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. 2011 Jan;17(1):134-44.
doi: 10.1261/rna.1210411. Epub 2010 Oct 29.

A nucleoporin, Nup60p, affects the nuclear and cytoplasmic localization of ASH1 mRNA in S. cerevisiae

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A nucleoporin, Nup60p, affects the nuclear and cytoplasmic localization of ASH1 mRNA in S. cerevisiae

Erin A Powrie et al. RNA. 2011 Jan.

Abstract

The biogenesis of a localization-competent mRNP begins in the nucleus. It is thought that the coordinated action of nuclear and cytoplasmic components of the localization machinery is required for the efficient export and subsequent subcellular localization of these mRNAs in the cytoplasm. Using quantitative poly(A)(+) and transcript-specific fluorescent in situ hybridization, we analyzed different nonessential nucleoporins and nuclear pore-associated proteins for their potential role in mRNA export and localization. We found that Nup60p, a nuclear pore protein located on the nucleoplasmic side of the nuclear pore complex, was required for the mRNA localization pathway. In a Δnup60 background, localized mRNAs were preferentially retained within the nucleus compared to nonlocalized transcripts. However, the export block was only partial and some transcripts could still reach the cytoplasm. Importantly, downstream processes were also affected. Localization of ASH1 and IST2 mRNAs to the bud was impaired in the Δnup60 background, suggesting that the assembly of a localization competent mRNP ("locasome") was inhibited when NUP60 was deleted. These results demonstrate transcript specificity of a nuclear mRNA retention defect and identify a specific nucleoporin as a functional component of the localization pathway in budding yeast.

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Figures

FIGURE 1.
FIGURE 1.
Differential poly(A)+ RNA retention in nonessential nucleoporin deletion strains. Fluorescent in situ hybridization using a Cy3 labeled oligo-dT probe of strains deleted for specific nuclear pore and associated proteins to determine poly(A)+ RNA distribution. Cells were grown at 30°C. (A) Wild type and mex67-5 (37°C). (B) Strains showing poly(A)+ retention phenotype. (C) Strains without significant nuclear retention of poly(A)+ RNA. (D) Histogram showing the relative amount of nuclear poly(A)+ RNA. The nuclear poly(A)+ signal was determined by segmenting nuclear and cytoplasmic boundaries and quantifying the ratio of nuclear-to-cytoplasmic signal. (Inset) Borders are drawn segmenting out the DAPI (magenta border) from the whole cell (yellow border). The line drawn at 10% retention indicates detectable enrichment of poly(A)+ signal in the nucleus. Scale bar is 5 μm.
FIGURE 2.
FIGURE 2.
Deletion of NUP60 leads to nuclear retention of localized transcripts. (A) In situ hybridization of ASH1 mRNA in wild-type and Δnup60 strains. Retention of transcripts in the nucleus is observed in the Δnup60 mother cell. Arrowheads indicate transcription sites. Retention is not seen in the Δnup60 daughter cell, where the ASH1 gene is not being transcribed. (B) Quantification of transcript-specific in situ hybridizations for seven genes with different expression profiles and cellular localization in wild-type and Δnup60 strains. Only the localized IST2, TPO1, and ASH1 mRNAs show enhanced accumulation of nuclear transcripts in Δnup60 over wild-type strains (*P = 0.011, **P = 0.0007, ***P = 0.0001). (C) Wild-type cells clear transcripts from the nucleus faster than Δnup60 cells. Number of total nuclear ASH1 mRNAs were counted in wild-type or Δnup60 cells (n = 61 cells for each strain). The percent of total cells containing one to 14 mRNAs per nucleus is shown.
FIGURE 3.
FIGURE 3.
Distribution of ASH1 transcription sites upon deletion of NUP60. FISH using ASH1 mRNA-specific probes were performed in wild-type and Δnup60 cells. 3D data sets (30 z-planes in 200 nm intervals) were acquired for ASH1 mRNA and the DAPI signal, merged, and the position of the transcription site relative to the border of the nucleus was determined in 3D. Representative images (z-projections) of the scoring phenotypes are shown on the bottom of the graph. One hundred and two wild-type and 90 Δnup60 transcription sites were scored (*P = 0.024, **P = 0.016).
FIGURE 4.
FIGURE 4.
mRNA abundance is affected upon deletion of NUP60. The total number of transcripts for genes indicated was measured on an individual cell level from FISH results in wild type (K699) and Δnup60 and plotted as a histogram. The localized mRNAs show significant decreases in transcripts comparing wild type with the Δnup60 strains; spliced mRNAs show increases. P-values are listed in order of decreasing significance: YRA1: P = 0.0001, ASH1: P = 0.0007, IST2: P = 0.0018, DBP2: P = 0.0033, TPO: P = 0.0048.
FIGURE 5.
FIGURE 5.
Deletion of NUP60 affects ASH1 mRNA localization. (A) Examples of the localization phenotypes scored by in situ hybridizations of ASH1 mRNA. “Tight crescent” shows the majority of ASH1 tightly localized in a crescent shape to the bud tip (top). “Bud localized” shows the majority of ASH1 mRNA contained within the bud, but no strong enrichment at the bud tip (middle). “Delocalized” phenotype shows ASH1 mRNA distributed throughout the mother and the daughter cells (bottom). ASH1 transcripts that were present in the nucleus (as determined by DAPI staining) were not scored for localization. (B) Histogram showing the change in localization of ASH1 in Δnup60 vs. wild-type strains (n = 51 wild type, n = 75 nup60).
FIGURE 6.
FIGURE 6.
NUP60 deletion decreases the asymmetric localization of Ash1p. (A) Examples of the localization phenotypes scored by immunofluorescence of Ash1p-myc in wild-type and Δnup60 cells. When Ash1p is exclusively localized in the bud nucleus it was considered asymmetric localization (top). Ash1p in both the mother and daughter nuclei was considered symmetric localization (middle). Ash1p is also found in the nuclei of single cells (bottom). Nuclei were stained with DAPI. (B) The effect of deleting NUP60 on the asymmetric distribution of Ash1p in budding yeast: percent of budding cells. P = 0.011 (C) The effect of deleting NUP60 on single cells: percent of single cells expressing Ash1p. P = 0.036 (n = 191 wild type, n = 287 Δnup60).

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