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Structural basis for the assembly and nucleic acid binding of the TREX-2 transcription-export complex.

Ellisdon AM, Dimitrova L, Hurt E, Stewart M - Nat. Struct. Mol. Biol. (2012)

Bottom Line: Sac3-Thp1-Sem1 forms a previously uncharacterized PCI-domain complex characterized by the juxtaposition of Sac3 and Thp1 winged helix domains, forming a platform that mediates nucleic acid binding.Our structure-guided mutations support the idea that the Thp1-Sac3 interaction is an essential requirement for mRNA binding and for the coupling of transcription and processing to mRNP assembly and export.These results provide insight into how newly synthesized transcripts are efficiently transferred from TREX-2 to the principal mRNA export factor, and they reveal how Sem1 stabilizes PCI domain-containing proteins and promotes complex assembly.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.

ABSTRACT
The conserved TREX-2 transcription-export complex integrates transcription and processing of many actively transcribed nascent mRNAs with the recruitment of export factors at nuclear pores and also contributes to transcriptional memory and genomic stability. We report the crystal structure of the Sac3-Thp1-Sem1 segment of Saccharomyces cerevisiae TREX-2 that interfaces with the gene expression machinery. Sac3-Thp1-Sem1 forms a previously uncharacterized PCI-domain complex characterized by the juxtaposition of Sac3 and Thp1 winged helix domains, forming a platform that mediates nucleic acid binding. Our structure-guided mutations support the idea that the Thp1-Sac3 interaction is an essential requirement for mRNA binding and for the coupling of transcription and processing to mRNP assembly and export. These results provide insight into how newly synthesized transcripts are efficiently transferred from TREX-2 to the principal mRNA export factor, and they reveal how Sem1 stabilizes PCI domain-containing proteins and promotes complex assembly.

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Structure-guided Thp1 and Sac3 mutants with impaired in vitro nucleic acid binding show cell growth and mRNA export defects, but do not inhibit TREX-2 assembly. (a) 10X serial dilutions of yeast thp1Δ expressing plasmid-borne THP1 (LEU2, YCplac111-THP1-FLAG-ProtA) wild-type or mutant alleles grown on SDC-leu plates. The thp1Δ cells were transformed with empty plasmid pRS315 (LEU2). (b) Poly(A)+ RNA export in thp1Δ cells expressing wild-type THP1 or mutant thp1 alleles, using the strains described in (a). The fraction of cells showing nuclear poly(A)+ accumulation is indicated (n=200). DNA stained with DAPI. (c) Tandem affinity-purification of plasmid-derived wild-type Thp1-FLAGPA and mutant Thp1-K414D-FLAGPA or Thp1-K427D K428D-FLAGPA from thp1Δ cells. Final eluates were analyzed by SDS-PAGE or Western blotting using anti-FLAG, anti-Sem1 and anti-Sac3 antibodies. Indicated bands were identified by mass spectrometry. # indicates Ssa1 plus Sac3; * bands not identified. (d) 10X serial dilutions of yeast sac3Δ expressing plasmid-borne SAC3(TRP1, pRS314-SAC3) wild-type or mutant alleles grown on SDC-trp1 plates. The sac3Δ cells were transformed with empty plasmid pRS314 (TRP1). (e) Poly(A)+ RNA export in sac3Δ cells expressing wild-type SAC3 or mutant sac3 alleles. The fraction of cells showing nuclear poly(A)+ accumulation is indicated (n=200). (f) Synthetically lethal interactions between sac3 mutant alleles that had impaired nucleic acid binding in vitro, and yra1-ΔRRM and mex67-5 alleles. pRS314-Sac3 wild-type and mutants were co-expressed with pRS315-yra1ΔRRM or pRS315-mex67-5 in sac3Δ yra1Δ cells (upper panel) or sac3Δ mex67Δ (lower panel) shuffle strains. Cells were grown in 10X serial dilutions on SDC-leu-trp or SDC+FOA plates.
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Figure 5: Structure-guided Thp1 and Sac3 mutants with impaired in vitro nucleic acid binding show cell growth and mRNA export defects, but do not inhibit TREX-2 assembly. (a) 10X serial dilutions of yeast thp1Δ expressing plasmid-borne THP1 (LEU2, YCplac111-THP1-FLAG-ProtA) wild-type or mutant alleles grown on SDC-leu plates. The thp1Δ cells were transformed with empty plasmid pRS315 (LEU2). (b) Poly(A)+ RNA export in thp1Δ cells expressing wild-type THP1 or mutant thp1 alleles, using the strains described in (a). The fraction of cells showing nuclear poly(A)+ accumulation is indicated (n=200). DNA stained with DAPI. (c) Tandem affinity-purification of plasmid-derived wild-type Thp1-FLAGPA and mutant Thp1-K414D-FLAGPA or Thp1-K427D K428D-FLAGPA from thp1Δ cells. Final eluates were analyzed by SDS-PAGE or Western blotting using anti-FLAG, anti-Sem1 and anti-Sac3 antibodies. Indicated bands were identified by mass spectrometry. # indicates Ssa1 plus Sac3; * bands not identified. (d) 10X serial dilutions of yeast sac3Δ expressing plasmid-borne SAC3(TRP1, pRS314-SAC3) wild-type or mutant alleles grown on SDC-trp1 plates. The sac3Δ cells were transformed with empty plasmid pRS314 (TRP1). (e) Poly(A)+ RNA export in sac3Δ cells expressing wild-type SAC3 or mutant sac3 alleles. The fraction of cells showing nuclear poly(A)+ accumulation is indicated (n=200). (f) Synthetically lethal interactions between sac3 mutant alleles that had impaired nucleic acid binding in vitro, and yra1-ΔRRM and mex67-5 alleles. pRS314-Sac3 wild-type and mutants were co-expressed with pRS315-yra1ΔRRM or pRS315-mex67-5 in sac3Δ yra1Δ cells (upper panel) or sac3Δ mex67Δ (lower panel) shuffle strains. Cells were grown in 10X serial dilutions on SDC-leu-trp or SDC+FOA plates.

