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Updated: Aug 14, 2026
Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae
Published on: June 3, 2017
Med9 Is a Novel eIF4E-Interacting Protein in Saccharomyces cerevisiae
Dora E Vélez1, Zorica Ristic2, Daniela Ross-Kaschitza2
1mRNA and Cancer Laboratory, Subdirección de Investigación Básica, National Institute of Cancer (Instituto Nacional de Cancerología, INCan), Mexico City 14080, Mexico.
Abstract:
The cap-binding protein eukaryotic initiation factor (eIF) 4E is a key protein for mRNA metabolism. The biological role of eIF4E is defined by the specific protein it interacts with. Thus, eIF4E binds to a constellation of partners across eukaryotes. The best-characterized role of eIF4E is to promote mRNA translation through interaction with the translation factor eIF4G. To seek new interactors in the ascomycete Saccharomyces cerevisiae, we performed a genomic yeast two-hybrid screen using eIF4E as bait. In addition to the already reported p20 and Eap1, we identified Med9, a component of the RNA polymerase II Mediator complex. A physical interaction between eIF4E and Med9 was confirmed using recombinant proteins prepared in E. coli and further isolating the eIF4E-Med9 complex both by size-exclusion chromatography and by m7GTP-Sepharose pull-down experiments. Moreover, the in vivo interaction was also detected. Surprisingly, the eIF4E W75A mutation, which impairs binding to eIF4G, p20, and Eap1, only slightly affected the interaction with Med9 in the two-hybrid system. We further performed random mutagenesis to identify the Med9 amino acids involved in eIF4E recognition. Mutants F65A/I66A and F65A/I66A/H68N did not interact with eIF4E. Our data suggest that the eIF4E-Med9 complex may be formed in both the cytoplasm and the nucleus.
Insights
Researchers discovered a new interaction between eukaryotic initiation factor 4E (eIF4E) and Med9, a component of the RNA polymerase II Mediator complex. This novel eIF4E-Med9 interaction occurs in both the cytoplasm and nucleus.
Area of Science:
- Molecular Biology
- Biochemistry
- Yeast Genetics
Background:
- The cap-binding protein eukaryotic initiation factor 4E (eIF4E) is crucial for mRNA metabolism and translation.
- eIF4E's biological functions are determined by its interaction partners.
- Its best-characterized role involves promoting mRNA translation via interaction with eIF4G.
Purpose of the Study:
- To identify novel interaction partners of eIF4E in the yeast *Saccharomyces cerevisiae*.
- To characterize the interaction between eIF4E and newly identified partners.
Main Methods:
- Genomic yeast two-hybrid screen using eIF4E as bait.
- Confirmation of physical interaction using recombinant proteins and co-immunoprecipitation.
- Site-directed mutagenesis to identify key amino acids in the interaction.
Main Results:
- Med9, a component of the RNA polymerase II Mediator complex, was identified as a novel eIF4E interactor.
- The eIF4E-Med9 interaction was confirmed both in vitro and in vivo.
- A W75A mutation in eIF4E, which disrupts interactions with known partners, had minimal effect on Med9 binding.
- Mutagenesis of Med9 identified specific amino acids (F65, I66, H68) crucial for eIF4E recognition.
Conclusions:
- eIF4E interacts with Med9, a component of the RNA polymerase II Mediator complex.
- This interaction is distinct from other known eIF4E interactions and may occur in both cellular compartments.
- The findings expand the known interactome of eIF4E and suggest a role in nuclear processes.
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