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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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Structural, Biochemical, and Genetic Characterization of TREX-2, Mediator, and Pol II, Related to Figures 1 and 2(A) Top view of the yeast Sac3(222-572)/Thp1(170-455)/Sem1 complex in ribbon representation, showing the electrostatic surface potential and the surface conservation of the trimeric complex (left to right). The winged-helix domains of Sac3 and Thp1 are encircled (black line). Positively charged Sac3 residues Lys467 and Lys468 that have been analyzed in mutational studies are indicated.(B) Structural alignment of Sac3 from this study (gray) and Sac3 (PDB: 3t5v) (orange). The interaction of Sac3 with its crystallographic neighbor (∗) is shown with α1 and α2 helices forming the crystallographic interface in both crystal forms.(C) The yeast Mediator complex was affinity-purified via TAP-tagged Med15 from wild-type and sac3Δ cells. Calmodulin eluates were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. Loss of Cdk8 occurs also when Med15 is used as a TAP-tagged bait.(D) Yeast RNA Polymerase II was affinity-purified via TAP-tagged Rpb3 from wild-type and sac3Δ cells. Calmodulin eluates were analyzed by SDS-PAGE and Coomassie staining (upper panel) and immunoblotting (lower panel) using the indicated antibodies. Subunits were assigned according to their calculated molecular weight. Tfg1 and Tfg2 are subunits of TFIIF. Asterisk indicates degradation product of Rpb1, which appeared to be more susceptible to proteolysis when hyperphosphorylated on Ser5.(E) Med7-TAP purifications of Mediator from wild-type and mutant cells. Calmodulin eluates were analyzed by SDS-PAGE and Coomassie staining. Open circles indicate Med13 (top) and Med12 (bottom).(F) Genetic interaction analysis shows negative synthetic links between MED31 and SAC3 R288D. The indicated genotypes were produced by transformation with the respective HIS plasmids into cells which also contain a SAC3 cover plasmid (URA3). Growth was followed on SDC-His and on SDC+5-fluoroorotic acid (5-FOA) plates to counterselect against the cover plasmid.
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figs2: Structural, Biochemical, and Genetic Characterization of TREX-2, Mediator, and Pol II, Related to Figures 1 and 2(A) Top view of the yeast Sac3(222-572)/Thp1(170-455)/Sem1 complex in ribbon representation, showing the electrostatic surface potential and the surface conservation of the trimeric complex (left to right). The winged-helix domains of Sac3 and Thp1 are encircled (black line). Positively charged Sac3 residues Lys467 and Lys468 that have been analyzed in mutational studies are indicated.(B) Structural alignment of Sac3 from this study (gray) and Sac3 (PDB: 3t5v) (orange). The interaction of Sac3 with its crystallographic neighbor (∗) is shown with α1 and α2 helices forming the crystallographic interface in both crystal forms.(C) The yeast Mediator complex was affinity-purified via TAP-tagged Med15 from wild-type and sac3Δ cells. Calmodulin eluates were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. Loss of Cdk8 occurs also when Med15 is used as a TAP-tagged bait.(D) Yeast RNA Polymerase II was affinity-purified via TAP-tagged Rpb3 from wild-type and sac3Δ cells. Calmodulin eluates were analyzed by SDS-PAGE and Coomassie staining (upper panel) and immunoblotting (lower panel) using the indicated antibodies. Subunits were assigned according to their calculated molecular weight. Tfg1 and Tfg2 are subunits of TFIIF. Asterisk indicates degradation product of Rpb1, which appeared to be more susceptible to proteolysis when hyperphosphorylated on Ser5.(E) Med7-TAP purifications of Mediator from wild-type and mutant cells. Calmodulin eluates were analyzed by SDS-PAGE and Coomassie staining. Open circles indicate Med13 (top) and Med12 (bottom).(F) Genetic interaction analysis shows negative synthetic links between MED31 and SAC3 R288D. The indicated genotypes were produced by transformation with the respective HIS plasmids into cells which also contain a SAC3 cover plasmid (URA3). Growth was followed on SDC-His and on SDC+5-fluoroorotic acid (5-FOA) plates to counterselect against the cover plasmid.