Mentions: sac3 and thp1 mutants that impair DNA or RNA binding in vitro caused significant growth and mRNA export defects in vivo, similar to those observed with the combination of sac3 and thp1 alleles that interfered with the Sac3–Thp1 interaction in vivo (Fig. 5). Importantly, TREX-2 assembly was not disrupted in the thp1 mutants with impaired nucleic acid binding (Fig. 5c), showing that these structure-guided mutations uncoupled TREX-2 nucleic acid binding from complex assembly. This result highlights the contribution made by the TREX-2-nucleic acid association to the yeast mRNA export pathway.


Structural basis for the assembly and nucleic acid binding of the TREX-2 transcription-export complex.

Ellisdon AM, Dimitrova L, Hurt E, Stewart M - Nat. Struct. Mol. Biol. (2012)

Structure-guided Thp1 and Sac3 mutants with impaired in vitro nucleic acid binding show cell growth and mRNA export defects, but do not inhibit TREX-2 assembly. (a) 10X serial dilutions of yeast thp1Δ expressing plasmid-borne THP1 (LEU2, YCplac111-THP1-FLAG-ProtA) wild-type or mutant alleles grown on SDC-leu plates. The thp1Δ cells were transformed with empty plasmid pRS315 (LEU2). (b) Poly(A)+ RNA export in thp1Δ cells expressing wild-type THP1 or mutant thp1 alleles, using the strains described in (a). The fraction of cells showing nuclear poly(A)+ accumulation is indicated (n=200). DNA stained with DAPI. (c) Tandem affinity-purification of plasmid-derived wild-type Thp1-FLAGPA and mutant Thp1-K414D-FLAGPA or Thp1-K427D K428D-FLAGPA from thp1Δ cells. Final eluates were analyzed by SDS-PAGE or Western blotting using anti-FLAG, anti-Sem1 and anti-Sac3 antibodies. Indicated bands were identified by mass spectrometry. # indicates Ssa1 plus Sac3; * bands not identified. (d) 10X serial dilutions of yeast sac3Δ expressing plasmid-borne SAC3(TRP1, pRS314-SAC3) wild-type or mutant alleles grown on SDC-trp1 plates. The sac3Δ cells were transformed with empty plasmid pRS314 (TRP1). (e) Poly(A)+ RNA export in sac3Δ cells expressing wild-type SAC3 or mutant sac3 alleles. The fraction of cells showing nuclear poly(A)+ accumulation is indicated (n=200). (f) Synthetically lethal interactions between sac3 mutant alleles that had impaired nucleic acid binding in vitro, and yra1-ΔRRM and mex67-5 alleles. pRS314-Sac3 wild-type and mutants were co-expressed with pRS315-yra1ΔRRM or pRS315-mex67-5 in sac3Δ yra1Δ cells (upper panel) or sac3Δ mex67Δ (lower panel) shuffle strains. Cells were grown in 10X serial dilutions on SDC-leu-trp or SDC+FOA plates.
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Figure 5: Structure-guided Thp1 and Sac3 mutants with impaired in vitro nucleic acid binding show cell growth and mRNA export defects, but do not inhibit TREX-2 assembly. (a) 10X serial dilutions of yeast thp1Δ expressing plasmid-borne THP1 (LEU2, YCplac111-THP1-FLAG-ProtA) wild-type or mutant alleles grown on SDC-leu plates. The thp1Δ cells were transformed with empty plasmid pRS315 (LEU2). (b) Poly(A)+ RNA export in thp1Δ cells expressing wild-type THP1 or mutant thp1 alleles, using the strains described in (a). The fraction of cells showing nuclear poly(A)+ accumulation is indicated (n=200). DNA stained with DAPI. (c) Tandem affinity-purification of plasmid-derived wild-type Thp1-FLAGPA and mutant Thp1-K414D-FLAGPA or Thp1-K427D K428D-FLAGPA from thp1Δ cells. Final eluates were