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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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Related in: MedlinePlus

Localization of Scp160-GFP in rpb1-1, mex67-5 and wild-type cells. The localization of Scp160-GFP was examined in wild-type, rpb1-1 and mex67-5 cells following a 1 h shift to 37°C. Results indicates that Scp160 is localized to the cytoplasm in both rpb1-1 and mex67-5 cells. As controls, we also localized NLS-NES-GFP and ΔRGG-Nab2-GFP. As expected, ΔRGG-Nab2-GFP, which is exported in an mRNA export-dependent manner, accumulates in the nuclei of both rpb1-1 and mex67-5 cells, but NLS-NES-GFP does not.
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Figure 3: Localization of Scp160-GFP in rpb1-1, mex67-5 and wild-type cells. The localization of Scp160-GFP was examined in wild-type, rpb1-1 and mex67-5 cells following a 1 h shift to 37°C. Results indicates that Scp160 is localized to the cytoplasm in both rpb1-1 and mex67-5 cells. As controls, we also localized NLS-NES-GFP and ΔRGG-Nab2-GFP. As expected, ΔRGG-Nab2-GFP, which is exported in an mRNA export-dependent manner, accumulates in the nuclei of both rpb1-1 and mex67-5 cells, but NLS-NES-GFP does not.

Mentions: No nuclear accumulation of Scp160 was observed in either the mex67-5 or rpb1-1 cells (Figure 3). As expected, the control mRNA binding protein, ΔRGG-Nab2-GFP, accumulated in the nucleus of both mex67-5 and rpb1-1 cells following a shift to the non-permissive temperature. No change in the localization of NLS-NES-GFP was observed. These results indicate that either Scp160 does not enter the nucleus or its export does not depend on ongoing mRNA synthesis or mRNA export.Figure 3.


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 rpb1-1, mex67-5 and wild-type cells. The localization of Scp160-GFP was examined in wild-type, rpb1-1 and mex67-5 cells following a 1 h shift to 37°C. Results indicates that Scp160 is localized to the cytoplasm in both rpb1-1 and mex67-5 cells. As controls, we also localized NLS-NES-GFP and ΔRGG-Nab2-GFP. As expected, ΔRGG-Nab2-GFP, which is exported in an mRNA export-dependent manner, accumulates in the nuclei of both rpb1-1 and mex67-5 cells, but NLS-NES-GFP does not.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: Localization of Scp160-GFP in rpb1-1, mex67-5 and wild-type cells. The localization of Scp160-GFP was examined in wild-type, rpb1-1 and mex67-5 cells following a 1 h shift to 37°C. Results indicates that Scp160 is localized to the cytoplasm in both rpb1-1 and mex67-5 cells. As controls, we also localized NLS-NES-GFP and ΔRGG-Nab2-GFP. As expected, ΔRGG-Nab2-GFP, which is exported in an mRNA export-dependent manner, accumulates in the nuclei of both rpb1-1 and mex67-5 cells, but NLS-NES-GFP does not.
Mentions: No nuclear accumulation of Scp160 was observed in either the mex67-5 or rpb1-1 cells (Figure 3). As expected, the control mRNA binding protein, ΔRGG-Nab2-GFP, accumulated in the nucleus of both mex67-5 and rpb1-1 cells following a shift to the non-permissive temperature. No change in the localization of NLS-NES-GFP was observed. These results indicate that either Scp160 does not enter the nucleus or its export does not depend on ongoing mRNA synthesis or mRNA export.Figure 3.

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