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HuR protein attenuates miRNA-mediated repression by promoting miRISC dissociation from the target RNA.

Kundu P, Fabian MR, Sonenberg N, Bhattacharyya SN, Filipowicz W - Nucleic Acids Res. (2012)

Bottom Line: Target mRNAs with regulatory AU-rich elements (AREs) in their 3'-untranslated regions (3'-UTR) can be relieved of miRNA repression under cellular stress in a process involving the embryonic lethal and altered vision family ARE-binding protein HuR.The HuR-mediated derepression occurred even when AREs were positioned at a considerable distance from the miRNA sites raising questions about the mechanism of HuR action.Further, we demonstrate that HuR association with AREs can also inhibit miRNA-mediated deadenylation of mRNA in the Krebs-2 ascites extract, in a manner likewise depending on the potential of HuR to oligomerize.

View Article: PubMed Central - PubMed

Affiliation: Friedrich Miescher Institute for Biomedical Research, PO Box 2543, 4002 Basel, Switzerland.

ABSTRACT
The microRNA (miRNA)-mediated repression of protein synthesis in mammalian cells is a reversible process. Target mRNAs with regulatory AU-rich elements (AREs) in their 3'-untranslated regions (3'-UTR) can be relieved of miRNA repression under cellular stress in a process involving the embryonic lethal and altered vision family ARE-binding protein HuR. The HuR-mediated derepression occurred even when AREs were positioned at a considerable distance from the miRNA sites raising questions about the mechanism of HuR action. Here, we show that the relief of miRNA-mediated repression involving HuR can be recapitulated in different in vitro systems in the absence of stress, indicating that HuR alone is sufficient to relieve the miRNA repression upon binding to RNA ARE. Using in vitro assays with purified miRISC and recombinant HuR and its mutants, we show that HuR, likely by its property to oligomerize along RNA, leads to the dissociation of miRISC from target RNA even when miRISC and HuR binding sites are positioned at a distance. Further, we demonstrate that HuR association with AREs can also inhibit miRNA-mediated deadenylation of mRNA in the Krebs-2 ascites extract, in a manner likewise depending on the potential of HuR to oligomerize.

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Effect of HuR and its mutants on miRNA-mediated deadenylation of target RNAs in Krebs-2 ascites extract. (A) Effect of HuR and its mutants on deadenylation of 6xB-ARD-3′UTR (upper row) and 6xB-3′-UTR (second row) RNAs as analyzed by PAGE. RNAs were incubated in the presence or absence of 250 nM of HuR or its mutants for indicated time. Positions of pA+ and pA− RNAs are marked on the right. (Bottom panels) Control assays performed in the absence of HuR with 6xBMut-ARD-3′-UTR and 6xB-ARD-3′-UTR RNAs, the latter assay containing anti-let-7 2′-O-methyl oligonucleotide. (B) Quantification of the deadenylation reactions of 6xB-ARD-3′-UTR (left panel) and 6xB-3′-UTR (right panel) shown in A.
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gks148-F6: Effect of HuR and its mutants on miRNA-mediated deadenylation of target RNAs in Krebs-2 ascites extract. (A) Effect of HuR and its mutants on deadenylation of 6xB-ARD-3′UTR (upper row) and 6xB-3′-UTR (second row) RNAs as analyzed by PAGE. RNAs were incubated in the presence or absence of 250 nM of HuR or its mutants for indicated time. Positions of pA+ and pA− RNAs are marked on the right. (Bottom panels) Control assays performed in the absence of HuR with 6xBMut-ARD-3′-UTR and 6xB-ARD-3′-UTR RNAs, the latter assay containing anti-let-7 2′-O-methyl oligonucleotide. (B) Quantification of the deadenylation reactions of 6xB-ARD-3′-UTR (left panel) and 6xB-3′-UTR (right panel) shown in A.

Mentions: We previously described an in vitro extract derived from Krebs-2 ascites cells that faithfully recapitulates both RNA deadenylation and translational repression induced by miRNAs (52,53). We tested whether addition of recombinant HuR or its mutants interferes with the miRNA-induced deadenylation of RNA bearing let-7 miRNA sites and the HuR binding region ARD, derived from the CAT-1 3′-UTR [6xB-ARD-3′-UTR; for detailed description of ARD, see ref. (11)]. We found that addition of wt HuR or its mutants HuRΔH and HuRΔ3, which have the potential to relieve repression in other assays (see above), markedly inhibited deadenylation of 6xB-ARD-3′-UTR RNA in vitro. In contrast, addition of the HuR mutant HuRΔH3 that was inactive in the derepression had no effect (Figures 6A, upper row and 6B, left panel). In control experiments, we verified that addition of any of the four forms of HuR had no major effect on deadenylation of RNA containing no ARD (6xB-3′-UTR; Figures 6A, second row and 6B, right panel). As expected, addition of anti-let-7 2′–O-methyl oligonucleotide blocked deadenylation of 6xB-ARD-3′-UTR and RNA bearing mutated let-7 sites (6xBMut-ARD-3′-UTR) did not undergo deadenylation (Figure 6A, two bottom panels).Figure 6.


