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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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Recombinant Ccr4-Not Specifically Removes Poly(A) Tails In Vitro(A) Deadenylation of an unstructured RNA substrate with 30 3′ adenosines (20-mer-A30) by recombinant S. pombe Ccr4-Not complex. The reaction was analyzed by denaturing PAGE. The sizes of the RNA substrate with and without the poly(A) tail are shown. The sequence of the 20-mer model RNA is shown above, with a green star representing the fluorescein fluorophore. See also Figures S2B–S2E and S3A.(B) Deadenylation of the 20-mer-A30 substrate with recombinant Caf1-Ccr4 nuclease module. See also Figure S4.(C and D) Deadenylation assays with recombinant Ccr4-Not complexes containing point mutations in the active sites of (C) Caf1 (D53A), Ccr4 (E387A), or (D) both.See also Figures S2–S4.
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fig3: Recombinant Ccr4-Not Specifically Removes Poly(A) Tails In Vitro(A) Deadenylation of an unstructured RNA substrate with 30 3′ adenosines (20-mer-A30) by recombinant S. pombe Ccr4-Not complex. The reaction was analyzed by denaturing PAGE. The sizes of the RNA substrate with and without the poly(A) tail are shown. The sequence of the 20-mer model RNA is shown above, with a green star representing the fluorescein fluorophore. See also Figures S2B–S2E and S3A.(B) Deadenylation of the 20-mer-A30 substrate with recombinant Caf1-Ccr4 nuclease module. See also Figure S4.(C and D) Deadenylation assays with recombinant Ccr4-Not complexes containing point mutations in the active sites of (C) Caf1 (D53A), Ccr4 (E387A), or (D) both.See also Figures S2–S4.

Mentions: To measure the deadenylation activity of recombinant Ccr4-Not, we tested its ability to remove a 3′ poly(A) tail from a model RNA. We used a synthetic 5′ fluorescently labeled substrate comprising a 20-mer RNA followed by 30 adenosines (20-mer-A30; Figure 3A). A similar RNA substrate has been used previously to study the in vitro activity of Caf1 and is predicted to be unstructured (Figure S3A) (Jonstrup et al., 2007). We assayed changes in the length of the poly(A) tail with denaturing PAGE.


Reconstitution of Targeted Deadenylation by the Ccr4-Not Complex and the YTH Domain Protein Mmi1
Recombinant Ccr4-Not Specifically Removes Poly(A) Tails In Vitro(A) Deadenylation of an unstructured RNA substrate with 30 3′ adenosines (20-mer-A30) by recombinant S. pombe Ccr4-Not complex. The reaction was analyzed by denaturing PAGE. The sizes of the RNA substrate with and without the poly(A) tail are shown. The sequence of the 20-mer model RNA is shown above, with a green star representing the fluorescein fluorophore. See also Figures S2B–S2E and S3A.(B) Deadenylation of the 20-mer-A30 substrate with recombinant Caf1-Ccr4 nuclease module. See also Figure S4.(C and D) Deadenylation assays with recombinant Ccr4-Not complexes containing point mutations in the active sites of (C) Caf1 (D53A), Ccr4 (E387A), or (D) both.See also Figures S2–S4.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120349&req=5

fig3: Recombinant Ccr4-Not Specifically Removes Poly(A) Tails In Vitro(A) Deadenylation of an unstructured RNA substrate with 30 3′ adenosines (20-mer-A30) by recombinant S. pombe Ccr4-Not complex. The reaction was analyzed by denaturing PAGE. The sizes of the RNA substrate with and without the poly(A) tail are shown. The sequence of the 20-mer model RNA is shown above, with a green star representing the fluorescein fluorophore. See also Figures S2B–S2E and S3A.(B) Deadenylation of the 20-mer-A30 substrate with recombinant Caf1-Ccr4 nuclease module. See also Figure S4.(C and D) Deadenylation assays with recombinant Ccr4-Not complexes containing point mutations in the active sites of (C) Caf1 (D53A), Ccr4 (E387A), or (D) both.See also Figures S2–S4.
Mentions: To measure the deadenylation activity of recombinant Ccr4-Not, we tested its ability to remove a 3′ poly(A) tail from a model RNA. We used a synthetic 5′ fluorescently labeled substrate comprising a 20-mer RNA followed by 30 adenosines (20-mer-A30; Figure 3A). A similar RNA substrate has been used previously to study the in vitro activity of Caf1 and is predicted to be unstructured (Figure S3A) (Jonstrup et al., 2007). We assayed changes in the length of the poly(A) tail with denaturing PAGE.

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