The DExD/H-box ATPase Dbp5 is essential for nuclear mRNA export, although its precise role in this process remains poorly understood. Here, we identify the nuclear pore protein Gle1 as a cellular activator of Dbp5. Dbp5 alone is unable to stably bind RNA or effectively hydrolyse ATP under physiological conditions, but addition of Gle1 dramatically stimulates these activities. A gle1 point mutant deficient for Dbp5 stimulation in vitro displays an mRNA export defect in vivo, indicating that activation of Dbp5 is an essential function of Gle1. Interestingly, Gle1 binds directly to inositol hexakisphosphate (InsP6) and InsP6 potentiates the Gle1-mediated stimulation of Dbp5. Dominant mutations in DBP5 and GLE1 that rescue mRNA export phenotypes associated with the lack of InsP6 mimic the InsP6 effects in vitro. Our results define specific functions for Gle1 and InsP6 in mRNA export and suggest that local activation of Dbp5 at the nuclear pore is critical for mRNA export.

Septins comprise a conserved family of proteins that are found primarily in fungi and animals. These GTP-binding proteins have several roles during cell division, cytoskeletal organization and membrane-remodelling events. One factor that is crucial for their functions is the ordered assembly of individual septins into oligomeric core complexes that, in turn, form higher-order structures such as filaments, rings and gauzes. The molecular details of these interactions and the mechanism by which septin-complex assembly is regulated have remained elusive. Recently, the first detailed structural views of the septin core have emerged, and these, along with studies of septin dynamics in vivo, have provided new insight into septin-complex assembly and septin function in vivo.

Septins are conserved GTPases that form heteromultimeric complexes and assemble into filaments that play a critical role in cell division and polarity. Results from budding and fission yeast indicate that septin complexes form around a tetrameric core. However, the molecular structure of the core and its influence on the polarity of septin complexes and filaments is poorly defined. The septin complex of the nematode Caenorhabditis elegans is formed entirely by the core septins UNC-59 and UNC-61. We show that UNC-59 and UNC-61 form a dimer of coiled-coil-mediated heterodimers. By electron microscopy, this heterotetramer appears as a linear arrangement of four densities representing the four septin subunits. Fusion of GFP to the N termini of UNC-59 and UNC-61 and subsequent electron microscopic visualization suggests that the sequence of septin subunits is UNC-59/UNC-61/UNC-61/ UNC-59. Visualization of GFP extensions fused to the extremity of the C-terminal coiled coils indicates that these extend laterally from the heterotetrameric core. Together, our study establishes that the septin core complex is symmetric, and suggests that septins form nonpolar filaments.

Nuclear export of mRNA in eukaryotic cells is mediated by soluble transport factors and components of the nuclear pore complex (NPC). The cytoplasmically oriented nuclear pore protein Nup159 plays a critical role in mRNA export through its conserved N-terminal domain (NTD). Here, we report the crystal structure of the Nup159 NTD, refined to 2.5 A. The structure reveals an unusually asymmetric seven-bladed beta-propeller that is structurally conserved throughout eukarya. Using structure-based conservation analysis, we have targeted specific surface residues for mutagenesis. Residue substitutions in a conserved loop of the NTD abolish in vitro binding to Dbp5, a DEAD box helicase required for mRNA export. In vivo, these mutations cause Dbp5 mislocalization and block mRNA export. These findings suggest that the Nup159 NTD functions in mRNA export as a binding platform, tethering shuttling Dbp5 molecules at the nuclear periphery and locally concentrating this mRNA remodeling factor at the cytoplasmic face of the NPC.

