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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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Functional analysis of Scp160-NLSmut. (A) An alignment of the predicted NLS sequence from Scp160 with functional NLS sequences from nucleoplasmin and p53 is shown. The lysine residues that were changed to alanines to create Scp160-NLSmut are indicated by asterisks. (B) To assess the functional importance of the predicted NLS within Scp160, we tested whether an Scp160 variant with a mutant NLS (Scp160-NLSmut) could replace wild-type Scp160 in Δscp160Δeap1 cells where Scp160 is required for viability (36). Cultures were grown to log phase and equal numbers of cells were serially diluted by orders of magnitude and spotted onto control plates lacking uracil and leucine (left) or test plates lacking leucine but containing 5-FOA (right). The top row shows cells carrying LEU2 vector (pRS315); the middle row shows cells containing a wild-type copy of Scp160; the bottom row shows cells that express Scp160-NLSmut as the sole copy of Scp160.
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Figure 5: Functional analysis of Scp160-NLSmut. (A) An alignment of the predicted NLS sequence from Scp160 with functional NLS sequences from nucleoplasmin and p53 is shown. The lysine residues that were changed to alanines to create Scp160-NLSmut are indicated by asterisks. (B) To assess the functional importance of the predicted NLS within Scp160, we tested whether an Scp160 variant with a mutant NLS (Scp160-NLSmut) could replace wild-type Scp160 in Δscp160Δeap1 cells where Scp160 is required for viability (36). Cultures were grown to log phase and equal numbers of cells were serially diluted by orders of magnitude and spotted onto control plates lacking uracil and leucine (left) or test plates lacking leucine but containing 5-FOA (right). The top row shows cells carrying LEU2 vector (pRS315); the middle row shows cells containing a wild-type copy of Scp160; the bottom row shows cells that express Scp160-NLSmut as the sole copy of Scp160.

Mentions: Based on its primary amino acid sequence, Scp160 has been predicted to contain a bipartite NLS (20). Like other previously characterized bipartite NLS sequences, Scp160 contains two stretches of basic amino acids separated by a short spacer region (Figure 5A). However, results of our localization studies suggest that the predicted NLS within Scp160 does not target the protein to the nucleus and, therefore, may not be functional. If this assessment is correct, amino acid substitutions within the key NLS residues in the sequence should not significantly impact the function of Scp160. To test this hypothesis, four basic residues within the predicted NLS of Scp160 were changed to alanines to create Scp160-NLSmut (residues changed are indicated by asterisks in Figure 5A). This Scp160 variant was then assayed for its ability to rescue the synthetic lethality of the Δscp160Δeap1 cells. Growth of Δscp160Δeap1 cells expressing Scp160-NLSmut was not significantly affected compared to growth of these cells expressing wild-type Scp160 (Figure 5B). In contrast, yeast-containing vector alone (pRS315) were unable to grow on 5-FOA medium. These results suggest that the predicted NLS does not affect the global in vivo function of Scp160.Figure 5.


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)

Functional analysis of Scp160-NLSmut. (A) An alignment of the predicted NLS sequence from Scp160 with functional NLS sequences from nucleoplasmin and p53 is shown. The lysine residues that were changed to alanines to create Scp160-NLSmut are indicated by asterisks. (B) To assess the functional importance of the predicted NLS within Scp160, we tested whether an Scp160 variant with a mutant NLS (Scp160-NLSmut) could replace wild-type Scp160 in Δscp160Δeap1 cells where Scp160 is required for viability (36). Cultures were grown to log phase and equal numbers of cells were serially diluted by orders of magnitude and spotted onto control plates lacking uracil and leucine (left) or test plates lacking leucine but containing 5-FOA (right). The top row shows cells carrying LEU2 vector (pRS315); the middle row shows cells containing a wild-type copy of Scp160; the bottom row shows cells that express Scp160-NLSmut as the sole copy of Scp160.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: Functional analysis of Scp160-NLSmut. (A) An alignment of the predicted NLS sequence from Scp160 with functional NLS sequences from nucleoplasmin and p53 is shown. The lysine residues that were changed to alanines to create Scp160-NLSmut are indicated by asterisks. (B) To assess the functional importance of the predicted NLS within Scp160, we tested whether an Scp160 variant with a mutant NLS (Scp160-NLSmut) could replace wild-type Scp160 in Δscp160Δeap1 cells where Scp160 is required for viability (36). Cultures were grown to log phase and equal numbers of cells were serially diluted by orders of magnitude and spotted onto control plates lacking uracil and leucine (left) or test plates lacking leucine but containing 5-FOA (right). The top row shows cells carrying LEU2 vector (pRS315); the middle row shows cells containing a wild-type copy of Scp160; the bottom row shows cells that express Scp160-NLSmut as the sole copy of Scp160.
Mentions: Based on its primary amino acid sequence, Scp160 has been predicted to contain a bipartite NLS (20). Like other previously characterized bipartite NLS sequences, Scp160 contains two stretches of basic amino acids separated by a short spacer region (Figure 5A). However, results of our localization studies suggest that the predicted NLS within Scp160 does not target the protein to the nucleus and, therefore, may not be functional. If this assessment is correct, amino acid substitutions within the key NLS residues in the sequence should not significantly impact the function of Scp160. To test this hypothesis, four basic residues within the predicted NLS of Scp160 were changed to alanines to create Scp160-NLSmut (residues changed are indicated by asterisks in Figure 5A). This Scp160 variant was then assayed for its ability to rescue the synthetic lethality of the Δscp160Δeap1 cells. Growth of Δscp160Δeap1 cells expressing Scp160-NLSmut was not significantly affected compared to growth of these cells expressing wild-type Scp160 (Figure 5B). In contrast, yeast-containing vector alone (pRS315) were unable to grow on 5-FOA medium. These results suggest that the predicted NLS does not affect the global in vivo function of Scp160.Figure 5.

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