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Hsp27 binding to the 3'UTR of bim mRNA prevents neuronal death during oxidative stress-induced injury: a novel cytoprotective mechanism.

Dávila D, Jiménez-Mateos EM, Mooney CM, Velasco G, Henshall DC, Prehn JH - Mol. Biol. Cell (2014)

Bottom Line: This effect could not be explained by proteasomal degradation of Bim or bim promoter inhibition; however, it was associated with a specific increase in the levels of bim mRNA and with its binding to Hsp27.Finally, we determined that enhanced Hsp27 expression in neurons exposed to H2O2 or glutamate prevented the translation of a reporter plasmid where bim-3'UTR mRNA sequence was cloned downstream of a luciferase gene.These results suggest that repression of bim mRNA translation through binding to the 3'UTR constitutes a novel cytoprotective mechanism of Hsp27 during stress in neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Medical Physics and RCSI Centre for the Study of Neurological Disorders, Royal College of Surgeons in Ireland, Dublin 2, Ireland Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain daviddav@ucm.es prehn@rcsi.ie.

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Hsp27 regulates bim-3′UTR sequence during oxidative stress. (A) Quantification of bim mRNA levels bound to the ribosomal protein RPL10a present at the polysome. CGNs were treated with H2O2 (37.5 μM). Hsp27 overexpression reduced bim mRNA levels coimmunoprecipitated with RPL10a when compared with controls (*p < 0.05; n = 3). (B) CGNs were cotransfected with a luciferase reporter plasmid containing the bim-3′UTR (divided into two sequences, named 1 and 2) and either pNEO-Hsp27 or a control construct. Bim-3′UTR (sequence 1) neurons showed a significant increase in the luciferase activity after H2O2 (37.5 μM) addition (*p < 0.05; n = 3). Coexpression of pNEO-Hsp27 prevented this effect, significantly reducing the luciferase activity (*p < 0.05; n = 3). (C) Bim-3′UTR (sequence 2) neurons did not show any modification of the luciferase activity induced by H2O2 treatment or pNEO-Hsp27 coexpression. (D) CGNs were coelectroporated with the luciferase reporter plasmid containing the bim-3′UTR (sequence 1) and either Hsp25 siRNA or control siRNA. Hsp25 siRNA neurons displayed significantly higher luciferase activity than control siRNA neurons (*p < 0.05; n = 3). (E) Quantification of bim mRNA levels bound to the endogenous Hsp25 protein and to the wild-type form of the human Hsp27 (pNEO-Hsp27 construct) overexpressed in CGNs. H2O2 (37.5 μM) treatment significantly increased bim mRNA levels coimmuno­precipitated with the Hsp25/Hsp27 compared to control neurons (*p < 0.05; n = 4). The coimmunoprecipitated bim mRNA levels were significantly increased in H2O2-treated cells when Hsp27 was overexpressed (*p < 0.05; n = 4). As positive and negative controls for the immuno­precipitation, a specific antibody for Argonaute-2 protein and an unspecific serum were used, respectively. Argonaute-2 levels were detected by Western blot. bim mRNA levels were not detected in the negative control.
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Figure 4: Hsp27 regulates bim-3′UTR sequence during oxidative stress. (A) Quantification of bim mRNA levels bound to the ribosomal protein RPL10a present at the polysome. CGNs were treated with H2O2 (37.5 μM). Hsp27 overexpression reduced bim mRNA levels coimmunoprecipitated with RPL10a when compared with controls (*p < 0.05; n = 3). (B) CGNs were cotransfected with a luciferase reporter plasmid containing the bim-3′UTR (divided into two sequences, named 1 and 2) and either pNEO-Hsp27 or a control construct. Bim-3′UTR (sequence 1) neurons showed a significant increase in the luciferase activity after H2O2 (37.5 μM) addition (*p < 0.05; n = 3). Coexpression of pNEO-Hsp27 prevented this effect, significantly reducing the luciferase activity (*p < 0.05; n = 3). (C) Bim-3′UTR (sequence 2) neurons did not show any modification of the luciferase activity induced by H2O2 treatment or pNEO-Hsp27 coexpression. (D) CGNs were coelectroporated with the luciferase reporter plasmid containing the bim-3′UTR (sequence 1) and either Hsp25 siRNA or control siRNA. Hsp25 siRNA neurons displayed significantly higher luciferase activity than control siRNA neurons (*p < 0.05; n = 3). (E) Quantification of bim mRNA levels bound to the endogenous Hsp25 protein and to the wild-type form of the human Hsp27 (pNEO-Hsp27 construct) overexpressed in CGNs. H2O2 (37.5 μM) treatment significantly increased bim mRNA levels coimmuno­precipitated with the Hsp25/Hsp27 compared to control neurons (*p < 0.05; n = 4). The coimmunoprecipitated bim mRNA levels were significantly increased in H2O2-treated cells when Hsp27 was overexpressed (*p < 0.05; n = 4). As positive and negative controls for the immuno­precipitation, a specific antibody for Argonaute-2 protein and an unspecific serum were used, respectively. Argonaute-2 levels were detected by Western blot. bim mRNA levels were not detected in the negative control.

