Limits...
Identification and functional analysis of NOL7 nuclear and nucleolar localization signals.

Zhou G, Doçi CL, Lingen MW - BMC Cell Biol. (2010)

Bottom Line: In support, targeting to the nucleolar compartment was dependent on the presence of RNA, as depletion of total RNA or rRNA resulted in a nucleoplasmic shift of NOL7.These results identify the minimal sequences required for the active targeting of NOL7 to the nucleus and nucleolus.Taken together, these results identify the requisite protein domains for NOL7 localization, the kinetics that drive this targeting, and suggest NOL7 may function in both the nucleus and nucleolus.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, The University of Chicago, Chicago, IL, USA.

ABSTRACT

Background: NOL7 is a candidate tumor suppressor that localizes to a chromosomal region 6p23. This locus is frequently lost in a number of malignancies, and consistent loss of NOL7 through loss of heterozygosity and decreased mRNA and protein expression has been observed in tumors and cell lines. Reintroduction of NOL7 into cells resulted in significant suppression of in vivo tumor growth and modulation of the angiogenic phenotype. Further, NOL7 was observed to localize to the nucleus and nucleolus of cells. However, the mechanisms regulating its subcellular localization have not been elucidated.

Results: An in vitro import assay demonstrated that NOL7 requires cytosolic machinery for active nuclear transport. Using sequence homology and prediction algorithms, four putative nuclear localization signals (NLSs) were identified. NOL7 deletion constructs and cytoplasmic pyruvate kinase (PK) fusion proteins confirmed the functionality of three of these NLSs. Site-directed mutagenesis of PK fusions and full-length NOL7 defined the minimal functional regions within each NLS. Further characterization revealed that NLS2 and NLS3 were critical for both the rate and efficiency of nuclear targeting. In addition, four basic clusters within NLS2 and NLS3 were independently capable of nucleolar targeting. The nucleolar occupancy of NOL7 revealed a complex balance of rapid nucleoplasmic shuttling but low nucleolar mobility, suggesting NOL7 may play functional roles in both compartments. In support, targeting to the nucleolar compartment was dependent on the presence of RNA, as depletion of total RNA or rRNA resulted in a nucleoplasmic shift of NOL7.

Conclusions: These results identify the minimal sequences required for the active targeting of NOL7 to the nucleus and nucleolus. Further, this work characterizes the relative contribution of each sequence to NOL7 nuclear and nucleolar dynamics, the subnuclear constituents that participate in this targeting, and suggests a functional role for NOL7 in both compartments. Taken together, these results identify the requisite protein domains for NOL7 localization, the kinetics that drive this targeting, and suggest NOL7 may function in both the nucleus and nucleolus.

Show MeSH

Related in: MedlinePlus

Basic residues within each of the NLSs are required for nuclear localization of full-length NOL7. (A) Schematic representing the different mutant constructs used to evaluate nuclear localization in the context of the full length protein. Results demonstrated in (B) are summarized in the column on the right, where "No" designates nucleolar localization, "Np" designates nucleoplasmic localization, and "C" designates cytoplasmic localization. (B) Localization of the GFP-tagged constructs in HeLa cells was confirmed by fluorescent microscopy and costaining of the nucleus with DAPI is shown in blue.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2957388&req=5

Figure 6: Basic residues within each of the NLSs are required for nuclear localization of full-length NOL7. (A) Schematic representing the different mutant constructs used to evaluate nuclear localization in the context of the full length protein. Results demonstrated in (B) are summarized in the column on the right, where "No" designates nucleolar localization, "Np" designates nucleoplasmic localization, and "C" designates cytoplasmic localization. (B) Localization of the GFP-tagged constructs in HeLa cells was confirmed by fluorescent microscopy and costaining of the nucleus with DAPI is shown in blue.

Mentions: While experiments using individual NLS fused to PK are useful, there are several limitations to these types of studies. For example, Burgess et al [59] demonstrated that EBNA3B has three functional NLSs when investigated in truncation experiments but only two were found to be functional in the context of the full-length protein. To determine the contributions of each NLS within full-length GFP-tagged NOL7, the arginine and lysine residues in each NLS were mutated to alanine (Figure 6A). The subcellular localization of the constructs was visualized using by GFP fluorescence, with DAPI costaining of the nucleus. Mutation of all three NLSs resulted in cytoplasmic localization of NOL7 but retention of only one NLS was sufficient for nuclear localization (Figure 6B). Taken together, these results demonstrate that NOL7 has three functional NLS that can independently cause translocation of full length NOL7.


