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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...

We have developed an ingenious method, termed Cas9 nickase-based amplification reaction (Cas9nAR), to amplify at arget fragment from genomic DNAa taconstant temperature of 37 8 8C. Cas9nAR employs as gRNA:Cas9n complex with as ingle-strand nicking property,astranddisplacing DNAp olymerase,a nd two primers bearing the cleavage sequence of Cas9n, to promote cycles of DNA replication through priming,e xtension, nicking,a nd displacement reaction steps.C as9nAR exhibits az eptomolar limit of detection (2 copies in 20 mLofreaction system) within 60 min and asingle-base discrimination capability.More importantly, the underlying principle of Cas9nAR offers simplicity in primer design and universality in application. Considering the superior sensitivity and specificity,a sw ell as the simple-toimplement, rapid, and isothermal features,C as9nAR holds great potential to become ar outine assayf or the quantitative detection of nucleic acids in basic and applied studies.