mRNA Export Complex Research Articles

Putative Arabidopsis THO/TREX mRNA export complex is involved in transgene and endogenous siRNA biosynthesis

RNA silencing in plants and some animals has a non-cell-autonomous effect due to an RNA signal that moves between cells or organs. To identify unique factors involved in this process, we analyzed a group of Arabidopsis mutants with defective spread of RNA silencing from a transgene expressed specifically in the phloem. These mutants accumulated reduced amounts of small interfering (si)RNA from the transgene locus and from endogenous loci TAS1, TAS2, and an inverted repeat locus IR71. The defect in TAS1 and TAS2 siRNA biogenesis is in the processing of a long siRNA precursor. We mapped the mutations to a gene encoding the Arabidopsis homolog of a protein, TEX1, which is involved in intracellular transport of RNA in animals. TEX1 is a component of the THO/TREX complex, and we show that the Arabidopsis TEX1 interacts with other predicted components of a THO/TREX complex. Correspondingly, we found at least two other components of the Arabidopsis THO core complex that are involved in RNA silencing. To reconcile the effect of these mutations on transgene and endogenous gene siRNA, we propose a mechanism in which THO/TREX processes or transports a long RNA molecule so that it can be a template for secondary siRNA production.

Multifunctional factor ENY2 is associated with the THO complex and promotes its recruitment onto nascent mRNA

Metazoan E(y)2/ENY2 is a multifunctional protein important for transcription activation and mRNA export, being a component of SAGA/TFTC and the mRNA export complex AMEX. Here, we show that ENY2 in Drosophila is also stably associated with THO, the complex involved in mRNP biogenesis. The ENY2-THO complex is required for normal Drosophila development, functioning independently on SAGA and AMEX. ENY2 and THO arrive on the transcribed region of the hsp70 gene after its activation, and ENY2 plays an important role in THO recruitment. ENY2 and THO show no direct association with elongating RNA polymerase II. Recruitment of ENY2 and THO occurs by their loading onto nascent mRNA, apparently immediately after its synthesis, while the AMEX component Xmas-2 is loaded onto mRNA at a later stage. Knockdown of either ENY2 or THO, but not SAGA or AMEX, affects the processing of the transcript's 3' end. Thus, ENY2, as a shared subunit of several protein complexes governing the sequential steps of gene expression, plays an important role in the coordination of these steps.

Arabidopsis homolog of the yeast TREX-2 mRNA export complex: components and anchoring nucleoporin

Nuclear pore complexes (NPCs) are vital to nuclear-cytoplasmic communication in eukaryotes. The yeast NPC-associated TREX-2 complex, also known as the Thp1-Sac3-Cdc31-Sus1 complex, is anchored on the NPC via the nucleoporin Nup1, and is essential for mRNA export. Here we report the identification and characterization of the putative Arabidopsis thaliana TREX-2 complex and its anchoring nucleoporin. Physical and functional evidence support the identification of the Arabidopsis orthologs of yeast Thp1 and Nup1. Of three Arabidopsis homologs of yeast Sac3, two are putative TREX-2 components, but, surprisingly, none are required for mRNA export as they are in yeast. Physical association of the two Cdc31 homologs, but not the Sus1 homolog, with the TREX-2 complex was observed. In addition to identification of these TREX-2 components, direct interactions of the Arabidopsis homolog of DSS1, which is an established proteasome component in yeast and animals, with both the TREX-2 complex and the proteasome were observed. This suggests the possibility of a link between the two complexes. Thus this work has identified the putative Arabidopsis TREX-2 complex and provides a foundation for future studies of nuclear export in Arabidopsis.

Mutational Uncoupling of the Role of Sus1 in Nuclear Pore Complex Targeting of an mRNA Export Complex and Histone H2B Deubiquitination

Sus1 is an evolutionary conserved protein that functions both in transcription and mRNA export and has been proposed to contribute to coupling these processes in yeast. Sus1 mediates its different roles as a component of both the histone H2B deubiquitinating module (Sus1-Sgf11-Ubp8-Sgf73) of the SAGA (Spt-Ada-Gcn5 acetyltransferase) transcriptional co-activator and the mRNA export complex, TREX-2 (Sus1-Sac3-Thp1-Cdc31). We have dissected the different functions of Sus1 with respect to its partitioning in transcription and export complexes using a mutational approach. Here we show that the sus1-10 (E18A, S19A, and G20A) and sus1-12 (V73A and D75A) alleles of Sus1 can be dissociated from TREX-2 while leaving its interaction with SAGA largely intact. Conversely, the binding to both TREX-2 and SAGA was impaired in the sus1-11 allele (G37A and W38A), in which two highly conserved residues were mutated. In vitro experiments demonstrated that dissociation of mutant Sus1 from its partners is caused by a reduced affinity toward the TREX-2 subunit, Sac3, and the SAGA factor, Sgf11, respectively. Consistent with the biochemical data, these sus1 mutant alleles showed differential genetic relationships with SAGA and mRNA export mutants. In vivo, all three sus1 mutants were impaired in targeting TREX-2 (i.e. Sac3) to the nuclear pore complexes and exhibited nuclear mRNA export defects. This study has implications for how Sus1, in combination with distinct interaction partners, can regulate diverse aspects of gene expression.

