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Reconstitution of Targeted Deadenylation by the Ccr4-Not Complex and the YTH Domain Protein Mmi1

View Article: PubMed Central - PubMed

ABSTRACT

Ccr4-Not is a conserved protein complex that shortens the 3′ poly(A) tails of eukaryotic mRNAs to regulate transcript stability and translation into proteins. RNA-binding proteins are thought to facilitate recruitment of Ccr4-Not to certain mRNAs, but lack of an in-vitro-reconstituted system has slowed progress in understanding the mechanistic details of this specificity. Here, we generate a fully recombinant Ccr4-Not complex that removes poly(A) tails from RNA substrates. The intact complex is more active than the exonucleases alone and has an intrinsic preference for certain RNAs. The RNA-binding protein Mmi1 is highly abundant in preparations of native Ccr4-Not. We demonstrate a high-affinity interaction between recombinant Ccr4-Not and Mmi1. Using in vitro assays, we show that Mmi1 accelerates deadenylation of target RNAs. Together, our results support a model whereby both RNA-binding proteins and the sequence context of mRNAs influence deadenylation rate to regulate gene expression.

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The DSR Sequence Is Required for Mmi1 Acceleration of Deadenylation(A–C) Fluorescence polarization experiments assaying binding of the YTH domain of Mmi1 to DSR RNAs. (A) RNA sequences with the hexanucleotide DSR sequence underlined. Green stars represent the fluorescein fluorophore. Mutated nucleotides are in bold. (B) The purified Mmi1 YTH construct specifically binds a DSR motif from the rec8 mRNA. Binding experiments in the presence of the same unlabeled RNA at the indicated concentrations are plotted in black. (C) Mutations of the central adenosines within the DSR motif reduce the affinity of the interaction. Plots in (B) and (C) show the change in fluorescence polarization signal of 10 nM fluorescently labeled RNA upon addition of purified protein. Error bars represent the SD of five independent experiments.(D) Deadenylation assays with recombinant Ccr4-Not (top panels) and Mmi1-Ccr4-Not (bottom panels) complexes show that mutation of either a single adenosine (left) or all three adenosines (right) within the DSR of the model substrate reduces the ability of Mmi1 to accelerate deadenylation by Ccr4–Not.See also Figures S3, S6E, and S7.
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fig6: The DSR Sequence Is Required for Mmi1 Acceleration of Deadenylation(A–C) Fluorescence polarization experiments assaying binding of the YTH domain of Mmi1 to DSR RNAs. (A) RNA sequences with the hexanucleotide DSR sequence underlined. Green stars represent the fluorescein fluorophore. Mutated nucleotides are in bold. (B) The purified Mmi1 YTH construct specifically binds a DSR motif from the rec8 mRNA. Binding experiments in the presence of the same unlabeled RNA at the indicated concentrations are plotted in black. (C) Mutations of the central adenosines within the DSR motif reduce the affinity of the interaction. Plots in (B) and (C) show the change in fluorescence polarization signal of 10 nM fluorescently labeled RNA upon addition of purified protein. Error bars represent the SD of five independent experiments.(D) Deadenylation assays with recombinant Ccr4-Not (top panels) and Mmi1-Ccr4-Not (bottom panels) complexes show that mutation of either a single adenosine (left) or all three adenosines (right) within the DSR of the model substrate reduces the ability of Mmi1 to accelerate deadenylation by Ccr4–Not.See also Figures S3, S6E, and S7.

Mentions: To test the sequence specificity of this activity, we first purified a construct containing the Mmi1 YTH domain (without its N-terminal low-complexity region) to characterize its RNA-binding properties (Figure S7A). We determined the dissociation constant (KD) for the Mmi1-RNA interaction by measuring the change in fluorescence polarization signal of a DSR RNA from the rec8 3′ UTR carrying a 3′ fluorescein label (rec8UUAAAC; Figure 6A). This revealed a strong interaction with a KD of 226 ± 15 nM (Figure 6B). Furthermore this interaction is specific because an unlabeled rec8UUAAAC RNA acts as a competitor to disrupt binding (Figure 6B, black curves).