Mentions: To determine whether Hsp27 was able to inhibit bim mRNA translation, we analyzed the amount of bim RNA present at the polysomes (the ribonucleoprotein particles where translation occurs) using the translating ribosome affinity purification (TRAP) methodology (Heiman et al., 2008; Kapeli and Yeo, 2012). Specifically, we determined by coimmunoprecipitation and subsequent qPCR analysis the levels of bim mRNA associated with the ribosomal protein RPL10a present at the polysome. In CGNs treated with H2O2 (37.5 μM), overexpression of Hsp27 significantly reduced the levels of bim mRNA associated with RPL10a and therefore present at the polysome (Figure 4A).


Hsp27 binding to the 3'UTR of bim mRNA prevents neuronal death during oxidative stress-induced injury: a novel cytoprotective mechanism.

Dávila D, Jiménez-Mateos EM, Mooney CM, Velasco G, Henshall DC, Prehn JH - Mol. Biol. Cell (2014)

Hsp27 regulates bim-3′UTR sequence during oxidative stress. (A) Quantification of bim mRNA levels bound to the ribosomal protein RPL10a present at the polysome. CGNs were treated with H2O2 (37.5 μM). Hsp27 overexpression reduced bim mRNA levels coimmunoprecipitated with RPL10a when compared with controls (*p < 0.05; n = 3). (B) CGNs were cotransfected with a luciferase reporter plasmid containing the bim-3′UTR (divided into two sequences, named 1 and 2) and either pNEO-Hsp27 or a control construct. Bim-3′UTR (sequence 1) neurons showed a significant increase in the luciferase activity after H2O2 (37.5 μM) addition (*p < 0.05; n = 3). Coexpression of pNEO-Hsp27 prevented this effect, significantly reducing the luciferase activity (*p < 0.05; n = 3). (C) Bim-3′UTR (sequence 2) neurons did not show any modification of the luciferase activity induced by H2O2 treatment or pNEO-Hsp27 coexpression. (D) CGNs were coelectroporated with the luciferase reporter plasmid containing the bim-3′UTR (sequence 1) and either Hsp25 siRNA or control siRNA. Hsp25 siRNA neurons displayed significantly higher luciferase activity than control siRNA neurons (*p < 0.05; n = 3). (E) Quantification of bim mRNA levels bound to the endogenous Hsp25 protein and to the wild-type form of the human Hsp27 (pNEO-Hsp27 construct) overexpressed in CGNs. H2O2 (37.5 μM) treatment significantly increased bim mRNA levels coimmuno­precipitated with the Hsp25/Hsp27 compared to control neurons (*p < 0.05; n = 4). The coimmunoprecipitated bim mRNA levels were significantly increased in H2O2-treated cells when Hsp27 was overexpressed (*p < 0.05; n = 4). As positive and negative controls for the immuno­precipitation, a specific antibody for Argonaute-2 protein and an unspecific serum were used, respectively. Argonaute-2 levels were detected by Western blot. bim mRNA levels were not detected in the negative control.
