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The Nuclear Pore-Associated TREX-2 Complex Employs Mediator to Regulate Gene Expression.

Schneider M, Hellerschmied D, Schubert T, Amlacher S, Vinayachandran V, Reja R, Pugh BF, Clausen T, Köhler A - Cell (2015)

Bottom Line: Transcriptome and phenotypic profiling confirm that TREX-2 and Med31 are functionally interdependent at specific genes.TREX-2 additionally uses its Mediator-interacting surface to regulate mRNA export suggesting a mechanism for coupling transcription initiation and early steps of mRNA processing.Our data provide mechanistic insight into how an NPC-associated adaptor complex accesses the core transcription machinery.

View Article: PubMed Central - PubMed

Affiliation: Max F. Perutz Laboratories, Medical University of Vienna, Vienna Biocenter Campus (VBC), Dr. Bohr-Gasse 9/3, 1030 Vienna, Austria.

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TREX-2 Interaction with Mediator Requires the Sac3 PCI Domain and Med31(A) TAP-tagged Med7 was purified from sac3Δ cells, which were transformed with plasmids carrying N-terminally myc-tagged wild-type or mutant alleles of SAC3. Calmodulin eluates and whole cell extracts were immunoblotted with the indicated antibodies. The faster migrating band of myc-Sac3 is a commonly observed degradation product. Med7 immunoblotting was used for normalization. Note that the mutant Sac3 proteins were expressed at similar levels as the wild-type protein.(B) Med7-TAP purifications from the indicated cells were analyzed by immunoblotting. For Cdk8 WCE levels see Figure 2C.(C) Mediator cartoon with the crystal structure of Med31/Med7N docked into the prominent protrusion on the “middle” module. Approximate position of the Pol II CTD binding site is marked and additional middle subunits are indicated. All data based on Tsai et al. (2014).(D) Med7-TAP was purified from the indicated strains, which were transformed with a myc-Sac3 plasmid and analyzed by immunoblotting.(E) Genetic interaction analysis. Single mutant strains were transformed with an empty vector and the double mutants with a SAC3 wild-type cover plasmid (URA3). Growth was followed on SDC and on SDC+5-FOA plates to shuffle out the cover plasmid.(F) Immunoblot analysis of the indicated Med7-TAP strains containing a myc-Sac3 plasmid.See also Figure S7B.
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fig3: TREX-2 Interaction with Mediator Requires the Sac3 PCI Domain and Med31(A) TAP-tagged Med7 was purified from sac3Δ cells, which were transformed with plasmids carrying N-terminally myc-tagged wild-type or mutant alleles of SAC3. Calmodulin eluates and whole cell extracts were immunoblotted with the indicated antibodies. The faster migrating band of myc-Sac3 is a commonly observed degradation product. Med7 immunoblotting was used for normalization. Note that the mutant Sac3 proteins were expressed at similar levels as the wild-type protein.(B) Med7-TAP purifications from the indicated cells were analyzed by immunoblotting. For Cdk8 WCE levels see Figure 2C.(C) Mediator cartoon with the crystal structure of Med31/Med7N docked into the prominent protrusion on the “middle” module. Approximate position of the Pol II CTD binding site is marked and additional middle subunits are indicated. All data based on Tsai et al. (2014).(D) Med7-TAP was purified from the indicated strains, which were transformed with a myc-Sac3 plasmid and analyzed by immunoblotting.(E) Genetic interaction analysis. Single mutant strains were transformed with an empty vector and the double mutants with a SAC3 wild-type cover plasmid (URA3). Growth was followed on SDC and on SDC+5-FOA plates to shuffle out the cover plasmid.(F) Immunoblot analysis of the indicated Med7-TAP strains containing a myc-Sac3 plasmid.See also Figure S7B.

Mentions: To find out whether the influence of TREX-2 on Mediator is direct, Mediator was purified from cells expressing N-terminally myc-tagged versions of Sac3 (Figure 3A). Indeed, the TREX-2 core subunit Sac3 could be readily detected in Mediator purifications. Notably, mutation of either Sac3 R256 or R288 resulted in significantly lower amounts of TREX-2 associated with Mediator. This effect was particularly pronounced when the charge of the side-chain was inverted (i.e., R256D and R288D). The sac3 R288D mutant, which displayed the weakest affinity toward Mediator partially recapitulated the deletion of the entire SAC3 gene with respect to Cdk8 disassembly, Pol II dissociation, and increased Ser5 phosphorylation levels. To investigate whether TREX-2 binds Mediator through the CKM, we tested whether deletions of kinase module subunits would impair TREX-2 binding. However, TREX-2 co-purified efficiently with Mediator even when the module was disrupted (Figure 3B). Thus, TREX-2 regulates association of the CKM with Mediator, yet, without using the CKM as a docking site.


