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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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Tethering Ago2 or TNRC6C to mRNAs is sufficient to recapitulate miRMD(a) Northern blots showing the effects of over-expressing λN-Ago2 (left) or λN-Ago2 (F2V2) (right) on deadenylation and decay of BBB+boxB. (b) Northern blots showing the effect of over-expressing λN-Ago2 (F2W2) (left) on deadenylation and decay of BBB+boxB and the effect of ectopically expressed Caf1 mutant (mut) on the decay of BBB+boxB tethered by λN-Ago2 (F2W2) (right). (c) Western blots showing the expression levels of the ectopically expressed proteins as indicated. α-tubulin served as a loading control. (d) Co-immunoprecipitation and Western blotting experiments showing that ectopically expressed Flag-TNRC6C and Flag-TNRC6B cannot effectively pull down ectopically expressed HA-Ago2(F2V2) or endogenous Caf1 deadenylase. Rabbit anti-Caf1 antibody was used at 1:4000 dilution. (e) Northern blots showing that tethering TNRC6C (upper right) to the otherwise stable BBB+boxB mRNA triggers highly processive deadenylation and rapid decay, which is impaired by over-expression of Caf1mut (lower left). Western blots (lower right) showing the expression levels of the ectopically expressed λN-TNRC6C and Caf1 mutant. α-tubulin served as a loading control. NIH3T3 B2A2 cells were transiently co-transfected with a Tet-promoter regulated plasmid encoding BBB+boxB and the plasmids encoding the proteins as indicated. Times correspond to hours after Tetracycline addition. The control mRNA (ctrl) and the preparation of Poly(A)− RNA (A−) were as described in the legend to Figure 1.
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Figure 4: Tethering Ago2 or TNRC6C to mRNAs is sufficient to recapitulate miRMD(a) Northern blots showing the effects of over-expressing λN-Ago2 (left) or λN-Ago2 (F2V2) (right) on deadenylation and decay of BBB+boxB. (b) Northern blots showing the effect of over-expressing λN-Ago2 (F2W2) (left) on deadenylation and decay of BBB+boxB and the effect of ectopically expressed Caf1 mutant (mut) on the decay of BBB+boxB tethered by λN-Ago2 (F2W2) (right). (c) Western blots showing the expression levels of the ectopically expressed proteins as indicated. α-tubulin served as a loading control. (d) Co-immunoprecipitation and Western blotting experiments showing that ectopically expressed Flag-TNRC6C and Flag-TNRC6B cannot effectively pull down ectopically expressed HA-Ago2(F2V2) or endogenous Caf1 deadenylase. Rabbit anti-Caf1 antibody was used at 1:4000 dilution. (e) Northern blots showing that tethering TNRC6C (upper right) to the otherwise stable BBB+boxB mRNA triggers highly processive deadenylation and rapid decay, which is impaired by over-expression of Caf1mut (lower left). Western blots (lower right) showing the expression levels of the ectopically expressed λN-TNRC6C and Caf1 mutant. α-tubulin served as a loading control. NIH3T3 B2A2 cells were transiently co-transfected with a Tet-promoter regulated plasmid encoding BBB+boxB and the plasmids encoding the proteins as indicated. Times correspond to hours after Tetracycline addition. The control mRNA (ctrl) and the preparation of Poly(A)− RNA (A−) were as described in the legend to Figure 1.

Mentions: Previously, it was reported that the human Ago2 protein lost its ability to repress translation when two conserved phenylalanines (F470 and F505) in its MID-domain were mutated to valines (F2V2 mutation) 32. However, it remains controversial as to why the F2V2 mutation renders Ago2 non-functional 18,32. Using the RNA-tethering assay, we found that the F2V2 mutation effectively abrogates the ability of Ago2 to promote rapid deadenylation and decay of BBB+boxB mRNA (Fig. 4a). On the other hand, a conservative mutation F2W2, which changed the two phenylalanines to tryptophans and was reported to not affect the translation repression activity of Ago2 32, did not impede the ability of Ago2 to promote rapid mRNA deadenylation and decay (Fig. 4b, left). Like let-7 miRMD (Fig. 1c) and the mRNA tethered by wild-type Ago2 (Fig. 3b), the rapid deadenylation and decay of BBB+boxB tethered by Ago2(F2W2) was nearly completely impaired by over-expression of the Caf1 mutant alone (Fig. 4b, right). These observations further indicate the importance of deadenylation in Ago2-directed mRNA decay and suggest that F470 and F505 in the MID-domain of Ago2 play a critical role in miRMD.