Reconstitution of Targeted Deadenylation by the Ccr4-Not Complex and the YTH Domain Protein Mmi1
The DSR Sequence Is Required for Mmi1 Acceleration of Deadenylation(A–C) Fluorescence polarization experiments assaying binding of the YTH domain of Mmi1 to DSR RNAs. (A) RNA sequences with the hexanucleotide DSR sequence underlined. Green stars represent the fluorescein fluorophore. Mutated nucleotides are in bold. (B) The purified Mmi1 YTH construct specifically binds a DSR motif from the rec8 mRNA. Binding experiments in the presence of the same unlabeled RNA at the indicated concentrations are plotted in black. (C) Mutations of the central adenosines within the DSR motif reduce the affinity of the interaction. Plots in (B) and (C) show the change in fluorescence polarization signal of 10 nM fluorescently labeled RNA upon addition of purified protein. Error bars represent the SD of five independent experiments.(D) Deadenylation assays with recombinant Ccr4-Not (top panels) and Mmi1-Ccr4-Not (bottom panels) complexes show that mutation of either a single adenosine (left) or all three adenosines (right) within the DSR of the model substrate reduces the ability of Mmi1 to accelerate deadenylation by Ccr4–Not.See also Figures S3, S6E, and S7.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5120349&req=5

fig6: The DSR Sequence Is Required for Mmi1 Acceleration of Deadenylation(A–C) Fluorescence polarization experiments assaying binding of the YTH domain of Mmi1 to DSR RNAs. (A) RNA sequences with the hexanucleotide DSR sequence underlined. Green stars represent the fluorescein fluorophore. Mutated nucleotides are in bold. (B) The purified Mmi1 YTH construct specifically binds a DSR motif from the rec8 mRNA. Binding experiments in the presence of the same unlabeled RNA at the indicated concentrations are plotted in black. (C) Mutations of the central adenosines within the DSR motif reduce the affinity of the interaction. Plots in (B) and (C) show the change in fluorescence polarization signal of 10 nM fluorescently labeled RNA upon addition of purified protein. Error bars represent the SD of five independent experiments.(D) Deadenylation assays with recombinant Ccr4-Not (top panels) and Mmi1-Ccr4-Not (bottom panels) complexes show that mutation of either a single adenosine (left) or all three adenosines (right) within the DSR of the model substrate reduces the ability of Mmi1 to accelerate deadenylation by Ccr4–Not.See also Figures S3, S6E, and S7.
Mentions: To test the sequence specificity of this activity, we first purified a construct containing the Mmi1 YTH domain (without its N-terminal low-complexity region) to characterize its RNA-binding properties (Figure S7A). We determined the dissociation constant (KD) for the Mmi1-RNA interaction by measuring the change in fluorescence polarization signal of a DSR RNA from the rec8 3′ UTR carrying a 3′ fluorescein label (rec8UUAAAC; Figure 6A). This revealed a strong interaction with a KD of 226 ± 15 nM (Figure 6B). Furthermore this interaction is specific because an unlabeled rec8UUAAAC RNA acts as a competitor to disrupt binding (Figure 6B, black curves).

View Article: PubMed Central - PubMed

ABSTRACT

Ccr4-Not is a conserved protein complex that shortens the 3′ poly(A) tails of eukaryotic mRNAs to regulate transcript stability and translation into proteins. RNA-binding proteins are thought to facilitate recruitment of Ccr4-Not to certain mRNAs, but lack of an in-vitro-reconstituted system has slowed progress in understanding the mechanistic details of this specificity. Here, we generate a fully recombinant Ccr4-Not complex that removes poly(A) tails from RNA substrates. The intact complex is more active than the exonucleases alone and has an intrinsic preference for certain RNAs. The RNA-binding protein Mmi1 is highly abundant in preparations of native Ccr4-Not. We demonstrate a high-affinity interaction between recombinant Ccr4-Not and Mmi1. Using in vitro assays, we show that Mmi1 accelerates deadenylation of target RNAs. Together, our results support a model whereby both RNA-binding proteins and the sequence context of mRNAs influence deadenylation rate to regulate gene expression.

No MeSH data available.


Related in: MedlinePlus