HuR protein attenuates miRNA-mediated repression by promoting miRISC dissociation from the target RNA.

Kundu P, Fabian MR, Sonenberg N, Bhattacharyya SN, Filipowicz W - Nucleic Acids Res. (2012)

Effect of HuR and its mutants on miRNA-mediated deadenylation of target RNAs in Krebs-2 ascites extract. (A) Effect of HuR and its mutants on deadenylation of 6xB-ARD-3′UTR (upper row) and 6xB-3′-UTR (second row) RNAs as analyzed by PAGE. RNAs were incubated in the presence or absence of 250 nM of HuR or its mutants for indicated time. Positions of pA+ and pA− RNAs are marked on the right. (Bottom panels) Control assays performed in the absence of HuR with 6xBMut-ARD-3′-UTR and 6xB-ARD-3′-UTR RNAs, the latter assay containing anti-let-7 2′-O-methyl oligonucleotide. (B) Quantification of the deadenylation reactions of 6xB-ARD-3′-UTR (left panel) and 6xB-3′-UTR (right panel) shown in A.
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Related In: Results  -  Collection

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gks148-F6: Effect of HuR and its mutants on miRNA-mediated deadenylation of target RNAs in Krebs-2 ascites extract. (A) Effect of HuR and its mutants on deadenylation of 6xB-ARD-3′UTR (upper row) and 6xB-3′-UTR (second row) RNAs as analyzed by PAGE. RNAs were incubated in the presence or absence of 250 nM of HuR or its mutants for indicated time. Positions of pA+ and pA− RNAs are marked on the right. (Bottom panels) Control assays performed in the absence of HuR with 6xBMut-ARD-3′-UTR and 6xB-ARD-3′-UTR RNAs, the latter assay containing anti-let-7 2′-O-methyl oligonucleotide. (B) Quantification of the deadenylation reactions of 6xB-ARD-3′-UTR (left panel) and 6xB-3′-UTR (right panel) shown in A.
Mentions: We previously described an in vitro extract derived from Krebs-2 ascites cells that faithfully recapitulates both RNA deadenylation and translational repression induced by miRNAs (52,53). We tested whether addition of recombinant HuR or its mutants interferes with the miRNA-induced deadenylation of RNA bearing let-7 miRNA sites and the HuR binding region ARD, derived from the CAT-1 3′-UTR [6xB-ARD-3′-UTR; for detailed description of ARD, see ref. (11)]. We found that addition of wt HuR or its mutants HuRΔH and HuRΔ3, which have the potential to relieve repression in other assays (see above), markedly inhibited deadenylation of 6xB-ARD-3′-UTR RNA in vitro. In contrast, addition of the HuR mutant HuRΔH3 that was inactive in the derepression had no effect (Figures 6A, upper row and 6B, left panel). In control experiments, we verified that addition of any of the four forms of HuR had no major effect on deadenylation of RNA containing no ARD (6xB-3′-UTR; Figures 6A, second row and 6B, right panel). As expected, addition of anti-let-7 2′–O-methyl oligonucleotide blocked deadenylation of 6xB-ARD-3′-UTR and RNA bearing mutated let-7 sites (6xBMut-ARD-3′-UTR) did not undergo deadenylation (Figure 6A, two bottom panels).Figure 6.

Bottom Line: Target mRNAs with regulatory AU-rich elements (AREs) in their 3'-untranslated regions (3'-UTR) can be relieved of miRNA repression under cellular stress in a process involving the embryonic lethal and altered vision family ARE-binding protein HuR.The HuR-mediated derepression occurred even when AREs were positioned at a considerable distance from the miRNA sites raising questions about the mechanism of HuR action.Further, we demonstrate that HuR association with AREs can also inhibit miRNA-mediated deadenylation of mRNA in the Krebs-2 ascites extract, in a manner likewise depending on the potential of HuR to oligomerize.

View Article: PubMed Central - PubMed

Affiliation: Friedrich Miescher Institute for Biomedical Research, PO Box 2543, 4002 Basel, Switzerland.

ABSTRACT
The microRNA (miRNA)-mediated repression of protein synthesis in mammalian cells is a reversible process. Target mRNAs with regulatory AU-rich elements (AREs) in their 3'-untranslated regions (3'-UTR) can be relieved of miRNA repression under cellular stress in a process involving the embryonic lethal and altered vision family ARE-binding protein HuR. The HuR-mediated derepression occurred even when AREs were positioned at a considerable distance from the miRNA sites raising questions about the mechanism of HuR action. Here, we show that the relief of miRNA-mediated repression involving HuR can be recapitulated in different in vitro systems in the absence of stress, indicating that HuR alone is sufficient to relieve the miRNA repression upon binding to RNA ARE. Using in vitro assays with purified miRISC and recombinant HuR and its mutants, we show that HuR, likely by its property to oligomerize along RNA, leads to the dissociation of miRISC from target RNA even when miRISC and HuR binding sites are positioned at a distance. Further, we demonstrate that HuR association with AREs can also inhibit miRNA-mediated deadenylation of mRNA in the Krebs-2 ascites extract, in a manner likewise depending on the potential of HuR to oligomerize.

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Related in: MedlinePlus