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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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Related in: MedlinePlus

HuR promotes dissociation of miRISC from target RNAs bearing either perfectly complementary or bulged let-7 sites. (A) Characterization of purified miRISC complexes containing Ago1, Ago2 or indicated Ago2 mutants. (Left panel) Western blot performed with anti-HA antibody, showing purification of miRISCs containing Ago1, Ago2 or indicated Ago2 mutants. ‘I’ denotes input cell extracts and ‘P’ denotes miRISCs purified on anti-FLAG M2 Affinity beads. (Right panel) miRISC cleavage assay demonstrating that only miRISC containing wt Ago2 is catalytically active. ‘Inp’ denotes input 5′-32P-labeled RNA substrate HBS_50_MBSp. (B) Schematic overview of experiments to determine the effect of HuR on association of miRISC with target RNA. MiRISC-bound beads were incubated with target RNA for 15 min at 23°C followed by additional 15 min incubation at 23°C in the absence (control) or presence of 150 nM HuR. The RNA remaining bound to beads was subjected to RT-qPCR (for details see ‘Materials and Methods’ section). (C) Quantification, by RT-qPCR, of the effect of HuR on association of indicated different forms (Ago2, Ago1, Ago2D669A and Ago2H634A) of miRISC with HBS_50_MBSp RNA. Values for RNA remaining bound with different miRISC forms upon addition of 150 nM HuR are normalized to values measured in the absence of HuR which are set to 1 (means ± SD; n ≥ 3) (D) (Right panel) Quantification, by qRT-PCR, of the effect of HuR or HuRΔH3 mutant on association of indicated forms (Ago2, Ago1 and Ago2D669A) of miRISC with RNA targets bearing bulged let-7 site positioned either downstream (HBS_50_MBSb) or upstream (MBSb_50_HBS) of the HuR binding site. Values for RNA remaining bound with different miRISC forms upon addition of 150 nM HuR or HuRΔH3 are normalized to values measured in the absence of HuR (means ± SD; n ≥ 3). (Left panel) Schemes of target RNAs used for the assay. ‘b’ denotes the bulged site.
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gks148-F5: HuR promotes dissociation of miRISC from target RNAs bearing either perfectly complementary or bulged let-7 sites. (A) Characterization of purified miRISC complexes containing Ago1, Ago2 or indicated Ago2 mutants. (Left panel) Western blot performed with anti-HA antibody, showing purification of miRISCs containing Ago1, Ago2 or indicated Ago2 mutants. ‘I’ denotes input cell extracts and ‘P’ denotes miRISCs purified on anti-FLAG M2 Affinity beads. (Right panel) miRISC cleavage assay demonstrating that only miRISC containing wt Ago2 is catalytically active. ‘Inp’ denotes input 5′-32P-labeled RNA substrate HBS_50_MBSp. (B) Schematic overview of experiments to determine the effect of HuR on association of miRISC with target RNA. MiRISC-bound beads were incubated with target RNA for 15 min at 23°C followed by additional 15 min incubation at 23°C in the absence (control) or presence of 150 nM HuR. The RNA remaining bound to beads was subjected to RT-qPCR (for details see ‘Materials and Methods’ section). (C) Quantification, by RT-qPCR, of the effect of HuR on association of indicated different forms (Ago2, Ago1, Ago2D669A and Ago2H634A) of miRISC with HBS_50_MBSp RNA. Values for RNA remaining bound with different miRISC forms upon addition of 150 nM HuR are normalized to values measured in the absence of HuR which are set to 1 (means ± SD; n ≥ 3) (D) (Right panel) Quantification, by qRT-PCR, of the effect of HuR or HuRΔH3 mutant on association of indicated forms (Ago2, Ago1 and Ago2D669A) of miRISC with RNA targets bearing bulged let-7 site positioned either downstream (HBS_50_MBSb) or upstream (MBSb_50_HBS) of the HuR binding site. Values for RNA remaining bound with different miRISC forms upon addition of 150 nM HuR or HuRΔH3 are normalized to values measured in the absence of HuR (means ± SD; n ≥ 3). (Left panel) Schemes of target RNAs used for the assay. ‘b’ denotes the bulged site.

Mentions: Concentration of active Ago2-miRISC was determined as described in ref. (50). In brief, the cleavage of labeled target RNA present in excess was quantified in a time course, the amount of product (y-axis) was plotted against time (x-axis), and slower steady-state cleavage rate line was extrapolated back to y-axis. The y-intercept at the zero time point denoted the amount of active miRISC. Since concentration of Ago2-mutant- and also Ago1-containing purified miRISCs could not be measured by the cleavage assay, calculation of their amounts was based on western analysis and comparison with the Ago2 miRISC (Figure 5A).