analyzed by SDS-PAGE or Western blotting using anti-FLAG, anti-Sem1 and anti-Sac3 antibodies. Indicated bands were identified by mass spectrometry. # indicates Ssa1 plus Sac3; * bands not identified. (d) 10X serial dilutions of yeast sac3Δ expressing plasmid-borne SAC3(TRP1, pRS314-SAC3) wild-type or mutant alleles grown on SDC-trp1 plates. The sac3Δ cells were transformed with empty plasmid pRS314 (TRP1). (e) Poly(A)+ RNA export in sac3Δ cells expressing wild-type SAC3 or mutant sac3 alleles. The fraction of cells showing nuclear poly(A)+ accumulation is indicated (n=200). (f) Synthetically lethal interactions between sac3 mutant alleles that had impaired nucleic acid binding in vitro, and yra1-ΔRRM and mex67-5 alleles. pRS314-Sac3 wild-type and mutants were co-expressed with pRS315-yra1ΔRRM or pRS315-mex67-5 in sac3Δ yra1Δ cells (upper panel) or sac3Δ mex67Δ (lower panel) shuffle strains. Cells were grown in 10X serial dilutions on SDC-leu-trp or SDC+FOA plates.
Mentions: sac3 and thp1 mutants that impair DNA or RNA binding in vitro caused significant growth and mRNA export defects in vivo, similar to those observed with the combination of sac3 and thp1 alleles that interfered with the Sac3–Thp1 interaction in vivo (Fig. 5). Importantly, TREX-2 assembly was not disrupted in the thp1 mutants with impaired nucleic acid binding (Fig. 5c), showing that these structure-guided mutations uncoupled TREX-2 nucleic acid binding from complex assembly. This result highlights the contribution made by the TREX-2-nucleic acid association to the yeast mRNA export pathway.

Bottom Line: Sac3-Thp1-Sem1 forms a previously uncharacterized PCI-domain complex characterized by the juxtaposition of Sac3 and Thp1 winged helix domains, forming a platform that mediates nucleic acid binding.Our structure-guided mutations support the idea that the Thp1-Sac3 interaction is an essential requirement for mRNA binding and for the coupling of transcription and processing to mRNP assembly and export.These results provide insight into how newly synthesized transcripts are efficiently transferred from TREX-2 to the principal mRNA export factor, and they reveal how Sem1 stabilizes PCI domain-containing proteins and promotes complex assembly.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.

ABSTRACT
The conserved TREX-2 transcription-export complex integrates transcription and processing of many actively transcribed nascent mRNAs with the recruitment of export factors at nuclear pores and also contributes to transcriptional memory and genomic stability. We report the crystal structure of the Sac3-Thp1-Sem1 segment of Saccharomyces cerevisiae TREX-2 that interfaces with the gene expression machinery. Sac3-Thp1-Sem1 forms a previously uncharacterized PCI-domain complex characterized by the juxtaposition of Sac3 and Thp1 winged helix domains, forming a platform that mediates nucleic acid binding. Our structure-guided mutations support the idea that the Thp1-Sac3 interaction is an essential requirement for mRNA binding and for the coupling of transcription and processing to mRNP assembly and export. These results provide insight into how newly synthesized transcripts are efficiently transferred from TREX-2 to the principal mRNA export factor, and they reveal how Sem1 stabilizes PCI domain-containing proteins and promotes complex assembly.

Show MeSH
Related in: MedlinePlus