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Ago-TNRC6 triggers microRNA-mediated decay by promoting two deadenylation steps.

Chen CY, Zheng D, Xia Z, Shyu AB - Nat. Struct. Mol. Biol. (2009)

Bottom Line: Combining the approaches of transcriptional pulsing, RNA tethering, overexpression of dominant-negative mutants, and siRNA-mediated gene knockdown, we show that let-7 miRNA-induced silencing complexes (miRISCs), which contain the proteins Argonaute (Ago) and TNRC6 (also known as GW182), trigger very rapid mRNA decay by inducing accelerated biphasic deadenylation mediated by Pan2-Pan3 and Ccr4-Caf1 deadenylase complexes followed by Dcp1-Dcp2 complex-directed decapping in mammalian cells.When tethered to mRNAs, all four human Ago proteins and TNRC6C are each able to recapitulate the two deadenylation steps.Two conserved human Ago2 phenylalanines (Phe470 and Phe505) are critical for recruiting TNRC6 to promote deadenylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The University of Texas Medical School, Houston, Texas, USA.

ABSTRACT
MicroRNAs (miRNAs) silence the expression of their mRNA targets mainly by promoting mRNA decay. The mechanism, kinetics and participating enzymes for miRNA-mediated decay in mammalian cells remain largely unclear. Combining the approaches of transcriptional pulsing, RNA tethering, overexpression of dominant-negative mutants, and siRNA-mediated gene knockdown, we show that let-7 miRNA-induced silencing complexes (miRISCs), which contain the proteins Argonaute (Ago) and TNRC6 (also known as GW182), trigger very rapid mRNA decay by inducing accelerated biphasic deadenylation mediated by Pan2-Pan3 and Ccr4-Caf1 deadenylase complexes followed by Dcp1-Dcp2 complex-directed decapping in mammalian cells. When tethered to mRNAs, all four human Ago proteins and TNRC6C are each able to recapitulate the two deadenylation steps. Two conserved human Ago2 phenylalanines (Phe470 and Phe505) are critical for recruiting TNRC6 to promote deadenylation. These findings indicate that promotion of biphasic deadenylation to trigger mRNA decay is an intrinsic property of miRISCs.

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A model for miRMD in mammalian cellsSee text for detailed discussion. miRNA-targeted mRNA decay is triggered by deadenylation followed by decapping. Pan2-Pan3 and Ccr4-Caf1 deadenylases form a super-complex that coordinates the two consecutive phases of deadenylation 35. For simplicity, cap-binding complex and its interaction with the 3' poly(A)-PABP complex are not depicted.
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Figure 5: A model for miRMD in mammalian cellsSee text for detailed discussion. miRNA-targeted mRNA decay is triggered by deadenylation followed by decapping. Pan2-Pan3 and Ccr4-Caf1 deadenylases form a super-complex that coordinates the two consecutive phases of deadenylation 35. For simplicity, cap-binding complex and its interaction with the 3' poly(A)-PABP complex are not depicted.

Mentions: Combining a transcriptional pulsing approach that allows monitoring of mRNA decay kinetics with approaches for compromising specific endogenous decay enzymes 33,40, we were able to trap intermediates derived from miRNA-mediated mRNA decay (miRMD). Our data show that miRISC targets mRNA for rapid decay by triggering deadenylation that exhibits biphasic kinetics and involves Pan2-Pan3 and Ccr4-Caf1 deadenylase complexes followed by decapping involving the Dcp1-Dcp2 complex (Fig. 5). Moreover, β-globin mRNA bearing let-7 recognition sites exhibits much faster deadenylation and decay kinetics than β-globin mRNA carrying a nonsense-codon or the AU-rich element (ARE) did 41,42.


Ago-TNRC6 triggers microRNA-mediated decay by promoting two deadenylation steps.

Chen CY, Zheng D, Xia Z, Shyu AB - Nat. Struct. Mol. Biol. (2009)

A model for miRMD in mammalian cellsSee text for detailed discussion. miRNA-targeted mRNA decay is triggered by deadenylation followed by decapping. Pan2-Pan3 and Ccr4-Caf1 deadenylases form a super-complex that coordinates the two consecutive phases of deadenylation 35. For simplicity, cap-binding complex and its interaction with the 3' poly(A)-PABP complex are not depicted.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2921184&req=5

Figure 5: A model for miRMD in mammalian cellsSee text for detailed discussion. miRNA-targeted mRNA decay is triggered by deadenylation followed by decapping. Pan2-Pan3 and Ccr4-Caf1 deadenylases form a super-complex that coordinates the two consecutive phases of deadenylation 35. For simplicity, cap-binding complex and its interaction with the 3' poly(A)-PABP complex are not depicted.
Mentions: Combining a transcriptional pulsing approach that allows monitoring of mRNA decay kinetics with approaches for compromising specific endogenous decay enzymes 33,40, we were able to trap intermediates derived from miRNA-mediated mRNA decay (miRMD). Our data show that miRISC targets mRNA for rapid decay by triggering deadenylation that exhibits biphasic kinetics and involves Pan2-Pan3 and Ccr4-Caf1 deadenylase complexes followed by decapping involving the Dcp1-Dcp2 complex (Fig. 5). Moreover, β-globin mRNA bearing let-7 recognition sites exhibits much faster deadenylation and decay kinetics than β-globin mRNA carrying a nonsense-codon or the AU-rich element (ARE) did 41,42.

Bottom Line: Combining the approaches of transcriptional pulsing, RNA tethering, overexpression of dominant-negative mutants, and siRNA-mediated gene knockdown, we show that let-7 miRNA-induced silencing complexes (miRISCs), which contain the proteins Argonaute (Ago) and TNRC6 (also known as GW182), trigger very rapid mRNA decay by inducing accelerated biphasic deadenylation mediated by Pan2-Pan3 and Ccr4-Caf1 deadenylase complexes followed by Dcp1-Dcp2 complex-directed decapping in mammalian cells.When tethered to mRNAs, all four human Ago proteins and TNRC6C are each able to recapitulate the two deadenylation steps.Two conserved human Ago2 phenylalanines (Phe470 and Phe505) are critical for recruiting TNRC6 to promote deadenylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The University of Texas Medical School, Houston, Texas, USA.

ABSTRACT
MicroRNAs (miRNAs) silence the expression of their mRNA targets mainly by promoting mRNA decay. The mechanism, kinetics and participating enzymes for miRNA-mediated decay in mammalian cells remain largely unclear. Combining the approaches of transcriptional pulsing, RNA tethering, overexpression of dominant-negative mutants, and siRNA-mediated gene knockdown, we show that let-7 miRNA-induced silencing complexes (miRISCs), which contain the proteins Argonaute (Ago) and TNRC6 (also known as GW182), trigger very rapid mRNA decay by inducing accelerated biphasic deadenylation mediated by Pan2-Pan3 and Ccr4-Caf1 deadenylase complexes followed by Dcp1-Dcp2 complex-directed decapping in mammalian cells. When tethered to mRNAs, all four human Ago proteins and TNRC6C are each able to recapitulate the two deadenylation steps. Two conserved human Ago2 phenylalanines (Phe470 and Phe505) are critical for recruiting TNRC6 to promote deadenylation. These findings indicate that promotion of biphasic deadenylation to trigger mRNA decay is an intrinsic property of miRISCs.

Show MeSH
Related in: MedlinePlus