry and Dental Research Institute,
2 Jonsson Comprehensive Cancer Center,
3 Molecular Biology Institute,
4 Division of Head & Neck Surgery/Otolaryngology, School of Medicine, and
5 Henry Samueli School of Engineering and Applied Science, University of California Los Angeles, Los Angeles, CA 90095-1668, USA
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Figure 1. Addition of AUUUA-containing competitor accelerates IL-8 RNA degradation in vitro. (A) Sequences of RNA used for the study. (B) Representative autoradiograms showing in vitro decay of full-length, radiolabeled IL-8 mRNA in the absence of competitor or in the presence of unlabeled 60-nt IL-8 ARE competitor (100 ng), unlabeled mutated IL-8 ARE competitor (100 ng), or a control 950-nt β-actin mRNA (100 ng). (C) Kinetics of transcript degradation. (D) Representative autoradiographs showing decay of 60-nt IL-8 ARE-containing fragments in which all 4 AREs were intact (IL-8 ARE), fragments in which a single ARE was mutated (3g mt), or fragments in which all AREs were mutated (All mt). (E) Kinetics of transcript degradation. The data are from three independent experiments; values are means ± SD.
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Figure 2. IL-8 mRNA associates with AUF1 and HuR in human saliva protein lysates. Salivary lysate was UV-cross-linked with IL-8 ARE, IL-8 ARE mutant, or β-actin. Cross-linked RNA-protein complexes were then immunoprecipitated with anti-AUF1 (lanes 4–6) or anti-HuR (lanes 8 and 9) and analyzed by SDS-PAGE. Lanes 1–3 show the cross-linked extract prior to immunoprecipitation. NS, non-specific serum.
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Figure 3. Recombinant HuR protects IL-8 mRNA against in vitro degradation by human saliva lysates. (A) Representative autoradiographs showing decay of IL-8 mRNA in the presence of increasing concentrations of recombinant HuR. (B) Autoradiographs showing decay in the presence of equal amounts of BSA or non-specific recombinant GST fusion protein. (C) Effect of recombinant HuR on the kinetics of transcript degradation. The data are from three independent experiments; values are means ± SD.
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Figure 4. AUF1 and HuR protein levels in human salivary glands and saliva. Western blot analysis of AUF1 isoforms and HuR in the submandibular gland (SM), the parotid gland (PA), and the saliva (SL).
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Journal of Dental Research, Vol. 87, No. 8,
772-776 (2008)
DOI: 10.1177/154405910808700803
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