Sus1, Cdc31, and the Sac3 CID Region Form a Conserved Interaction Platform that Promotes Nuclear Pore Association and mRNA Export

SummaryThe yeast Sac3:Cdc31:Sus1:Thp1 (TREX-2) complex facilitates the repositioning and association of actively transcribing genes with nuclear pores (NPCs)—“gene gating”—that is central to integrating transcription, processing, and mRNA nuclear export. We present here the crystal structure of Sus1 and Cdc31 bound to a central region of Sac3 (the CID domain) that is crucial for its function. Sac3CID forms a long, gently undulating α helix around which one Cdc31 and two Sus1 chains are wrapped. Sus1 has an articulated helical hairpin fold that facilitates its wrapping around Sac3. In vivo studies using engineered mutations that selectively disrupted binding of individual chains to Sac3 indicated that Sus1 and Cdc31 function synergistically to promote NPC association of TREX-2 and mRNA nuclear export. These data indicate Sac3CID provides a scaffold within TREX-2 to integrate interactions between protein complexes to facilitate the coupling of transcription and mRNA export during gene expression.

The Anchor-Away Technique: Rapid, Conditional Establishment of Yeast Mutant Phenotypes

The anchor-away (AA) technique depletes the nucleus of Saccharomyces cerevisiae of a protein of interest (the target) by conditional tethering to an abundant cytoplasmic protein (the anchor) by appropriate gene tagging and rapamycin-dependent heterodimerization. Taking advantage of the massive flow of ribosomal proteins through the nucleus during maturation, a protein of the large subunit was chosen as the anchor. Addition of rapamycin, due to formation of the ternary complex, composed of the anchor, rapamycin, and the target, then results in the rapid depletion of the target from the nucleus. All 43 tested genes displayed on rapamycin plates the expected defective growth phenotype. In addition, when examined functionally, specific mutant phenotypes were obtained within minutes. These are genes involved in protein import, RNA export, transcription, sister chromatid cohesion, and gene silencing. The AA technique is a powerful tool for nuclear biology to dissect the function of individual or gene pairs in synthetic, lethal situations.

Nap1 Links Transcription Elongation, Chromatin Assembly, and Messenger RNP Complex Biogenesis

Chromatin remodeling is central to the regulation of transcription elongation. We demonstrate that the conserved Saccharomyces cerevisiae histone chaperone Nap1 associates with chromatin. We show that Nap1 regulates transcription of PHO5, and the increase in transcript level and the higher phosphatase activity plateau observed for Deltanap1 cells suggest that the net function of Nap1 is to facilitate nucleosome reassembly during transcription elongation. To further our understanding of histone chaperones in transcription elongation, we identified factors that regulate the function of Nap1 in this process. One factor investigated is an essential mRNA export and TREX complex component, Yra1. Nap1 interacts directly with Yra1 and genetically with other TREX complex components and the mRNA export factor Mex67. Additionally, we show that the recruitment of Nap1 to the coding region of actively transcribed genes is Yra1 dependent and that its recruitment to promoters is TREX complex independent. These observations suggest that Nap1 functions provide a new connection between transcription elongation, chromatin assembly, and messenger RNP complex biogenesis.

Action of FMIP/THOC5 and THOC7, members of the THO mRNA export-complex.

Action of FMIP/THOC5 and THOC7, members of the THO mRNA export-complex.

Yra1p, a conserved nuclear RNA-binding protein, interacts directly with Mex67p and is required for mRNA export.

Mex67p and Mtr2p constitute an essential mRNA export complex that interacts with poly(A)+ RNA and nuclear pore proteins. We have identified Yra1p, an intranuclear protein with in vitro RNA-RNA annealing activity, which directly binds to Mex67p. The complex between Yra1p and Mex67p was reconstituted in vitro and shown by UV-crosslinking to bind directly to RNA. Mutants of YRA1 are impaired in nuclear poly(A)+ RNA export at restrictive growth conditions. ALY, the mouse homologue of Yra1p and a transcriptional coactivator, can bind in vitro to yeast and human Mex67p and partly complements the otherwise non-viable yra1 null mutant. Thus, Yra1p is the first RNA-binding protein characterized, which bridges the shuttling Mex67p/Mtr2p exporter to intranuclear mRNA transport cargoes.

Nuclear mRNA export requires complex formation between Mex67p and Mtr2p at the nuclear pores.

We have identified between Mex67p and Mtr2p a complex which is essential for mRNA export. This complex, either isolated from yeast or assembled in Escherichia coli, can bind in vitro to RNA through Mex67p. In vivo, Mex67p requires Mtr2p for association with the nuclear pores, which can be abolished by mutating either MEX67 or MTR2. In all cases, detachment of Mex67p from the pores into the cytoplasm correlates with a strong inhibition of mRNA export. At the nuclear pores, Nup85p represents one of the targets with which the Mex67p-Mtr2p complex interacts. Thus, Mex67p and Mtr2p constitute a novel mRNA export complex which can bind to RNA via Mex67p and which interacts with nuclear pores via Mtr2p.