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Published on: December 28, 2016
A eukaryotic translation initiation factor 4E-binding protein promotes mRNA decapping and is required for PUF
Nathan H Blewett1, Aaron C Goldstrohm
1Cellular and Molecular Biology Training Program, University of Michigan Medical School, Ann Arbor, Michigan, USA.
Abstract:
PUF proteins are eukaryotic RNA-binding proteins that repress specific mRNAs. The mechanisms and corepressors involved in PUF repression remain to be fully identified. Here, we investigated the mode of repression by Saccharomyces cerevisiae Puf5p and Puf4p and found that Puf5p specifically requires Eap1p to repress mRNAs, whereas Puf4p does not. Surprisingly, we observed that Eap1p, which is a member of the eukaryotic translation initiation factor 4E (eIF4E)-binding protein (4E-BP) class of translational inhibitors, does not inhibit the efficient polyribosome association of a Puf5p target mRNA. Rather, we found that Eap1p accelerates mRNA degradation by promoting decapping, and the ability of Eap1p to interact with eIF4E facilitates this activity. Deletion of EAP1 dramatically reduces decapping, resulting in accumulation of deadenylated, capped mRNA. In support of this phenotype, Eap1p associates both with Puf5p and the Dhh1p decapping factor. Furthermore, recruitment of Eap1p to downregulated mRNA is mediated by Puf5p. On the basis of these results, we propose that Puf5p promotes decapping by recruiting Eap1p and associated decapping factors to mRNAs. The implication of these findings is that a 4E-BP can repress protein expression by promoting specific mRNA degradation steps in addition to or in lieu of inhibiting translation initiation.
Insights
Saccharomyces cerevisiae Puf5p uses Eap1p to degrade specific mRNAs by promoting decapping, not translation inhibition. This reveals a new mRNA decay mechanism for eukaryotic RNA-binding proteins.
Area of Science:
- Molecular Biology
- Gene Regulation
- RNA Metabolism
Background:
- PUF proteins are eukaryotic RNA-binding proteins that regulate gene expression by repressing specific messenger RNAs (mRNAs).
- The precise mechanisms and corepressors utilized by PUF proteins for mRNA repression are not fully understood.
- Eukaryotic translation initiation factor 4E (eIF4E)-binding proteins (4E-BPs) are known translational inhibitors.
Purpose of the Study:
- To investigate the repression mechanisms of Saccharomyces cerevisiae Puf5p and Puf4p.
- To identify corepressors involved in Puf5p-mediated mRNA repression.
- To elucidate the role of Eap1p, a 4E-BP, in Puf5p-mediated gene silencing.
Main Methods:
- Investigated Puf5p and Puf4p repression mechanisms in Saccharomyces cerevisiae.
- Assessed the requirement of Eap1p for Puf5p-mediated mRNA repression.
- Analyzed the effect of Eap1p on polyribosome association of target mRNAs.
- Studied mRNA degradation kinetics, specifically decapping and deadenylation.
- Examined protein-protein interactions between Eap1p, Puf5p, and Dhh1p.
Main Results:
- Puf5p requires Eap1p for mRNA repression, while Puf4p does not.
- Eap1p promotes mRNA degradation by accelerating decapping, rather than inhibiting translation initiation.
- Eap1p's interaction with eIF4E is crucial for its decapping-promoting activity.
- Deletion of EAP1 significantly reduces mRNA decapping, leading to mRNA accumulation.
- Eap1p interacts with both Puf5p and the decapping factor Dhh1p.
- Puf5p mediates the recruitment of Eap1p to target mRNAs.
Conclusions:
- Puf5p represses mRNAs by recruiting Eap1p and associated decapping factors, thereby promoting mRNA degradation.
- This study reveals a novel mechanism where a 4E-BP promotes specific mRNA decay pathways.
- Eap1p functions in mRNA degradation by facilitating decapping, expanding the known roles of 4E-BPs beyond translational inhibition.
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