Abstract
Heat shock protein 70 (Hsp70) is well documented to possess general cytoprotective properties in protecting the cell against stressful and noxious stimuli. We have recently shown that expression of the stress-inducible Hsp70.3 gene in the myocardium in response to ischemic preconditioning is NF-κB-dependent and necessary for the resulting late phase cardioprotection against a subsequent ischemia/reperfusion injury. Here we show that the Hsp70.3 gene product is subject to post-transcriptional regulation through parallel regulatory processes involving microRNAs and alternative polyadenylation of the mRNA transcript. First, we show that cardiac ischemic preconditioning of the in vivo mouse heart results in decreased levels of two Hsp70.3-targeting microRNAs: miR-378* and miR-711. Furthermore, an ischemic or heat shock stimulus induces alternative polyadenylation of the expressed Hsp70.3 transcript that results in the accumulation of transcripts with a shortened 3'-UTR. This shortening of the 3'-UTR results in the loss of the binding site for the suppressive miR-378* and thus renders the alternatively polyadenylated transcript insusceptible to miR-378*-mediated suppression. Results also suggest that the alternative polyadenylation-mediated shortening of the Hsp70.3 3'-UTR relieves translational suppression observed in the long 3'-UTR variant, allowing for a more robust increase in protein expression. These results demonstrate alternative polyadenylation of Hsp70.3 in parallel with ischemic or heat shock-induced up-regulation of mRNA levels and implicate the importance of this process in post-transcriptional control of Hsp70.3 expression.
Highlights
Studied of the heat shock proteins, has been shown to be induced by many cardiac preconditioning stimuli, and plays a necessary role in the second window of protection [3,4,5,6]
Heat shock protein 70 (Hsp70).3 Expression Is Subject to Post-transcriptional Regulation in the Myocardium—We previously showed that Hsp70.3 mRNA expression was induced 26.5-fold in the myocardium after late ischemic preconditioning (IPC) in wild-type mice and 13.8-fold in 2M NF-B dominant-negative mice [6]
The suppressive effect of the Hsp70.3 3Ј-untranslated region (3Ј-UTR) sequence was observed with both a CMV promoter (Fig. 2) and the Hsp70.3 promoter, indicating that this suppressive effect is independent of promoter regulation and specific to the 3Ј-UTR sequence
Summary
Animals and Surgical Models—All mice were maintained in accordance with institutional guidelines, the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, revised 1996), and the University of Cincinnati Institutional Animal Care and Use Committee. The Hsp70.3 promoter was PCR-purified from genomic DNA of C57/Bl6 mice and cloned into the Promega pGL4.10 luciferase reporter vector. Cells were rinsed with sterile PBS, 40 l of cell culture lysis buffer (Promega) was added per well, and plates were incubated at room temperature for 5 min. For assessment of Hsp70.3 transcript levels, total RNA was isolated (RNeasy kit; Qiagen), and cDNA was synthesized (high capacity RNA-to-cDNA kit; Applied Biosystems) per the manufacturer’s instructions. RNAi (miRNA, siRNA, or negative control RNAi; Qiagen) was transfected at the indicated molar doses using Lipofectamine 2000 at the recommended ratio of 1.0 l/20 pmol of RNAi. All transfections were done in reduced serum Opti-MEM, an equal volume of DMEM supplemented with 20% FBS was added 4 h following transfection, and cells were cultured overnight prior to experimental treatment. Unpaired Student’s t tests were performed to assess differences between groups, and statistical significance was considered at p Յ 0.05
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