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Figure 4: Hsp27 regulates bim-3′UTR sequence during oxidative stress. (A) Quantification of bim mRNA levels bound to the ribosomal protein RPL10a present at the polysome. CGNs were treated with H2O2 (37.5 μM). Hsp27 overexpression reduced bim mRNA levels coimmunoprecipitated with RPL10a when compared with controls (*p < 0.05; n = 3). (B) CGNs were cotransfected with a luciferase reporter plasmid containing the bim-3′UTR (divided into two sequences, named 1 and 2) and either pNEO-Hsp27 or a control construct. Bim-3′UTR (sequence 1) neurons showed a significant increase in the luciferase activity after H2O2 (37.5 μM) addition (*p < 0.05; n = 3). Coexpression of pNEO-Hsp27 prevented this effect, significantly reducing the luciferase activity (*p < 0.05; n = 3). (C) Bim-3′UTR (sequence 2) neurons did not show any modification of the luciferase activity induced by H2O2 treatment or pNEO-Hsp27 coexpression. (D) CGNs were coelectroporated with the luciferase reporter plasmid containing the bim-3′UTR (sequence 1) and either Hsp25 siRNA or control siRNA. Hsp25 siRNA neurons displayed significantly higher luciferase activity than control siRNA neurons (*p < 0.05; n = 3). (E) Quantification of bim mRNA levels bound to the endogenous Hsp25 protein and to the wild-type form of the human Hsp27 (pNEO-Hsp27 construct) overexpressed in CGNs. H2O2 (37.5 μM) treatment significantly increased bim mRNA levels coimmuno­precipitated with the Hsp25/Hsp27 compared to control neurons (*p < 0.05; n = 4). The coimmunoprecipitated bim mRNA levels were significantly increased in H2O2-treated cells when Hsp27 was overexpressed (*p < 0.05; n = 4). As positive and negative controls for the immuno­precipitation, a specific antibody for Argonaute-2 protein and an unspecific serum were used, respectively. Argonaute-2 levels were detected by Western blot. bim mRNA levels were not detected in the negative control.
Mentions: To determine whether Hsp27 was able to inhibit bim mRNA translation, we analyzed the amount of bim RNA present at the polysomes (the ribonucleoprotein particles where translation occurs) using the translating ribosome affinity purification (TRAP) methodology (Heiman et al., 2008; Kapeli and Yeo, 2012). Specifically, we determined by coimmunoprecipitation and subsequent qPCR analysis the levels of bim mRNA associated with the ribosomal protein RPL10a present at the polysome. In CGNs treated with H2O2 (37.5 μM), overexpression of Hsp27 significantly reduced the levels of bim mRNA associated with RPL10a and therefore present at the polysome (Figure 4A).

Bottom Line: This effect could not be explained by proteasomal degradation of Bim or bim promoter inhibition; however, it was associated with a specific increase in the levels of bim mRNA and with its binding to Hsp27.Finally, we determined that enhanced Hsp27 expression in neurons exposed to H2O2 or glutamate prevented the translation of a reporter plasmid where bim-3'UTR mRNA sequence was cloned downstream of a luciferase gene.These results suggest that repression of bim mRNA translation through binding to the 3'UTR constitutes a novel cytoprotective mechanism of Hsp27 during stress in neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Medical Physics and RCSI Centre for the Study of Neurological Disorders, Royal College of Surgeons in Ireland, Dublin 2, Ireland Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain daviddav@ucm.es prehn@rcsi.ie.

Show MeSH
Related in: MedlinePlus