The Nuclear Pore-Associated TREX-2 Complex Employs Mediator to Regulate Gene Expression.

Schneider M, Hellerschmied D, Schubert T, Amlacher S, Vinayachandran V, Reja R, Pugh BF, Clausen T, Köhler A - Cell (2015)

TREX-2 Interaction with Mediator Requires the Sac3 PCI Domain and Med31(A) TAP-tagged Med7 was purified from sac3Δ cells, which were transformed with plasmids carrying N-terminally myc-tagged wild-type or mutant alleles of SAC3. Calmodulin eluates and whole cell extracts were immunoblotted with the indicated antibodies. The faster migrating band of myc-Sac3 is a commonly observed degradation product. Med7 immunoblotting was used for normalization. Note that the mutant Sac3 proteins were expressed at similar levels as the wild-type protein.(B) Med7-TAP purifications from the indicated cells were analyzed by immunoblotting. For Cdk8 WCE levels see Figure 2C.(C) Mediator cartoon with the crystal structure of Med31/Med7N docked into the prominent protrusion on the “middle” module. Approximate position of the Pol II CTD binding site is marked and additional middle subunits are indicated. All data based on Tsai et al. (2014).(D) Med7-TAP was purified from the indicated strains, which were transformed with a myc-Sac3 plasmid and analyzed by immunoblotting.(E) Genetic interaction analysis. Single mutant strains were transformed with an empty vector and the double mutants with a SAC3 wild-type cover plasmid (URA3). Growth was followed on SDC and on SDC+5-FOA plates to shuffle out the cover plasmid.(F) Immunoblot analysis of the indicated Med7-TAP strains containing a myc-Sac3 plasmid.See also Figure S7B.
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fig3: TREX-2 Interaction with Mediator Requires the Sac3 PCI Domain and Med31(A) TAP-tagged Med7 was purified from sac3Δ cells, which were transformed with plasmids carrying N-terminally myc-tagged wild-type or mutant alleles of SAC3. Calmodulin eluates and whole cell extracts were immunoblotted with the indicated antibodies. The faster migrating band of myc-Sac3 is a commonly observed degradation product. Med7 immunoblotting was used for normalization. Note that the mutant Sac3 proteins were expressed at similar levels as the wild-type protein.(B) Med7-TAP purifications from the indicated cells were analyzed by immunoblotting. For Cdk8 WCE levels see Figure 2C.(C) Mediator cartoon with the crystal structure of Med31/Med7N docked into the prominent protrusion on the “middle” module. Approximate position of the Pol II CTD binding site is marked and additional middle subunits are indicated. All data based on Tsai et al. (2014).(D) Med7-TAP was purified from the indicated strains, which were transformed with a myc-Sac3 plasmid and analyzed by immunoblotting.(E) Genetic interaction analysis. Single mutant strains were transformed with an empty vector and the double mutants with a SAC3 wild-type cover plasmid (URA3). Growth was followed on SDC and on SDC+5-FOA plates to shuffle out the cover plasmid.(F) Immunoblot analysis of the indicated Med7-TAP strains containing a myc-Sac3 plasmid.See also Figure S7B.
Mentions: To find out whether the influence of TREX-2 on Mediator is direct, Mediator was purified from cells expressing N-terminally myc-tagged versions of Sac3 (Figure 3A). Indeed, the TREX-2 core subunit Sac3 could be readily detected in Mediator purifications. Notably, mutation of either Sac3 R256 or R288 resulted in significantly lower amounts of TREX-2 associated with Mediator. This effect was particularly pronounced when the charge of the side-chain was inverted (i.e., R256D and R288D). The sac3 R288D mutant, which displayed the weakest affinity toward Mediator partially recapitulated the deletion of the entire SAC3 gene with respect to Cdk8 disassembly, Pol II dissociation, and increased Ser5 phosphorylation levels. To investigate whether TREX-2 binds Mediator through the CKM, we tested whether deletions of kinase module subunits would impair TREX-2 binding. However, TREX-2 co-purified efficiently with Mediator even when the module was disrupted (Figure 3B). Thus, TREX-2 regulates association of the CKM with Mediator, yet, without using the CKM as a docking site.

Bottom Line: Transcriptome and phenotypic profiling confirm that TREX-2 and Med31 are functionally interdependent at specific genes.TREX-2 additionally uses its Mediator-interacting surface to regulate mRNA export suggesting a mechanism for coupling transcription initiation and early steps of mRNA processing.Our data provide mechanistic insight into how an NPC-associated adaptor complex accesses the core transcription machinery.

View Article: PubMed Central - PubMed

Affiliation: Max F. Perutz Laboratories, Medical University of Vienna, Vienna Biocenter Campus (VBC), Dr. Bohr-Gasse 9/3, 1030 Vienna, Austria.

Show MeSH
Related in: MedlinePlus