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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 to mRNAs triggers biphasic deadenylation and decay(a) Diagram showing BBB and BBB+boxB mRNAs. The Ago protein N-terminally fused to a peptide derived from the N protein of bacteriophage-λ (λN-Ago) binds with high affinity to four boxB elements in the 3' UTR of BBB+boxB mRNA. (b) Northern blots showing that tethering Ago2 (middle) to the otherwise stable BBB+boxB mRNA triggers highly processive deadenylation and rapid decay, which is impaired by over-expression of Caf1mut (right). (c) Northern blots showing that expression of the control λN-lacZ did not enhance deadenylation or decay of BBB+boxB mRNA (left) and that deadenylation and decay of BBB mRNA lacking boxB was not accelerated in the presence of λN-Ago2 (right). (d) Northern blot showing that tethering Ago2 (H634P) mutant that lacks endonuclease activity enhances the deadenylation and decay of BBB+boxB mRNA. (e) Western blots showing the expression levels of the ectopically expressed λN-lacZ and λN-Ago2 or its mutant derivatives. α-tubulin served as a loading control. For panels b, c and d: times correspond to hr 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 3: Tethering Ago2 to mRNAs triggers biphasic deadenylation and decay(a) Diagram showing BBB and BBB+boxB mRNAs. The Ago protein N-terminally fused to a peptide derived from the N protein of bacteriophage-λ (λN-Ago) binds with high affinity to four boxB elements in the 3' UTR of BBB+boxB mRNA. (b) Northern blots showing that tethering Ago2 (middle) to the otherwise stable BBB+boxB mRNA triggers highly processive deadenylation and rapid decay, which is impaired by over-expression of Caf1mut (right). (c) Northern blots showing that expression of the control λN-lacZ did not enhance deadenylation or decay of BBB+boxB mRNA (left) and that deadenylation and decay of BBB mRNA lacking boxB was not accelerated in the presence of λN-Ago2 (right). (d) Northern blot showing that tethering Ago2 (H634P) mutant that lacks endonuclease activity enhances the deadenylation and decay of BBB+boxB mRNA. (e) Western blots showing the expression levels of the ectopically expressed λN-lacZ and λN-Ago2 or its mutant derivatives. α-tubulin served as a loading control. For panels b, c and d: times correspond to hr 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: To elucidate the role of Ago proteins in miRMD, we employed a RNA-tethering assay to see whether Ago proteins can promote biphasic deadenylation and thus down-regulate the expression of mRNAs when directly tethered to them. This assay 37 involves co-expression of a reporter mRNA whose 3’ UTR contains four boxB elements (BBB+boxB) and an Ago protein fused to a peptide derived from the N protein of bacteriophage-λ (λN-Ago), which binds with high affinity and specificity to the boxB elements (Fig. 3a). To facilitate detection of λN-Ago proteins, a hemagglutinin (HA)-epitope tag was fused immediately after the λN peptide to create λN-HA-Ago proteins 38. Our results (Supplementary Fig. 3a) showed that tethering any one of the Ago 1– 4 proteins greatly reduced the level of BBB+boxB mRNA to about 30% of that observed when tethered to the lacZ control protein. Moreover, the reduction in BBB+box mRNA level that resulted from tethering to Ago proteins was appreciably lessened by over-expressing the Caf1 mutant that blocks deadenylation but not by the Dcp2 mutant (Supplementary Fig. 3a). When both Dcp2 and Caf1 mutants were co-expressed, the increase in the level of BBB+boxB mRNA was similar to that resulted from over-expressing the Caf1 mutant alone (Supplementary Fig. 3a). Taken together, these results suggest that all four mammalian Ago proteins can promote deadenylation and decay when tethered to mRNAs.


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 to mRNAs triggers biphasic deadenylation and decay(a) Diagram showing BBB and BBB+boxB mRNAs. The Ago protein N-terminally fused to a peptide derived from the N protein of bacteriophage-λ (λN-Ago) binds with high affinity to four boxB elements in the 3' UTR of BBB+boxB mRNA. (b) Northern blots showing that tethering Ago2 (middle) to the otherwise stable BBB+boxB mRNA triggers highly processive deadenylation and rapid decay, which is impaired by over-expression of Caf1mut (right). (c) Northern blots showing that expression of the control λN-lacZ did not enhance deadenylation or decay of BBB+boxB mRNA (left) and that deadenylation and decay of BBB mRNA lacking boxB was not accelerated in the presence of λN-Ago2 (right). (d) Northern blot showing that tethering Ago2 (H634P) mutant that lacks endonuclease activity enhances the deadenylation and decay of BBB+boxB mRNA. (e) Western blots showing the expression levels of the ectopically expressed λN-lacZ and λN-Ago2 or its mutant derivatives. α-tubulin served as a loading control. For panels b, c and d: times correspond to hr 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 3: Tethering Ago2 to mRNAs triggers biphasic deadenylation and decay(a) Diagram showing BBB and BBB+boxB mRNAs. The Ago protein N-terminally fused to a peptide derived from the N protein of bacteriophage-λ (λN-Ago) binds with high affinity to four boxB elements in the 3' UTR of BBB+boxB mRNA. (b) Northern blots showing that tethering Ago2 (middle) to the otherwise stable BBB+boxB mRNA triggers highly processive deadenylation and rapid decay, which is impaired by over-expression of Caf1mut (right). (c) Northern blots showing that expression of the control λN-lacZ did not enhance deadenylation or decay of BBB+boxB mRNA (left) and that deadenylation and decay of BBB mRNA lacking boxB was not accelerated in the presence of λN-Ago2 (right). (d) Northern blot showing that tethering Ago2 (H634P) mutant that lacks endonuclease activity enhances the deadenylation and decay of BBB+boxB mRNA. (e) Western blots showing the expression levels of the ectopically expressed λN-lacZ and λN-Ago2 or its mutant derivatives. α-tubulin served as a loading control. For panels b, c and d: times correspond to hr 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: To elucidate the role of Ago proteins in miRMD, we employed a RNA-tethering assay to see whether Ago proteins can promote biphasic deadenylation and thus down-regulate the expression of mRNAs when directly tethered to them. This assay 37 involves co-expression of a reporter mRNA whose 3’ UTR contains four boxB elements (BBB+boxB) and an Ago protein fused to a peptide derived from the N protein of bacteriophage-λ (λN-Ago), which binds with high affinity and specificity to the boxB elements (Fig. 3a). To facilitate detection of λN-Ago proteins, a hemagglutinin (HA)-epitope tag was fused immediately after the λN peptide to create λN-HA-Ago proteins 38. Our results (Supplementary Fig. 3a) showed that tethering any one of the Ago 1– 4 proteins greatly reduced the level of BBB+boxB mRNA to about 30% of that observed when tethered to the lacZ control protein. Moreover, the reduction in BBB+box mRNA level that resulted from tethering to Ago proteins was appreciably lessened by over-expressing the Caf1 mutant that blocks deadenylation but not by the Dcp2 mutant (Supplementary Fig. 3a). When both Dcp2 and Caf1 mutants were co-expressed, the increase in the level of BBB+boxB mRNA was similar to that resulted from over-expressing the Caf1 mutant alone (Supplementary Fig. 3a). Taken together, these results suggest that all four mammalian Ago proteins can promote deadenylation and decay when tethered to mRNAs.

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