Mentions: We hypothesized that the ability of TREX-2 to regulate both transcription and mRNA export is encoded in its PCI portion that represents the evolutionary most conserved part of the complex. Among PCI proteins two different types can be discerned: the TREX-2 subunit Thp1 contains a “typical” PCI, whereas Sac3 contains a less common “atypical” PCI, which features an additional N-terminal extension (Pick et al., 2009). Multiple sequence alignments of Sac3 revealed a conserved region (Sac3 aa 200–300), which corresponds to the atypical PCI extension (Figure S1A). Despite being highly conserved, this region was not functionally characterized in a recent study reporting the crystal structure of the yeast Sac3(253–551)/Thp1/Sem1 complex (PDB: 3t5v) (Ellisdon et al., 2012). To address the function of the most conserved region of Sac3 we determined the crystal structure of a Saccharomyces cerevisiae TREX-2 sub-complex containing an N-terminally extended variant of Sac3 (Sac3(222–572)/Thp1(170–455)/Sem1) at 3.1 Å resolution revealing an overall organization that was highly similar to the reported structure (root-mean-square deviation [rmsd] values: Sac3 0.8 Å, Thp1 1.4 Å, and Sem1 2.8 Å for 299, 450, and 56 equivalent Ca atoms, respectively) (Figure 1B). Despite their conservation, Sac3 residues 222–252 were not defined by electron density, most likely because this segment is inherently flexible. Strikingly, mapping the sequence conservation onto the TREX-2 PCI structure highlighted a distinct surface patch in the atypical part of the Sac3 PCI domain as the largest conserved region of the entire Sac3/Thp1/Sem1 complex (Figure 1C). This region includes Sac3 helices α1, α2, and α4 and consists of two clusters of positively charged residues centered around R256 and R288 (Figure 1D). To analyze the function of this region, we phenotypically characterized the impact of point mutations in residues that are identical between yeast and human (Figures 1E and S1A). Interestingly, both sac3 R256D and R288D mutations, exhibited pronounced growth defects with the R288D mutation being more severely affected on galactose-containing medium, a condition that requires highly inducible transcription of the NPC-targeted GAL1 gene (Figure 1E). The respective alanine substitutions resulted in weaker phenotypes, while other mutations (e.g., D351R) showed no readily detectable growth defects. The observed phenotypes do not result from Sac3 protein instability, as the mutant proteins assembled into stable complexes with the other TREX-2 subunits (Figures S1B and S1C) and localized to NPCs in vivo (Figure S1D). Sac3 residues K467 and K468, located in the remote winged helix subdomain, were reported to be crucial for nucleotide binding and mRNA export (Ellisdon et al., 2012). However, upon mutation (sac3 K467D/K468D) we detected comparatively minor growth phenotypes under the conditions of our assay, consistent with these residues being poorly conserved (Figures S1A and S2A). Thus, our analysis assigns a critical function to the N-terminal region of the atypical Sac3 PCI domain.


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)

Structural, Biochemical, and Genetic Characterization of TREX-2, Mediator, and Pol II, Related to Figures 1 and 2(A) Top view of the yeast Sac3(222-572)/Thp1(170-455)/Sem1 complex in ribbon representation, showing the electrostatic surface potential and the surface conservation of the trimeric complex (left to right). The winged-helix domains of Sac3 and Thp1 are encircled (black line). Positively charged Sac3 residues Lys467 and Lys468 that have been analyzed in mutational studies are indicated.(B) Structural alignment of Sac3 from this study (gray) and Sac3 (PDB: 3t5v) (orange). The interaction of Sac3 with its crystallographic neighbor (∗) is shown with α1 and α2 helices forming the crystallographic interface in both crystal forms.(C) The yeast Mediator complex was affinity-purified via TAP-tagged Med15 from wild-type and sac3Δ cells. Calmodulin eluates were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. Loss of Cdk8 occurs also when Med15 is used as a TAP-tagged bait.(D) Yeast RNA Polymerase II was affinity-purified via TAP-tagged Rpb3 from wild-type and sac3Δ cells. Calmodulin eluates were analyzed by SDS-PAGE and Coomassie staining (upper panel) and immunoblotting (lower panel) using the indicated antibodies. Subunits were assigned according to their calculated molecular weight. Tfg1 and Tfg2 are subunits of TFIIF. Asterisk indicates degradation product of Rpb1, which appeared to be more susceptible to proteolysis when hyperphosphorylated on Ser5.(E) Med7-TAP purifications of Mediator from wild-type and mutant cells. Calmodulin eluates were analyzed by SDS-PAGE and Coomassie staining. Open circles indicate Med13 (top) and Med12 (bottom).(F) Genetic interaction analysis shows negative synthetic links between MED31 and SAC3 R288D. The indicated genotypes were produced by transformation with the respective HIS plasmids into cells which also contain a SAC3 cover plasmid (URA3). Growth was followed on SDC-His and on SDC+5-fluoroorotic acid (5-FOA) plates to counterselect against the cover plasmid.
