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Analysis of a predicted nuclear localization signal: implications for the intracellular localization and function of the Saccharomyces cerevisiae RNA-binding protein Scp160.

Brykailo MA, McLane LM, Fridovich-Keil J, Corbett AH - Nucleic Acids Res. (2007)

Bottom Line: We exploited a variety of yeast export mutants to capture any potential nuclear accumulation of Scp160 and found no evidence that Scp160 enters the nucleus.These localization studies were complemented by a mutational analysis of the predicted NLS.Results indicate that key basic residues within the predicted NLS of Scp160 can be altered without severely affecting Scp160 function.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics and Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA.

ABSTRACT
Gene expression is controlled by RNA-binding proteins that modulate the synthesis, processing, transport and stability of various classes of RNA. Some RNA-binding proteins shuttle between the nucleus and cytoplasm and are thought to bind to RNA transcripts in the nucleus and remain bound during translocation to the cytoplasm. One RNA-binding protein that has been hypothesized to function in this manner is the Saccharomyces cerevisiae Scp160 protein. Although the steady-state localization of Scp160 is cytoplasmic, previous studies have identified putative nuclear localization (NLS) and nuclear export (NES) signals. The goal of this study was to test the hypothesis that Scp160 is a nucleocytoplasmic shuttling protein. We exploited a variety of yeast export mutants to capture any potential nuclear accumulation of Scp160 and found no evidence that Scp160 enters the nucleus. These localization studies were complemented by a mutational analysis of the predicted NLS. Results indicate that key basic residues within the predicted NLS of Scp160 can be altered without severely affecting Scp160 function. This finding has important implications for understanding the function of Scp160, which is likely limited to the cytoplasm. Additionally, our results provide strong evidence that the presence of a predicted nuclear localization signal within the sequence of a protein should not lead to the assumption that the protein enters the nucleus in the absence of additional experimental evidence.

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Localization of Scp160-GFP in rat7-1 and wild-type cells. The localization of Scp160-GFP was examined in rat7-1 cells following a 30 min shift to 37°C where all known transport pathways are blocked. As shown, Scp160 is localized to the cytoplasm in rat7-1 cells. As controls, we also visualized NLS-NES-GFP and ΔRGG-Nab2-GFP. As expected both control proteins accumulate in the nuclei of rat7-1 cells.
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Figure 4: Localization of Scp160-GFP in rat7-1 and wild-type cells. The localization of Scp160-GFP was examined in rat7-1 cells following a 30 min shift to 37°C where all known transport pathways are blocked. As shown, Scp160 is localized to the cytoplasm in rat7-1 cells. As controls, we also visualized NLS-NES-GFP and ΔRGG-Nab2-GFP. As expected both control proteins accumulate in the nuclei of rat7-1 cells.

Mentions: As a final test of whether Scp160 enters the nucleus, we exploited a nuclear pore mutant that blocks all known nuclear export pathways, rat7-1 (32,45–47). We hypothesized that if Scp160 enters the nucleus it should accumulate in the nucleus of rat7-1 cells. Although RAT7 (NUP159) is essential, the rat7-1 allele is a temperature-sensitive allele that can be shifted to the non-permissive temperature to inactivate Rat7/Nup159 and block export pathways (46). We examined the localization of Scp160-GFP and control proteins, NLS-NES GFP and ΔRGG-Nab2-GFP, in rat7-1 cells. Following the shift to the non-permissive temperature, Scp160-GFP remained cytoplasmic in rat7-1 cells similar to wild-type cells (Figure 4). In contrast, NLS-NES-GFP and ΔRGG-Nab2-GFP both accumulated in the nuclei of rat7-1 cells. These results taken together with localization of Scp160 in other export mutants suggest that Scp160 does not enter the nucleus, at least under normal growth conditions.Figure 4.


Analysis of a predicted nuclear localization signal: implications for the intracellular localization and function of the Saccharomyces cerevisiae RNA-binding protein Scp160.

Brykailo MA, McLane LM, Fridovich-Keil J, Corbett AH - Nucleic Acids Res. (2007)

Localization of Scp160-GFP in rat7-1 and wild-type cells. The localization of Scp160-GFP was examined in rat7-1 cells following a 30 min shift to 37°C where all known transport pathways are blocked. As shown, Scp160 is localized to the cytoplasm in rat7-1 cells. As controls, we also visualized NLS-NES-GFP and ΔRGG-Nab2-GFP. As expected both control proteins accumulate in the nuclei of rat7-1 cells.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175298&req=5

Figure 4: Localization of Scp160-GFP in rat7-1 and wild-type cells. The localization of Scp160-GFP was examined in rat7-1 cells following a 30 min shift to 37°C where all known transport pathways are blocked. As shown, Scp160 is localized to the cytoplasm in rat7-1 cells. As controls, we also visualized NLS-NES-GFP and ΔRGG-Nab2-GFP. As expected both control proteins accumulate in the nuclei of rat7-1 cells.
Mentions: As a final test of whether Scp160 enters the nucleus, we exploited a nuclear pore mutant that blocks all known nuclear export pathways, rat7-1 (32,45–47). We hypothesized that if Scp160 enters the nucleus it should accumulate in the nucleus of rat7-1 cells. Although RAT7 (NUP159) is essential, the rat7-1 allele is a temperature-sensitive allele that can be shifted to the non-permissive temperature to inactivate Rat7/Nup159 and block export pathways (46). We examined the localization of Scp160-GFP and control proteins, NLS-NES GFP and ΔRGG-Nab2-GFP, in rat7-1 cells. Following the shift to the non-permissive temperature, Scp160-GFP remained cytoplasmic in rat7-1 cells similar to wild-type cells (Figure 4). In contrast, NLS-NES-GFP and ΔRGG-Nab2-GFP both accumulated in the nuclei of rat7-1 cells. These results taken together with localization of Scp160 in other export mutants suggest that Scp160 does not enter the nucleus, at least under normal growth conditions.Figure 4.

Bottom Line: We exploited a variety of yeast export mutants to capture any potential nuclear accumulation of Scp160 and found no evidence that Scp160 enters the nucleus.These localization studies were complemented by a mutational analysis of the predicted NLS.Results indicate that key basic residues within the predicted NLS of Scp160 can be altered without severely affecting Scp160 function.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics and Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA.

ABSTRACT
Gene expression is controlled by RNA-binding proteins that modulate the synthesis, processing, transport and stability of various classes of RNA. Some RNA-binding proteins shuttle between the nucleus and cytoplasm and are thought to bind to RNA transcripts in the nucleus and remain bound during translocation to the cytoplasm. One RNA-binding protein that has been hypothesized to function in this manner is the Saccharomyces cerevisiae Scp160 protein. Although the steady-state localization of Scp160 is cytoplasmic, previous studies have identified putative nuclear localization (NLS) and nuclear export (NES) signals. The goal of this study was to test the hypothesis that Scp160 is a nucleocytoplasmic shuttling protein. We exploited a variety of yeast export mutants to capture any potential nuclear accumulation of Scp160 and found no evidence that Scp160 enters the nucleus. These localization studies were complemented by a mutational analysis of the predicted NLS. Results indicate that key basic residues within the predicted NLS of Scp160 can be altered without severely affecting Scp160 function. This finding has important implications for understanding the function of Scp160, which is likely limited to the cytoplasm. Additionally, our results provide strong evidence that the presence of a predicted nuclear localization signal within the sequence of a protein should not lead to the assumption that the protein enters the nucleus in the absence of additional experimental evidence.

Show MeSH
Related in: MedlinePlus