Identification and functional analysis of NOL7 nuclear and nucleolar localization signals.

Zhou G, Doçi CL, Lingen MW - BMC Cell Biol. (2010)

Basic residues within each of the NLSs are required for nuclear localization of full-length NOL7. (A) Schematic representing the different mutant constructs used to evaluate nuclear localization in the context of the full length protein. Results demonstrated in (B) are summarized in the column on the right, where "No" designates nucleolar localization, "Np" designates nucleoplasmic localization, and "C" designates cytoplasmic localization. (B) Localization of the GFP-tagged constructs in HeLa cells was confirmed by fluorescent microscopy and costaining of the nucleus with DAPI is shown in blue.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2957388&req=5

Figure 6: Basic residues within each of the NLSs are required for nuclear localization of full-length NOL7. (A) Schematic representing the different mutant constructs used to evaluate nuclear localization in the context of the full length protein. Results demonstrated in (B) are summarized in the column on the right, where "No" designates nucleolar localization, "Np" designates nucleoplasmic localization, and "C" designates cytoplasmic localization. (B) Localization of the GFP-tagged constructs in HeLa cells was confirmed by fluorescent microscopy and costaining of the nucleus with DAPI is shown in blue.
Mentions: While experiments using individual NLS fused to PK are useful, there are several limitations to these types of studies. For example, Burgess et al [59] demonstrated that EBNA3B has three functional NLSs when investigated in truncation experiments but only two were found to be functional in the context of the full-length protein. To determine the contributions of each NLS within full-length GFP-tagged NOL7, the arginine and lysine residues in each NLS were mutated to alanine (Figure 6A). The subcellular localization of the constructs was visualized using by GFP fluorescence, with DAPI costaining of the nucleus. Mutation of all three NLSs resulted in cytoplasmic localization of NOL7 but retention of only one NLS was sufficient for nuclear localization (Figure 6B). Taken together, these results demonstrate that NOL7 has three functional NLS that can independently cause translocation of full length NOL7.

Bottom Line: In support, targeting to the nucleolar compartment was dependent on the presence of RNA, as depletion of total RNA or rRNA resulted in a nucleoplasmic shift of NOL7.These results identify the minimal sequences required for the active targeting of NOL7 to the nucleus and nucleolus.Taken together, these results identify the requisite protein domains for NOL7 localization, the kinetics that drive this targeting, and suggest NOL7 may function in both the nucleus and nucleolus.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, The University of Chicago, Chicago, IL, USA.

ABSTRACT

Background: NOL7 is a candidate tumor suppressor that localizes to a chromosomal region 6p23. This locus is frequently lost in a number of malignancies, and consistent loss of NOL7 through loss of heterozygosity and decreased mRNA and protein expression has been observed in tumors and cell lines. Reintroduction of NOL7 into cells resulted in significant suppression of in vivo tumor growth and modulation of the angiogenic phenotype. Further, NOL7 was observed to localize to the nucleus and nucleolus of cells. However, the mechanisms regulating its subcellular localization have not been elucidated.

Results: An in vitro import assay demonstrated that NOL7 requires cytosolic machinery for active nuclear transport. Using sequence homology and prediction algorithms, four putative nuclear localization signals (NLSs) were identified. NOL7 deletion constructs and cytoplasmic pyruvate kinase (PK) fusion proteins confirmed the functionality of three of these NLSs. Site-directed mutagenesis of PK fusions and full-length NOL7 defined the minimal functional regions within each NLS. Further characterization revealed that NLS2 and NLS3 were critical for both the rate and efficiency of nuclear targeting. In addition, four basic clusters within NLS2 and NLS3 were independently capable of nucleolar targeting. The nucleolar occupancy of NOL7 revealed a complex balance of rapid nucleoplasmic shuttling but low nucleolar mobility, suggesting NOL7 may play functional roles in both compartments. In support, targeting to the nucleolar compartment was dependent on the presence of RNA, as depletion of total RNA or rRNA resulted in a nucleoplasmic shift of NOL7.

Conclusions: These results identify the minimal sequences required for the active targeting of NOL7 to the nucleus and nucleolus. Further, this work characterizes the relative contribution of each sequence to NOL7 nuclear and nucleolar dynamics, the subnuclear constituents that participate in this targeting, and suggests a functional role for NOL7 in both compartments. Taken together, these results identify the requisite protein domains for NOL7 localization, the kinetics that drive this targeting, and suggest NOL7 may function in both the nucleus and nucleolus.

Show MeSH
Related in: MedlinePlus