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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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Oligonucleotides complementary to the spacer separating HBS and MBS sites prevent the HuR effect on miRISC cleavage in vitro. (A) Schemes of the HBS_50_MBSp target RNA hybridized to complementary 50-mer DNA and 27-mer LNA oligonucleotides. (B) Prehybridization of DNA and LNA oligonucleotides interferes with the alleviating effect of HuR on miRISC cleavage. (Upper panels) Effect of increasing concentrations of HuR on cleavage of control HBS_50_MBSp RNA or its duplexes with either DNA or LNA oligonucleotides. (Lower panel) Quantification (means ± SD; n = 3) of experiments similar to those shown in upper panels. (C) Prehybridization of 50-nt DNA oligonucleotide complementary to the spacer region of HBS_50_MBSp RNA interferes with HuR oligomerization along RNA as visualized by EMSA. Although comparison of the left (no oligonucleotide) and right (complementary oligonucleotide added) panels reveals the decrease in a number of HuR-associated bands in response to hybridizing the oligonucletide, the precise number and nature of HuR molecules associated with each EMSA band is difficult to assign. The IL-1β HBS element itself is 33-nt long and is likely to accommodate two HuR molecules; in addition, HuR may associate with RNA as either monomer or homodimer (36,56).
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gks148-F3: Oligonucleotides complementary to the spacer separating HBS and MBS sites prevent the HuR effect on miRISC cleavage in vitro. (A) Schemes of the HBS_50_MBSp target RNA hybridized to complementary 50-mer DNA and 27-mer LNA oligonucleotides. (B) Prehybridization of DNA and LNA oligonucleotides interferes with the alleviating effect of HuR on miRISC cleavage. (Upper panels) Effect of increasing concentrations of HuR on cleavage of control HBS_50_MBSp RNA or its duplexes with either DNA or LNA oligonucleotides. (Lower panel) Quantification (means ± SD; n = 3) of experiments similar to those shown in upper panels. (C) Prehybridization of 50-nt DNA oligonucleotide complementary to the spacer region of HBS_50_MBSp RNA interferes with HuR oligomerization along RNA as visualized by EMSA. Although comparison of the left (no oligonucleotide) and right (complementary oligonucleotide added) panels reveals the decrease in a number of HuR-associated bands in response to hybridizing the oligonucletide, the precise number and nature of HuR molecules associated with each EMSA band is difficult to assign. The IL-1β HBS element itself is 33-nt long and is likely to accommodate two HuR molecules; in addition, HuR may associate with RNA as either monomer or homodimer (36,56).

Mentions: To obtain additional evidence that HuR oligomerization may be important for interference with the miRISC-mediated target cleavage, we tested the effect of pre-hybridizing the oligonucleotides complementary to the spacer separating HBS and MBSp sequences in the target RNA. Hybridization of a 50-nt-long oligodeoxynucleotide complementary to the entire spacer of the HBS_50_MBSp RNA or a 27-mer oligonucleotide bearing several LNA nucleotides (Figure 3A) and forming a duplex of stability comparable to that formed with the 50-mer DNA oligonucleotide, almost entirely eliminated the inhibitory effect of HuR on RNA cleavage by miRISC (Figure 3B). Similar result was obtained with HBS_20_MBSp RNA, having the 20-nt-long spacer separating HBS and MBSp sites. Annealing of the 20-mer LNA oligonucleotide complementary to the HBS_20_MBSp spacer completely eliminated the effect of HuR (Supplementary Figure S4). We have verified that hybridization of complementary 50-mer DNA oligonucleotide to the HBS_50_MBSp RNA strongly reduced a potential of the duplex to form multimeric complexes with HuR, as analyzed by EMSA (Figure 3C). It was also noted that the IL-1β HBS element is 33-nt long and probably able to accommodate two HuR molecules, either monomers or dimers (36,56). Incubation of HuR with duplexes of HBS_50_MBSp RNA and LNA oligonucleotide resulted in formation of complexes that did not enter the non-denaturing gel, thus preventing analysis of the LNA effect on HuR oligomerization by EMSA (data not shown).Figure 3.


