mRNA Processing Body (P-Body) is a specialized cytoplasmic structure that functions as a major site for post-transcriptional and trnanslational repression. Target mRNA is guided into P-Body through base-pairing with its micro-RNA (miRNA), where they further bind to P-Body components such as GW182 and AGO2; thus get retained away from translation machinery or degraded. Our previous study detected an increase of a key component of P-Body, miRNA, in heat-shock (HS) protected hearts. To gain further insights into the protective mechanism, we hypothesized that the miRNA-associated protection is mediated through P-Body formation. This hypothesis was tested by measuring three key components of P-Body, i.e., miRNA (target seeker), GW182 (marker of P-Body, also called GW-Body) and AGO2 (mRNA catalytic enzyme).
METHODS
: ICR mice were either HS-preconditioned (15 min, 42°C, anal temperature) or kept at room temperature (controls). miRNA and proteins were extracted 2 hour after HS. miRNA induction was verified by RT-PCR. P-Body formation was evaluated by measuring the binding of GW182 and AGO2, using a combination of immunopricipitation and Western blotting techniques. To study the role of miRNA in P-Body formation, identical experiments were also repeated in mice treated with miRNA-1 inhibitor (antisense miRNA-1 with 2′-O-methyl base at every nucleotide).
RESULTS
: Compared to the control, HS-preconditioning significantly induced miRNA-1 (150 ± 11%, mean ± SEM), miRNA-21 (71 ± 10%) and miRNA-24 (68 ± 14%). GW182 (109 ± 16%) and AGO2 (50 ± 16%) were also increased in the HS-group. More importantly, there was an increase (39 ± 11%) in co-immunopricipitation between GW182 and AGO2 in the HS-group than in the control, indicating more binding of the two key P-Body components. However, this co-immunoprecipitation was significantly reduced (−68 ± 8%) in the mice treated with the miRNA-1 inhibitor after HS.
CONCLUSION
: The formation of P-Body following HS-preconditioning represents a novel protective pathway against ischemic injury. The pharmaceutical potential of P-Body formation may offer a new strategy in cardiac preconditioning.
Negative correlation between the ratio of K+to Na+and proline accumulation in tobacco suspension cells