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 promotes dissociation of miRISC from target RNAs bearing either perfectly complementary or bulged let-7 sites. (A) Characterization of purified miRISC complexes containing Ago1, Ago2 or indicated Ago2 mutants. (Left panel) Western blot performed with anti-HA antibody, showing purification of miRISCs containing Ago1, Ago2 or indicated Ago2 mutants. ‘I’ denotes input cell extracts and ‘P’ denotes miRISCs purified on anti-FLAG M2 Affinity beads. (Right panel) miRISC cleavage assay demonstrating that only miRISC containing wt Ago2 is catalytically active. ‘Inp’ denotes input 5′-32P-labeled RNA substrate HBS_50_MBSp. (B) Schematic overview of experiments to determine the effect of HuR on association of miRISC with target RNA. MiRISC-bound beads were incubated with target RNA for 15 min at 23°C followed by additional 15 min incubation at 23°C in the absence (control) or presence of 150 nM HuR. The RNA remaining bound to beads was subjected to RT-qPCR (for details see ‘Materials and Methods’ section). (C) Quantification, by RT-qPCR, of the effect of HuR on association of indicated different forms (Ago2, Ago1, Ago2D669A and Ago2H634A) of miRISC with HBS_50_MBSp RNA. Values for RNA remaining bound with different miRISC forms upon addition of 150 nM HuR are normalized to values measured in the absence of HuR which are set to 1 (means ± SD; n ≥ 3) (D) (Right panel) Quantification, by qRT-PCR, of the effect of HuR or HuRΔH3 mutant on association of indicated forms (Ago2, Ago1 and Ago2D669A) of miRISC with RNA targets bearing bulged let-7 site positioned either downstream (HBS_50_MBSb) or upstream (MBSb_50_HBS) of the HuR binding site. Values for RNA remaining bound with different miRISC forms upon addition of 150 nM HuR or HuRΔH3 are normalized to values measured in the absence of HuR (means ± SD; n ≥ 3). (Left panel) Schemes of target RNAs used for the assay. ‘b’ denotes the bulged site.
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Related In: Results  -  Collection

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gks148-F5: HuR promotes dissociation of miRISC from target RNAs bearing either perfectly complementary or bulged let-7 sites. (A) Characterization of purified miRISC complexes containing Ago1, Ago2 or indicated Ago2 mutants. (Left panel) Western blot performed with anti-HA antibody, showing purification of miRISCs containing Ago1, Ago2 or indicated Ago2 mutants. ‘I’ denotes input cell extracts and ‘P’ denotes miRISCs purified on anti-FLAG M2 Affinity beads. (Right panel) miRISC cleavage assay demonstrating that only miRISC containing wt Ago2 is catalytically active. ‘Inp’ denotes input 5′-32P-labeled RNA substrate HBS_50_MBSp. (B) Schematic overview of experiments to determine the effect of HuR on association of miRISC with target RNA. MiRISC-bound beads were incubated with target RNA for 15 min at 23°C followed by additional 15 min incubation at 23°C in the absence (control) or presence of 150 nM HuR. The RNA remaining bound to beads was subjected to RT-qPCR (for details see ‘Materials and Methods’ section). (C) Quantification, by RT-qPCR, of the effect of HuR on association of indicated different forms (Ago2, Ago1, Ago2D669A and Ago2H634A) of miRISC with HBS_50_MBSp RNA. Values for RNA remaining bound with different miRISC forms upon addition of 150 nM HuR are normalized to values measured in the absence of HuR which are set to 1 (means ± SD; n ≥ 3) (D) (Right panel) Quantification, by qRT-PCR, of the effect of HuR or HuRΔH3 mutant on association of indicated forms (Ago2, Ago1 and Ago2D669A) of miRISC with RNA targets bearing bulged let-7 site positioned either downstream (HBS_50_MBSb) or upstream (MBSb_50_HBS) of the HuR binding site. Values for RNA remaining bound with different miRISC forms upon addition of 150 nM HuR or HuRΔH3 are normalized to values measured in the absence of HuR (means ± SD; n ≥ 3). (Left panel) Schemes of target RNAs used for the assay. ‘b’ denotes the bulged site.
Mentions: Concentration of active Ago2-miRISC was determined as described in ref. (50). In brief, the cleavage of labeled target RNA present in excess was quantified in a time course, the amount of product (y-axis) was plotted against time (x-axis), and slower steady-state cleavage rate line was extrapolated back to y-axis. The y-intercept at the zero time point denoted the amount of active miRISC. Since concentration of Ago2-mutant- and also Ago1-containing purified miRISCs could not be measured by the cleavage assay, calculation of their amounts was based on western analysis and comparison with the Ago2 miRISC (Figure 5A).

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