Reversal of microRNA mediated translational repression in human cells

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Reversal of microRNA mediated translational repression in human cells
HFSP Long-Term Fellow Suvendra Bhattacharyya and colleagues
MicroRNAs, 21-22 nucleotide small RNAs, are negative regulators of gene expression in metazoa and plants. In animals, miRNA repress translation of target genes by imperfect base-pairing with the mRNA 3';UTR. Components of miRNA machinery are concentrated in dynamic cellular structures named P-bodies (PBs) originally discovered in yeast as the sites of mRNA degradation. Reporter mRNAs repressed by miRNAs also localize to these structures in human cells (Liu et al, 2005; Pillai et al, 2005). The fate of miRNA-repressed mRNAs localized to PBs is not clear. Likewise, it is not known whether miRNA-mediated repression is a reversible process and whether mRNAs localized in mammalian PBs can return to active translation. Different forms of cellular stress lead to the increased expression of genes required for cellular survival under stress. In mammalian cells, expression of a large fraction of these stress-induced genes is regulated at post-transcription level.
We have now demonstrated that a liver specific miRNA, miR-122, represses expression of a stress gene encoding cationic amino acid transporter-1 (CAT-1) in a human hepatocarcinoma cell line Huh7. When Huh7 cells are grown in a medium rich in amino acids the expression of CAT-1 is repressed but amino acid starvation and other types of stress induce expression of CAT-1 protein by relieving the CAT-1 mRNA from translational repression by miR-122. The CAT-1 mRNA localizes to PBs in a miR-122-dependent manner in non stressed Huh7 cells and the stress induced derepression is accompanied its re-localization from PBs (Fig.1) and recruitment to polysomes. Chimeric reporter mRNAs containing CAT-1 3';UTR showed similar behaviour in Huh7 cells. HuR, a member of ELAV family of RNA binding proteins, was found to be essential for both derepression of translation and re-localization of CAT-1 mRNA from PBs in stressed cells. This indicates that mRNA translation and stability may be regulated by miRNPs and ELAV proteins in a reciprocal way in mammalian cells and be modulated by different physiological conditions.
This works demonstrates that mammalian PBs could serve as storage sites for miRNA repressed mRNAs and repression of translation by miRNAs in human cells is a reversible process. Binding of HuR to target mRNA is responsible for relocalization of repressed mRNAs from PBs in response to stress. The data favor a model where multiple elements in the mRNA 3’UTR determine the fate of the mRNA by binding different trans-acting factors which function either in cooperative or antagonistic way (Fig.1).


Figure legend Stress-induced derepression of CAT-1 mRNA translation in Huh7 cells. (A) Stress-induced relocalization of CAT-1 mRNA from PBs. In situ analysis for CAT-1 mRNA in Huh7 cells, incubated for 2 h in a medium contains (Fed) or devoid (starved) of amino acids. CAT-1 RNA signals are in red and PBs are visualized by expressing GFP-tagged Dcp1a protein, a marker of PBs. One PB with localized CAT-1 mRNAs is shown by arrow head in a non stressed (fed) cell. (B) A model of HuR-mediated relief of mRNA from miRNP-dependent repression in mammalian cells subjected to stress.

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Commentary in Cell
Reference:
Relief of microRNA-mediated translational repression in human cells subjected to stress. Bhattacharyya SN., Habermacher R., Martine U., Closs E.I., Filipowicz W. (2006), Cell, 125:1111-1124.
Additional References:
1. MicroRNA dependent localization of targeted mRNAs to mammalian P-bodies. Liu, J., Valencia-Sanchez, MA, Hannon, GJ, and Parker, R. (2005), Nat. Cell Biol., 7( ): 719-723.
2. Inhibition of translational initiation by Let-7 MicroRNA in human cells. Pillai, RS., Bhattacharyya, SN., Artus, CG., Zoller, T., Cougot, N., Basyuk, E., Bertrand, E., and Filipowicz, W. (2005), Science, 309( ):1573-1576.