The nuclear pore complex and the DEAD box protein Rat8p/Dbp5p have nonessential features which appear to facilitate mRNA export following heat shock

Abstract

Nuclear pore complexes (NPCs) play an essential role in RNA export. Nucleoporins required for mRNA export in Saccharomyces cerevisiae are found in the Nup84p and Nup82p subcomplexes of the NPC. The Nup82p subcomplex contains Nup82p, Rat7p/Nup159p, Nsp1p, Gle1p/Rss1p, and Rip1p/Nup42p and is found only on the cytoplasmic face of NPCs. Both Rat7p and Gle1p contain binding sites for Rat8p/Dbp5p, an essential DEAD box protein and putative RNA helicase. Rip1p interacts directly with Gle1p and is the only protein known to be essential for mRNA export after heat shock but not under normal growth conditions. We report that in cells lacking Rip1p, both Gle1p and Rat8p dissociate from NPCs following heat shock at 42 degrees C. Rat8p but not Gle1p was retained at NPCs if rip1Delta cells were first shifted to 37 degrees C and then to 42 degrees C, and this was correlated with preserving mRNA export in heat-shocked rip1Delta cells. Export following ethanol shock was less dependent on the presence of Rip1p. Exposure to 10% ethanol led to dissociation of Rat8p from NPCs in both wild-type and rip1Delta cells. Following this treatment, Rat8p was primarily nuclear in wild-type cells but primarily cytoplasmic in rip1Delta cells. We also determined that efficient export of heat shock mRNA after heat shock depends upon a novel 6-amino-acid element within Rat8p. This motif is not required under normal growth conditions or following ethanol shock. These studies suggest that the molecular mechanism responsible for the defect in export of heat shock mRNAs in heat-shocked rip1Delta cells is dissociation of Rat8p from NPCs. These studies also suggest that both nuclear pores and Rat8p have features not required for mRNA export in growing cells but which enhance the ability of mRNAs to be exported following heat shock.

FIG. 1.

In rip1Δ cells, the loss of Rat8p and Gle1p from the nuclear rim at 42°C can be preserved for Rat8p after induction of thermotolerance. Live cell images of wild-type and rip1Δ cells expressing GFP-tagged Rat8p (A) and Gle1p (B) are shown. Cells in early exponential growth were shifted to the indicated temperatures. Where indicated, cells were shifted to 37°C for 1 h and then to 42°C for another hour. DIC images of the same cells visualized for Rat8p-GFP and Gle1p-GFP are also shown.

FIG. 2.

The SSA4 mRNA export defect in rip1Δ cells correlates with the loss of Rat8p-GFP from the nuclear rim. In situ hybridization was performed to monitor the localization of SSA4 mRNA, and flow cytometry was used to analyze the Ssa4p-GFP protein production. (A) Wild-type cells; (B) rip1Δ cells. The Ssa4p-GFP protein signal and distribution of SSA4 mRNA were detected in cells shifted to 37 or 42°C for 1 h. Where indicated, cells shifted to 37°C for 1 h were shifted to 42°C for another hour. Cells were also stained with DAPI to permit visualization of nuclei.

FIG. 3.

RAT8 mutations lead to a defect in export of SSA4 mRNA at 42°C. In situ hybridization and flow cytometry were used to examine the distribution of SSA4 mRNA and production of Ssa4p-GFP in rat8-2 (A) and rat8Δ6 (B) cells incubated as indicated.

FIG. 4.

The SSA4 mRNA export defect at 42°C in rat7ΔN cells can be suppressed by expression of Rat8p from a high-copy plasmid. (A and B) In situ hybridization and flow cytometry were used to examine the distribution of SSA4 mRNA and production of Ssa4p-GFP. (A) rat7ΔN cells and rat7ΔN cells overexpressing Rat8p and in exponential growth phase at 23°C are shown. (B) rat7ΔN cells and rat7ΔN cells overexpressing Rat8p shifted to 42°C are shown. Cells examined by in situ hybridization were also stained with DAPI to permit visualization of nuclei. (C) Live cell images of the location of Rat8p-GFP, expressed from the RAT8 chromosomal locus, in rat7ΔN cells incubated at 23°C or shifted to 42°C. DIC images of the same cells visualized for Rat8p-GFP are also shown.

FIG. 5.

Rat8p-GFP but not Gle1p-GFP is mislocalized in cells exposed to 10% ethanol. Live cell images of the location of Rat8p-GFP (A) and Gle1p-GFP (B) in wild-type and rip1Δ cells expressing GFP-tagged Rat8p and Gle1p are shown. Where indicated, cells were incubated with 5 or 10% ethanol for 1 h. Live cell DIC images of the same cells visualized for Rat8p-GFP and Gle1p-GFP are also shown.

FIG. 6.

Rip1p is not required for export of SSA4 mRNA following ethanol shock. In situ hybridization and flow cytometry were used to analyze SSA4 RNA export and Ssa4p-GFP protein production in wild-type (A) and rip1Δ (B) cells exposed to 5 or 10% ethanol. Cells were also stained with DAPI to permit visualization of nuclei.

FIG. 7.

RAT8 mutants have a distinct response to ethanol shock. In situ hybridization and a flow cytometry assay were performed on rat8-2 (A) and rat8Δ6 (B) cells following exposure to 5 or 10% ethanol. The temperature-sensitive mutant rat8-2 was shifted to 37°C for 0.5 h followed by 5 or 10% ethanol treatment for 1 h at 37°C. rat8Δ6 cells were treated with 5 or 10% ethanol for 1 h at room temperature. Cells were also stained with DAPI to permit visualization of nuclei.