To understand the role of chromatin-remodeling activities in transcription, it is necessary to understand how they interact with transcriptional activators in vivo to regulate the different steps of transcription. Human heat shock factor 1 (HSF1) stimulates both transcriptional initiation and elongation. We replaced mouse HSF1 in fibroblasts with wild-type and mutant human HSF1 constructs and characterized regulation of an endogenous mouse hsp70 gene. A mutation that diminished transcriptional initiation led to twofold reductions in hsp70 mRNA induction and recruitment of a SWI/SNF remodeling complex. In contrast, a mutation that diminished transcriptional elongation abolished induction of full-length mRNA, SWI/SNF recruitment, and chromatin remodeling, but minimally impaired initiation from the hsp70 promoter. Another remodeling factor, SNF2h, is constitutively present at the promoter irrespective of the genotype of HSF1. These data suggest that localized recruitment of SWI/SNF drives a specialized remodeling reaction necessary for the production of full-length hsp70 mRNA. Transcriptional activators regulate gene expression by a variety of mechanisms. They stimulate many steps in the process of transcription, including formation of the preinitiation complex, promoter opening, initiation, promoter clearance, and elongation. Activators direct these events through interactions with the basal transcription machinery, general transcription factors, and chromatinremodeling factors. Chromatin-remodeling activities can be divided into two broad categories: those that use the energy of ATP to change the association of DNA with core histones and those that alter DNA-histone interactions through covalent modifications of histone tails. Both activities are involved in transcriptional regulation, and mammalian cells contain several complexes of each kind (for review, see Becker and Horz 2002;Berger 2002). Transcriptional activators can target both histone tailmodification complexes and ATP-dependent remodeling complexes (Utley et al. 1998;Yudkovsky et al. 1999; for review, see Narlikar et al. 2002).Mammalian cells contain a variety of ATP-dependent chromatin-remodeling complexes that appear to play a role in regulating several key nuclear processes. The three major families of ATP-dependent remodeling complexes are characterized by their ATPase subunits: SWI/ SNF, ISWI, and Mi-2. Whereas SWI/SNF and ISWI family complexes function in both transcriptional activation and repression, the Mi-2 family appears to function solely in repression (for review, see Narlikar et al. 2002). Mammalian SWI/SNF is an ∼2-MD complex consisting of either the BRM or BRG1 ATPase and several additional subunits, whereas the mammalian ISWI homolog SNF2h participates in several chromatographically distinct complexes. SNF2h, hBRG1, and hBRM can all remodel chromatin in the absence of the other subunits Phelan et al. 1999;Aalfs et al. 2001). The primary mechanism by which SNF2h remodels chromatin is translational repositioning of the histone octamer a...

Chromatin remodeling complexes such as SWI/SNF use the energy of ATP hydrolysis to remodel nucleosomal DNA and increase transcription of nucleosomal templates. Human heat shock factor one (hHSF1) is a tightly regulated activator that stimulates transcriptional initiation and elongation using different portions of its activation domains. Here we demonstrate that hHSF1 associates with BRG1, the ATPase subunit of human SWI/SNF (hSWI/SNF) at endogenous protein concentrations. We also show that hHSF1 activation domains recruit hSWI/SNF to a chromatin template in a purified system. Mutation of hHSF1 residues responsible for activation of transcriptional elongation has the most severe effect on recruitment of SWI/SNF and association of hHSF1 with BRG1, suggesting that recruitment of chromatin remodeling activity might play a role in stimulation of elongation.Efficient function of transcriptional activators requires interactions with the general transcription machinery and with complexes that alter the chromatin template. Complexes that alter the chromatin template can be divided broadly into those that covalently modify the template (e.g., acetyltransferases, kinases, and methylases) and those that utilize the energy of ATP hydrolysis to "remodel" nucleosome structure and/or position (reviewed in references 22, 24, and 41). While genetic and biochemical studies have demonstrated a role for ATPdependent chromatin remodeling complexes in transcriptional activation (2, 24, 41), the mechanisms by which these complexes are targeted and the steps in the transcription process that they activate are poorly understood. The heat stress-induced transcriptional activator, heat shock factor (HSF), is responsible for coordinating the rapid transcriptional response of many heat shock protein genes, including the well-studied hsp70 gene (reviewed in reference 13). This transcriptional activator has provided a good model system for understanding mechanisms of activation.The heat shock response is regulated via activation of HSF (reviewed in references 30, 33, and 34). HSF1 is the primary stress-activated member of a family of vertebrate HSFs. Prior to heat shock, human heat shock factor 1 (hHSF1) is found throughout the cell as an inactive monomer. Upon heat shock, it becomes hyperphosphorylated, concentrates in the nucleus, trimerizes, and binds DNA. Transactivation is controlled by HSF's regulatory domain, located in the center of the protein, which is able to repress the two C-terminal activation domains (AD1 and AD2) and render them heat inducible (see Fig. 1A) (16). AD1 and AD2 each contain residues that are important for both transcriptional initiation and elongation. Mutations in acidic residues in both AD1 and AD2 preferentially affect the ability of HSF1 to stimulate transcriptional initiation, while mutations in phenylalanine residues in both activation domains preferentially affect stimulation of elongation (5).In the nucleus, DNA is packaged into nucleosomes, which frequently inhibit transcription. Remodeling complexes act ...

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