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)

Oligonucleotides complementary to the spacer separating HBS and MBS sites prevent the HuR effect on miRISC cleavage in vitro. (A) Schemes of the HBS_50_MBSp target RNA hybridized to complementary 50-mer DNA and 27-mer LNA oligonucleotides. (B) Prehybridization of DNA and LNA oligonucleotides interferes with the alleviating effect of HuR on miRISC cleavage. (Upper panels) Effect of increasing concentrations of HuR on cleavage of control HBS_50_MBSp RNA or its duplexes with either DNA or LNA oligonucleotides. (Lower panel) Quantification (means ± SD; n = 3) of experiments similar to those shown in upper panels. (C) Prehybridization of 50-nt DNA oligonucleotide complementary to the spacer region of HBS_50_MBSp RNA interferes with HuR oligomerization along RNA as visualized by EMSA. Although comparison of the left (no oligonucleotide) and right (complementary oligonucleotide added) panels reveals the decrease in a number of HuR-associated bands in response to hybridizing the oligonucletide, the precise number and nature of HuR molecules associated with each EMSA band is difficult to assign. The IL-1β HBS element itself is 33-nt long and is likely to accommodate two HuR molecules; in addition, HuR may associate with RNA as either monomer or homodimer (36,56).
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Related In: Results  -  Collection

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gks148-F3: Oligonucleotides complementary to the spacer separating HBS and MBS sites prevent the HuR effect on miRISC cleavage in vitro. (A) Schemes of the HBS_50_MBSp target RNA hybridized to complementary 50-mer DNA and 27-mer LNA oligonucleotides. (B) Prehybridization of DNA and LNA oligonucleotides interferes with the alleviating effect of HuR on miRISC cleavage. (Upper panels) Effect of increasing concentrations of HuR on cleavage of control HBS_50_MBSp RNA or its duplexes with either DNA or LNA oligonucleotides. (Lower panel) Quantification (means ± SD; n = 3) of experiments similar to those shown in upper panels. (C) Prehybridization of 50-nt DNA oligonucleotide complementary to the spacer region of HBS_50_MBSp RNA interferes with HuR oligomerization along RNA as visualized by EMSA. Although comparison of the left (no oligonucleotide) and right (complementary oligonucleotide added) panels reveals the decrease in a number of HuR-associated bands in response to hybridizing the oligonucletide, the precise number and nature of HuR molecules associated with each EMSA band is difficult to assign. The IL-1β HBS element itself is 33-nt long and is likely to accommodate two HuR molecules; in addition, HuR may associate with RNA as either monomer or homodimer (36,56).
Mentions: To obtain additional evidence that HuR oligomerization may be important for interference with the miRISC-mediated target cleavage, we tested the effect of pre-hybridizing the oligonucleotides complementary to the spacer separating HBS and MBSp sequences in the target RNA. Hybridization of a 50-nt-long oligodeoxynucleotide complementary to the entire spacer of the HBS_50_MBSp RNA or a 27-mer oligonucleotide bearing several LNA nucleotides (Figure 3A) and forming a duplex of stability comparable to that formed with the 50-mer DNA oligonucleotide, almost entirely eliminated the inhibitory effect of HuR on RNA cleavage by miRISC (Figure 3B). Similar result was obtained with HBS_20_MBSp RNA, having the 20-nt-long spacer separating HBS and MBSp sites. Annealing of the 20-mer LNA oligonucleotide complementary to the HBS_20_MBSp spacer completely eliminated the effect of HuR (Supplementary Figure S4). We have verified that hybridization of complementary 50-mer DNA oligonucleotide to the HBS_50_MBSp RNA strongly reduced a potential of the duplex to form multimeric complexes with HuR, as analyzed by EMSA (Figure 3C). It was also noted that the IL-1β HBS element is 33-nt long and probably able to accommodate two HuR molecules, either monomers or dimers (36,56). Incubation of HuR with duplexes of HBS_50_MBSp RNA and LNA oligonucleotide resulted in formation of complexes that did not enter the non-denaturing gel, thus preventing analysis of the LNA effect on HuR oligomerization by EMSA (data not shown).Figure 3.

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