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

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

Tethering Ago2 or TNRC6C to mRNAs is sufficient to recapitulate miRMD(a) Northern blots showing the effects of over-expressing λN-Ago2 (left) or λN-Ago2 (F2V2) (right) on deadenylation and decay of BBB+boxB. (b) Northern blots showing the effect of over-expressing λN-Ago2 (F2W2) (left) on deadenylation and decay of BBB+boxB and the effect of ectopically expressed Caf1 mutant (mut) on the decay of BBB+boxB tethered by λN-Ago2 (F2W2) (right). (c) Western blots showing the expression levels of the ectopically expressed proteins as indicated. α-tubulin served as a loading control. (d) Co-immunoprecipitation and Western blotting experiments showing that ectopically expressed Flag-TNRC6C and Flag-TNRC6B cannot effectively pull down ectopically expressed HA-Ago2(F2V2) or endogenous Caf1 deadenylase. Rabbit anti-Caf1 antibody was used at 1:4000 dilution. (e) Northern blots showing that tethering TNRC6C (upper right) to the otherwise stable BBB+boxB mRNA triggers highly processive deadenylation and rapid decay, which is impaired by over-expression of Caf1mut (lower left). Western blots (lower right) showing the expression levels of the ectopically expressed λN-TNRC6C and Caf1 mutant. α-tubulin served as a loading control. NIH3T3 B2A2 cells were transiently co-transfected with a Tet-promoter regulated plasmid encoding BBB+boxB and the plasmids encoding the proteins as indicated. Times correspond to hours after Tetracycline addition. The control mRNA (ctrl) and the preparation of Poly(A)− RNA (A−) were as described in the legend to Figure 1.
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Related In: Results  -  Collection

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Figure 4: Tethering Ago2 or TNRC6C to mRNAs is sufficient to recapitulate miRMD(a) Northern blots showing the effects of over-expressing λN-Ago2 (left) or λN-Ago2 (F2V2) (right) on deadenylation and decay of BBB+boxB. (b) Northern blots showing the effect of over-expressing λN-Ago2 (F2W2) (left) on deadenylation and decay of BBB+boxB and the effect of ectopically expressed Caf1 mutant (mut) on the decay of BBB+boxB tethered by λN-Ago2 (F2W2) (right). (c) Western blots showing the expression levels of the ectopically expressed proteins as indicated. α-tubulin served as a loading control. (d) Co-immunoprecipitation and Western blotting experiments showing that ectopically expressed Flag-TNRC6C and Flag-TNRC6B cannot effectively pull down ectopically expressed HA-Ago2(F2V2) or endogenous Caf1 deadenylase. Rabbit anti-Caf1 antibody was used at 1:4000 dilution. (e) Northern blots showing that tethering TNRC6C (upper right) to the otherwise stable BBB+boxB mRNA triggers highly processive deadenylation and rapid decay, which is impaired by over-expression of Caf1mut (lower left). Western blots (lower right) showing the expression levels of the ectopically expressed λN-TNRC6C and Caf1 mutant. α-tubulin served as a loading control. NIH3T3 B2A2 cells were transiently co-transfected with a Tet-promoter regulated plasmid encoding BBB+boxB and the plasmids encoding the proteins as indicated. Times correspond to hours after Tetracycline addition. The control mRNA (ctrl) and the preparation of Poly(A)− RNA (A−) were as described in the legend to Figure 1.
Mentions: Previously, it was reported that the human Ago2 protein lost its ability to repress translation when two conserved phenylalanines (F470 and F505) in its MID-domain were mutated to valines (F2V2 mutation) 32. However, it remains controversial as to why the F2V2 mutation renders Ago2 non-functional 18,32. Using the RNA-tethering assay, we found that the F2V2 mutation effectively abrogates the ability of Ago2 to promote rapid deadenylation and decay of BBB+boxB mRNA (Fig. 4a). On the other hand, a conservative mutation F2W2, which changed the two phenylalanines to tryptophans and was reported to not affect the translation repression activity of Ago2 32, did not impede the ability of Ago2 to promote rapid mRNA deadenylation and decay (Fig. 4b, left). Like let-7 miRMD (Fig. 1c) and the mRNA tethered by wild-type Ago2 (Fig. 3b), the rapid deadenylation and decay of BBB+boxB tethered by Ago2(F2W2) was nearly completely impaired by over-expression of the Caf1 mutant alone (Fig. 4b, right). These observations further indicate the importance of deadenylation in Ago2-directed mRNA decay and suggest that F470 and F505 in the MID-domain of Ago2 play a critical role in miRMD.

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