Coordinated Post-transcriptional Regulation of Hsp70.3 Gene Expression by MicroRNA and Alternative Polyadenylation

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-B-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.Heat shock (HS) 3 and the subsequent expression of heat shock proteins have long been known to possess cytoprotective properties and provide cardioprotection against ischemia/reperfusion (I/R) injury (1-3). The Hsp70 family is among the best 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 (3-6). Hsp70.1 and Hsp70.3 are two nearly identical stress-inducible Hsp70 genes present in the murine heart. The two protein products differ by only a single amino acid, but curiously, the sequence of the two genes is divergent in the regulatory regions of the gene promoter (beyond the first 270 amino acids proximal to the transcriptional start site) and within the 3Ј-untranslated region (3Ј-UTR) of the mRNA transcript. Historically, these two genes are considered to be functionally redundant.We have recently shown that NF-B-dependent expression of heat shock protein 70.3 (Hsp70.3), but not the closely related Hsp70.1, is necessary for the late phase or second window of ischemic preconditioning (IPC) cardioprotection against acute I/R in the heart (6). In fact, Hsp70.3 and Hsp70.1 appear to contribute differing functions to cell survival in the myocardium (6, 7). Thus, it is important to understand the regulation of these two genes independently. We previously reported that NF-B in...

The Use of microRNAs to Modulate Redox and Immune Response to Stroke

“…miR-1, which exacerbates cardiac ischemia-reperfusion injury, decreases expression of HSP60 (135). Cardiac ischemia preconditioning, which protects against ischemic injury, decreases miR-378* and miR-711, both of which target the HSP70 family (170). Overexpression of miR-320 in cardiomyocytes enhances ischemia-reperfusion injury, in part, via downregulation of HSP20 (146).…”

Non-coding RNAs turn up the heat: An emerging layer of novel regulators in the mammalian heat shock response

“…In rat ventricular cells, miR-1 also functions to promote apoptosis during oxidative stress by targeting both HSPD1/HSP60 and HSPA1A/HSP70 . Conversely, ischemia preconditioning in the murine heart decreases levels of miR-378* and miR-711, which aid in tissue protection by increasing levels of downstream target HSPA1A/Hsp70.3 (Tranter et al 2011). In addition, depletion of miR-320 enhances levels of its downstream target HSPB6/ HSP20 to protect cardiomyocytes from I/R-induced death (Ren et al 2009).…”

B-chromosome effects on Hsp70 gene expression does not occur at transcriptional level in the grasshopper Eyprepocnemis plorans

“…The remaining samples showed no significant difference associated with the presence of B chromosomes, suggesting that the most important regulation events of Hsp70, at this respect, could take place at post-transcriptional level. This may include any of the battery of mRNA modification processes (Tranter et al 2011). Such a diversity of processes could make this regulation level prone to be affected by the presence of B chromosomes.…”