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. 2018 Aug 30;8(9):2991-3003.
doi: 10.1534/g3.118.200447.

Identification of the Novel Nup188-brr7 Allele in a Screen for Cold-Sensitive mRNA Export Mutants in Saccharomyces cerevisiae

Anne de Bruyn Kops  1 , Christine Guthrie  1
Affiliations

Identification of the Novel Nup188-brr7 Allele in a Screen for Cold-Sensitive mRNA Export Mutants in Saccharomyces cerevisiae

Anne de Bruyn Kops et al. G3 (Bethesda). .

Abstract

The maturation and export of mRNA from the nucleus through the nuclear pore complex is critical for maintaining an appropriate proteome in all eukaryotic cells. Here we summarize a previously unpublished screen in S. cerevisiae that utilized an established dT50 in situ hybridization assay to identify cold-sensitive mutants that accumulated bulk poly A RNA in the nucleus. The screen identified seven mutants in six complementation groups, including the brr6-1 strain that we described previously. In addition to brr6-1, we identified novel alleles of the key transport gene GLE1 and NUP188, a component of the Nic96 nucleoporin complex. Notably, we show that the nup188-brr7 allele causes defects in select protein import pathways as well as mRNA export. Given recent structural and functional evidence linking the Nic96 complex to transport components, this mutant may be particularly useful to the transport community.

Keywords: GLE1; NUP188; brr3; brr7; mRNA export.

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Figures

Figure 1
Figure 1
mRNA Localization in candidate cs mRNA export mutants. Shown are the mRNA localization patterns in the cs mutants incubated for 2 hr at 16° C and assayed by in situ hybridization with a digoxygenin-labeled oligo dT50 probe. Nuclear location was confirmed by DAPI staining to identify DNA.
Figure 2
Figure 2
Poly A tail length in candidate cs mRNA export mutants. Shown are the results of poly A tail length assays carried out using RNA from cells grown at 30° C (u) or incubated at 16° C for 3 hr (s). The brr3-1 strain shows a population of longer poly A tails (approximately 80-90 nt) not seen in RNA samples from wild-type cells or the other cs mutant.
Figure 3
Figure 3
mRNA localization phenotype in nup188-brr7. Panel A: shows electron microscopy images of wild type and nup188-brr7 1 nuclei. Black arrow indicates nuclear envelope abnormalities. Panel B: shows mRNA accumulation detected by dT50 in situ hybridization in nup188-brr7 at early times (0 min- 30’) following a shift to 16° C. DAPI staining shows the location of the cell nuclei. Panel C: shows two enlarged examples of the nuclear rim-staining pattern observed in nup188-brr7 at early times. At later times (2-6 hr), the mRNA staining becomes nucleoplasmic (Figure 1).
Figure 4
Figure 4
Growth and mRNA localization phenotypes in nup188∆::LEU2. Panel A: shows the growth phenotypes of YPH399 wild type (a,b), nup188-brr7 (c,d) and nup188∆::LEU2 (e,f) strains at 30° and 16° C. The strains carried either vector alone (a,c,e) or a wild-type NUP188 plasmid (b,d,f). Panel B: shows the nuclear mRNA accumulation phenotype observed in the nup188∆::LEU2 deletion strain assayed by dT50 in situ hybridization following a 2 hr incubation at 16° C. The mRNA export defect was observed in approximately 30–70% of the cells in different experiments. DAPI staining shows the location of the cell nuclei.
Figure 5
Figure 5
SV40 NLS-GFP and Npl3-GFP Localization in nup188-brr7 and nup188∆::LEU2. Panel A: shows GFP localization in live nup188-brr7 and nup188∆::LEU2 cells containing low copy SV40 NLS-GFP (left) and Npl3-GFP with the Npl3 promoter (right) constructs. Cells were grown at 30° C in selective media and incubated at 16° C for 2 hr. Quantitation of cells showing cytoplasmic accumulation based on scoring ≥100 cells per condition are shown below. Panel B: Shows immunofluorescence with an anti-Srp1 antibody in fixed cells (left) and GFP localization in live cells containing a low copy plasmid carrying Mtr10-GFP (right) under the same conditions.
Figure 6
Figure 6
Nab2-GFP and L25 NLS-GFP Localization. Panel A: shows Nab2-GFP (left) and L25 NLS-GFP (right) fusion protein localization in separate live experiments using YPH399, nup188-brr7 and nup188∆::LEU2 strains containing low copy constructs. Cells were grown at 30° C and then shifted to 16°C for 2 hr. Panel B: shows control experiments in which Nab2-GFP (top) and L25 NLS-GFP (bottom) were localized in kap104-16 and ∆kap123/pse1-1 strains respectively. Cells were grown at room temperature (RT) and imaged after a shift to 37° C for 30’ (∆kap123/pse1-1 mutant, and isogenic wild type) or 30° for 1 hr (kap104-16, ∆nup188::LEU2/kap104-16 double mutant, and wild type isogenic to kap104-16) or 16°C for 2h (kap104-16 and ∆nup188::LEU2/kap104-16 double mutant). Both reporters show striking cytoplasmic accumulation in these controls.
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