DEAD-box helicase eIF4A2 inhibits CNOT7 deadenylation activity.

Hedda A Meijer,Claudia Langlais,Ania Wilczynska,Sarah L Gillen,Cornelia H De Moor,Tobias Schmidt,Rebekah Jukes-Jones,Kelvin Cain,Martin Bushell

doi:10.1093/nar/gkz509

Hedda A Meijer, Claudia Langlais + Show 7 more

Open Access

https://doi.org/10.1093/nar/gkz509

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Abstract

The CCR4–NOT complex plays an important role in the translational repression and deadenylation of mRNAs. However, little is known about the specific roles of interacting factors. We demonstrate that the DEAD-box helicases eIF4A2 and DDX6 interact directly with the MA3 and MIF domains of CNOT1 and compete for binding. Furthermore, we now show that incorporation of eIF4A2 into the CCR4–NOT complex inhibits CNOT7 deadenylation activity in contrast to DDX6 which enhances CNOT7 activity. Polyadenylation tests (PAT) on endogenous mRNAs determined that eIF4A2 bound mRNAs have longer poly(A) tails than DDX6 bound mRNAs. Immunoprecipitation experiments show that eIF4A2 does not inhibit CNOT7 association with the CCR4–NOT complex but instead inhibits CNOT7 activity. We identified a CCR4–NOT interacting factor, TAB182, that modulates helicase recruitment into the CCR4–NOT complex, potentially affecting the outcome for the targeted mRNA. Together, these data show that the fate of an mRNA is dependent on the specific recruitment of either eIF4A2 or DDX6 to the CCR4–NOT complex which results in different pathways for translational repression and mRNA deadenylation.

Highlights

The poly(A) tail at the 3 end of mRNAs plays a critical role in the life-cycle of an mRNA
When an mRNA is targeted for deadenylation and decay, PABP can interact with the CCR4–NOT complex which is critical for the removal of the poly(A) tail [5,6]
The CCR4–NOT complex plays an important role in many aspects of eukaryotic gene expression, but it is best known for its role in the translational repression and deadenylation of mRNAs [7]

Summary

Introduction

The poly(A) tail at the 3 end of mRNAs plays a critical role in the life-cycle of an mRNA. To investigate the interaction between the CCR4–NOT complex and the DEAD-box helicases eIF4A1, eIF4A2 and DDX6 we performed endogenous CNOT1 immunoprecipitation experiments (Figure 2A). When combining the helicase knockdown with the CNOT7 binding mutant full translational activity was restored, demonstrating that eIF4A2, DDX6 and CNOT7 are required and sufficient for translational repression via the CNOT1-MIF domain (Figure 4B, Supplementary Figure S5D–F).

Results

Conclusion