Human DDX6 effects miRNA-mediated gene silencing via direct binding to CNOT1
Christopher Rouya1, Nadeem Siddiqui1, Masahiro Morita1
1Department of Biochemistry, McGill University, Montreal, Quebec, H3G 1Y6, Canada Goodman Cancer Research Centre, McGill University, Montreal, Quebec, H3A 1A3, Canada.
Abstract:
MicroRNAs (miRNAs) play critical roles in a variety of biological processes through widespread effects on protein synthesis. Upon association with the miRNA-induced silencing complex (miRISC), miRNAs repress target mRNA translation and accelerate mRNA decay. Degradation of the mRNA is initiated by shortening of the poly(A) tail by the CCR4-NOT deadenylase complex followed by the removal of the 5' cap structure and exonucleolytic decay of the mRNA. Here, we report a direct interaction between the large scaffolding subunit of CCR4-NOT, CNOT1, with the translational repressor and decapping activator protein, DDX6. DDX6 binds to a conserved CNOT1 subdomain in a manner resembling the interaction of the translation initiation factor eIF4A with eIF4G. Importantly, mutations that disrupt the DDX6-CNOT1 interaction impair miRISC-mediated gene silencing in human cells. Thus, CNOT1 facilitates recruitment of DDX6 to miRNA-targeted mRNAs, placing DDX6 as a downstream effector in the miRNA silencing pathway.
Insights
MicroRNAs (miRNAs) regulate gene expression by inhibiting protein synthesis. This study reveals CNOT1 directly interacts with DDX6, a key step in miRNA-mediated gene silencing.
Area of Science:
- Molecular Biology
- Gene Regulation
- RNA Biology
Background:
- MicroRNAs (miRNAs) are crucial regulators of gene expression, impacting protein synthesis.
- miRNAs function via the miRNA-induced silencing complex (miRISC) to repress mRNA translation and promote decay.
- mRNA degradation involves deadenylation by CCR4-NOT, followed by decapping and exonucleolytic decay.
Purpose of the Study:
- To investigate the molecular mechanisms underlying miRNA-mediated gene silencing.
- To identify direct interactions between components of the CCR4-NOT complex and other silencing factors.
- To elucidate the role of CNOT1 and DDX6 in the miRNA pathway.
Main Methods:
- Biochemical assays to detect direct protein-protein interactions.
- Analysis of conserved protein domains and interaction interfaces.
- Functional studies in human cells using mutagenesis to assess impact on gene silencing.
Main Results:
- A direct interaction between CNOT1, the scaffolding subunit of CCR4-NOT, and DDX6, a translational repressor, was identified.
- DDX6 binds to a conserved CNOT1 subdomain, mimicking the eIF4A-eIF4G interaction.
- Disruption of the DDX6-CNOT1 interaction significantly impairs miRNA-mediated gene silencing in human cells.
Conclusions:
- CNOT1 directly recruits DDX6 to miRNA-targeted mRNAs.
- DDX6 acts as a downstream effector in the miRNA silencing pathway, facilitated by CNOT1.
- This interaction is critical for efficient miRISC-mediated gene silencing.
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