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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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Activity of different HuR mutants in alleviating miRISC cleavage. (A) Schemes of wt HuR and its deletion mutants. (B) Coomassie blue-stained SDS–polyacrylamide gel showing purification of different HuR mutant HuR. Lane M, protein size markers (in kDa). (C) Representative in vitro cleavage reactions of HBS_50_MBSp RNA in the presence of increasing concentrations of indicated HuR mutants. (D) Quantification of the effect of different HuR mutants on HBS_50_MBSp RNA cleavage. Values represent means ± SD; n ≥ 3.
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gks148-F2: Activity of different HuR mutants in alleviating miRISC cleavage. (A) Schemes of wt HuR and its deletion mutants. (B) Coomassie blue-stained SDS–polyacrylamide gel showing purification of different HuR mutant HuR. Lane M, protein size markers (in kDa). (C) Representative in vitro cleavage reactions of HBS_50_MBSp RNA in the presence of increasing concentrations of indicated HuR mutants. (D) Quantification of the effect of different HuR mutants on HBS_50_MBSp RNA cleavage. Values represent means ± SD; n ≥ 3.

Mentions: HuR and related proteins, such as HuB, HuD and Drosophila ELAV, are known to oligomerize along RNA substrates (35–40). Furthermore, for HuR and Drosophila ELAV, the hinge separating RRM2 and RRM3, and the RRM3 were identified as domains contributing to the formation of cooperative HuR–RNA complexes (36,40). We investigated whether HuR oligomerization might play a role in miRISC interference of RNA cleavage. To this end, we have purified three different HuR mutants: HuRΔH (devoid of a hinge region separating RRM2 and RRM3), HuRΔ3 (RRM3 deleted) and HuRΔH3 (missing both the hinge region and RRM3) (Figure 2A and B). Consistent with previous findings (36,40), a full-length HuR, and HuRΔH and HuRΔ3 mutants formed complexes with HBS_50_MBSp RNA, gradually increasing in size in a concentration-dependent manner. In contrast, the potential of HuRΔH3 to oligomerize was clearly diminished as seen in Supplementary Figure S3 (36). We then compared activity of different HuR mutants to interfere with the miRISC function. Interestingly, the two mutants able to oligomerize, HuRΔH and HuRΔ3, inhibited miRISC-mediated cleavage of HBS_50_MBSp RNA, although at a concentration higher than the full-length HuR. In contrast, the cleavage of target RNA remained unaffected in the presence of the mutant HuRΔH3, which is defective in oligomerization (Figure 2C and D).Figure 2.


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)

Activity of different HuR mutants in alleviating miRISC cleavage. (A) Schemes of wt HuR and its deletion mutants. (B) Coomassie blue-stained SDS–polyacrylamide gel showing purification of different HuR mutant HuR. Lane M, protein size markers (in kDa). (C) Representative in vitro cleavage reactions of HBS_50_MBSp RNA in the presence of increasing concentrations of indicated HuR mutants. (D) Quantification of the effect of different HuR mutants on HBS_50_MBSp RNA cleavage. Values represent means ± SD; n ≥ 3.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3367187&req=5

gks148-F2: Activity of different HuR mutants in alleviating miRISC cleavage. (A) Schemes of wt HuR and its deletion mutants. (B) Coomassie blue-stained SDS–polyacrylamide gel showing purification of different HuR mutant HuR. Lane M, protein size markers (in kDa). (C) Representative in vitro cleavage reactions of HBS_50_MBSp RNA in the presence of increasing concentrations of indicated HuR mutants. (D) Quantification of the effect of different HuR mutants on HBS_50_MBSp RNA cleavage. Values represent means ± SD; n ≥ 3.
Mentions: HuR and related proteins, such as HuB, HuD and Drosophila ELAV, are known to oligomerize along RNA substrates (35–40). Furthermore, for HuR and Drosophila ELAV, the hinge separating RRM2 and RRM3, and the RRM3 were identified as domains contributing to the formation of cooperative HuR–RNA complexes (36,40). We investigated whether HuR oligomerization might play a role in miRISC interference of RNA cleavage. To this end, we have purified three different HuR mutants: HuRΔH (devoid of a hinge region separating RRM2 and RRM3), HuRΔ3 (RRM3 deleted) and HuRΔH3 (missing both the hinge region and RRM3) (Figure 2A and B). Consistent with previous findings (36,40), a full-length HuR, and HuRΔH and HuRΔ3 mutants formed complexes with HBS_50_MBSp RNA, gradually increasing in size in a concentration-dependent manner. In contrast, the potential of HuRΔH3 to oligomerize was clearly diminished as seen in Supplementary Figure S3 (36). We then compared activity of different HuR mutants to interfere with the miRISC function. Interestingly, the two mutants able to oligomerize, HuRΔH and HuRΔ3, inhibited miRISC-mediated cleavage of HBS_50_MBSp RNA, although at a concentration higher than the full-length HuR. In contrast, the cleavage of target RNA remained unaffected in the presence of the mutant HuRΔH3, which is defective in oligomerization (Figure 2C and D).Figure 2.

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