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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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HuR inhibits cleavage by the miRISC pre-bound to RNA. (A) Overview of the experimental set up for cleavage assay with miRISC pre-bound to target RNA. RNA was pre-bound to miRISC for 10 min in the presence of EDTA to ensure blocking of the cleavage (time-point A1), followed by incubation on ice for 5 min (time-point A2) after simultaneous addition of HuR and Mg2+. The cleavage reaction was incubated for 15 min at 30°C. (B) Representative in vitro cleavage reactions of HBS_20_MBSp and HBS_50_MBSp RNAs (upper panels) and ΔHBS_20_MBSp and MutHBS_20_MBSp RNAs (lower panels), as a function of increasing concentration of HuR. Reactions followed the experimental set up presented in A. The RNA remains uncleaved when incubated with miRISC in the presence of EDTA at 30°C (lane A1) or upon addition of Mg2+ when the incubation is performed on ice (lane A2). (C) Quantification of reactions similar to the one shown in B (mean ± SD; n = 3).
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gks148-F4: HuR inhibits cleavage by the miRISC pre-bound to RNA. (A) Overview of the experimental set up for cleavage assay with miRISC pre-bound to target RNA. RNA was pre-bound to miRISC for 10 min in the presence of EDTA to ensure blocking of the cleavage (time-point A1), followed by incubation on ice for 5 min (time-point A2) after simultaneous addition of HuR and Mg2+. The cleavage reaction was incubated for 15 min at 30°C. (B) Representative in vitro cleavage reactions of HBS_20_MBSp and HBS_50_MBSp RNAs (upper panels) and ΔHBS_20_MBSp and MutHBS_20_MBSp RNAs (lower panels), as a function of increasing concentration of HuR. Reactions followed the experimental set up presented in A. The RNA remains uncleaved when incubated with miRISC in the presence of EDTA at 30°C (lane A1) or upon addition of Mg2+ when the incubation is performed on ice (lane A2). (C) Quantification of reactions similar to the one shown in B (mean ± SD; n = 3).

Mentions: In the experiments described so far, miRISC and HuR were always mixed together prior to addition of target mRNA. We investigated whether the addition of HuR can inhibit miRISC or dissociate it from the target even when it has been pre-bound to the RNA. First, we tested the effect of HuR addition on the cleavage activity of miRISC pre-incubated with target RNAs. Pre-incubation of miRISC with either HBS_20_MBSp or HBS_50_MBSp was performed in the presence of EDTA to inhibit Mg2+-dependent cleavage of RNAs containing perfectly complementary let-7 sites. Following a 10-min incubation at 30°C, Mg2+ and HuR were added and the incubation continued initially at 4°C (to inhibit miRISC cleavage but to allow HuR binding) and then at 30°C (Figure 4A). We have verified that inclusion of EDTA or incubation at 4°C effectively prevents substrate RNA cleavage by miRISC (Figure 4A and B, conditions A1 and A2). Importantly, as shown in Figure 4B and C, inclusion of HuR led to concentration-dependent inhibition of target cleavage, indicating that HuR can interfere with the activity of miRISC pre-bound to RNA. The observed effect was dependent on the presence of functional HuR-binding site since addition of HuR had no effect on activity of miRISC pre-bound to RNA bearing either mutated HBS or having no HBS (Figure 4B and C).Figure 4.


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)

HuR inhibits cleavage by the miRISC pre-bound to RNA. (A) Overview of the experimental set up for cleavage assay with miRISC pre-bound to target RNA. RNA was pre-bound to miRISC for 10 min in the presence of EDTA to ensure blocking of the cleavage (time-point A1), followed by incubation on ice for 5 min (time-point A2) after simultaneous addition of HuR and Mg2+. The cleavage reaction was incubated for 15 min at 30°C. (B) Representative in vitro cleavage reactions of HBS_20_MBSp and HBS_50_MBSp RNAs (upper panels) and ΔHBS_20_MBSp and MutHBS_20_MBSp RNAs (lower panels), as a function of increasing concentration of HuR. Reactions followed the experimental set up presented in A. The RNA remains uncleaved when incubated with miRISC in the presence of EDTA at 30°C (lane A1) or upon addition of Mg2+ when the incubation is performed on ice (lane A2). (C) Quantification of reactions similar to the one shown in B (mean ± SD; n = 3).
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gks148-F4: HuR inhibits cleavage by the miRISC pre-bound to RNA. (A) Overview of the experimental set up for cleavage assay with miRISC pre-bound to target RNA. RNA was pre-bound to miRISC for 10 min in the presence of EDTA to ensure blocking of the cleavage (time-point A1), followed by incubation on ice for 5 min (time-point A2) after simultaneous addition of HuR and Mg2+. The cleavage reaction was incubated for 15 min at 30°C. (B) Representative in vitro cleavage reactions of HBS_20_MBSp and HBS_50_MBSp RNAs (upper panels) and ΔHBS_20_MBSp and MutHBS_20_MBSp RNAs (lower panels), as a function of increasing concentration of HuR. Reactions followed the experimental set up presented in A. The RNA remains uncleaved when incubated with miRISC in the presence of EDTA at 30°C (lane A1) or upon addition of Mg2+ when the incubation is performed on ice (lane A2). (C) Quantification of reactions similar to the one shown in B (mean ± SD; n = 3).
Mentions: In the experiments described so far, miRISC and HuR were always mixed together prior to addition of target mRNA. We investigated whether the addition of HuR can inhibit miRISC or dissociate it from the target even when it has been pre-bound to the RNA. First, we tested the effect of HuR addition on the cleavage activity of miRISC pre-incubated with target RNAs. Pre-incubation of miRISC with either HBS_20_MBSp or HBS_50_MBSp was performed in the presence of EDTA to inhibit Mg2+-dependent cleavage of RNAs containing perfectly complementary let-7 sites. Following a 10-min incubation at 30°C, Mg2+ and HuR were added and the incubation continued initially at 4°C (to inhibit miRISC cleavage but to allow HuR binding) and then at 30°C (Figure 4A). We have verified that inclusion of EDTA or incubation at 4°C effectively prevents substrate RNA cleavage by miRISC (Figure 4A and B, conditions A1 and A2). Importantly, as shown in Figure 4B and C, inclusion of HuR led to concentration-dependent inhibition of target cleavage, indicating that HuR can interfere with the activity of miRISC pre-bound to RNA. The observed effect was dependent on the presence of functional HuR-binding site since addition of HuR had no effect on activity of miRISC pre-bound to RNA bearing either mutated HBS or having no HBS (Figure 4B and C).Figure 4.

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.

Show MeSH
Related in: MedlinePlus