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Show All Figures
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figs2: Structural, Biochemical, and Genetic Characterization of TREX-2, Mediator, and Pol II, Related to Figures 1 and 2(A) Top view of the yeast Sac3(222-572)/Thp1(170-455)/Sem1 complex in ribbon representation, showing the electrostatic surface potential and the surface conservation of the trimeric complex (left to right). The winged-helix domains of Sac3 and Thp1 are encircled (black line). Positively charged Sac3 residues Lys467 and Lys468 that have been analyzed in mutational studies are indicated.(B) Structural alignment of Sac3 from this study (gray) and Sac3 (PDB: 3t5v) (orange). The interaction of Sac3 with its crystallographic neighbor (∗) is shown with α1 and α2 helices forming the crystallographic interface in both crystal forms.(C) The yeast Mediator complex was affinity-purified via TAP-tagged Med15 from wild-type and sac3Δ cells. Calmodulin eluates were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. Loss of Cdk8 occurs also when Med15 is used as a TAP-tagged bait.(D) Yeast RNA Polymerase II was affinity-purified via TAP-tagged Rpb3 from wild-type and sac3Δ cells. Calmodulin eluates were analyzed by SDS-PAGE and Coomassie staining (upper panel) and immunoblotting (lower panel) using the indicated antibodies. Subunits were assigned according to their calculated molecular weight. Tfg1 and Tfg2 are subunits of TFIIF. Asterisk indicates degradation product of Rpb1, which appeared to be more susceptible to proteolysis when hyperphosphorylated on Ser5.(E) Med7-TAP purifications of Mediator from wild-type and mutant cells. Calmodulin eluates were analyzed by SDS-PAGE and Coomassie staining. Open circles indicate Med13 (top) and Med12 (bottom).(F) Genetic interaction analysis shows negative synthetic links between MED31 and SAC3 R288D. The indicated genotypes were produced by transformation with the respective HIS plasmids into cells which also contain a SAC3 cover plasmid (URA3). Growth was followed on SDC-His and on SDC+5-fluoroorotic acid (5-FOA) plates to counterselect against the cover plasmid.
Mentions: We hypothesized that the ability of TREX-2 to regulate both transcription and mRNA export is encoded in its PCI portion that represents the evolutionary most conserved part of the complex. Among PCI proteins two different types can be discerned: the TREX-2 subunit Thp1 contains a “typical” PCI, whereas Sac3 contains a less common “atypical” PCI, which features an additional N-terminal extension (Pick et al., 2009). Multiple sequence alignments of Sac3 revealed a conserved region (Sac3 aa 200–300), which corresponds to the atypical PCI extension (Figure S1A). Despite being highly conserved, this region was not functionally characterized in a recent study reporting the crystal structure of the yeast Sac3(253–551)/Thp1/Sem1 complex (PDB: 3t5v) (Ellisdon et al., 2012). To address the function of the most conserved region of Sac3 we determined the crystal structure of a Saccharomyces cerevisiae TREX-2 sub-complex containing an N-terminally extended variant of Sac3 (Sac3(222–572)/Thp1(170–455)/Sem1) at 3.1 Å resolution revealing an overall organization that was highly similar to the reported structure (root-mean-square deviation [rmsd] values: Sac3 0.8 Å, Thp1 1.4 Å, and Sem1 2.8 Å for 299, 450, and 56 equivalent Ca atoms, respectively) (Figure 1B). Despite their conservation, Sac3 residues 222–252 were not defined by electron density, most likely because this segment is inherently flexible. Strikingly, mapping the sequence conservation onto the TREX-2 PCI structure highlighted a distinct surface patch in the atypical part of the Sac3 PCI domain as the largest conserved region of the entire Sac3/Thp1/Sem1 complex (Figure 1C). This region includes Sac3 helices α1, α2, and α4 and consists of two clusters of positively charged residues centered around R256 and R288 (Figure 1D). To analyze the function of this region, we phenotypically characterized the impact of point mutations in residues that are identical between yeast and human (Figures 1E and S1A). Interestingly, both sac3 R256D and R288D mutations, exhibited pronounced growth defects with the R288D mutation being more severely affected on galactose-containing medium, a condition that requires highly inducible transcription of the NPC-targeted GAL1 gene (Figure 1E). The respective alanine substitutions resulted in weaker phenotypes, while other mutations (e.g., D351R) showed no readily detectable growth defects. The observed phenotypes do not result from Sac3 protein instability, as the mutant proteins assembled into stable complexes with the other TREX-2 subunits (Figures S1B and S1C) and localized to NPCs in vivo (Figure S1D). Sac3 residues K467 and K468, located in the remote winged helix subdomain, were reported to be crucial for nucleotide binding and mRNA export (Ellisdon et al., 2012). However, upon mutation (sac3 K467D/K468D) we detected comparatively minor growth phenotypes under the conditions of our assay, consistent with these residues being poorly conserved (Figures S1A and S2A). Thus, our analysis assigns a critical function to the N-terminal region of the atypical Sac3 PCI domain.

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