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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.

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Each NLS contributes differently to the rate and efficiency of NOL7 nuclear localization. The steady-state efficiency and rate of import for NLS mutants was evaluated to determine their relative contribution to the subcellular localization of NOL7 in HeLa cells. (A) Twenty hours after transfection, mutants were imaged by immunofluorescence against the HA tags and costained with DAPI and WGA to delineate the nucleus and cytoplasm. Using ImageJ, the nuclear-to-total cell fluorescence ratio was calculated for twenty cells per construct. Error bars represent standard error. (B) Cells were transfected with the different NOL7 NLS constructs and imaged at 5, 6, 7, and 8 hours post-transfection. The nuclear accumulation was measured by α-HA immunofluorescence and the rates were calculated as the change in nuclear signal over time. Bars represent the average rate for ten cells and error bars are representative of the standard error.
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Figure 7: Each NLS contributes differently to the rate and efficiency of NOL7 nuclear localization. The steady-state efficiency and rate of import for NLS mutants was evaluated to determine their relative contribution to the subcellular localization of NOL7 in HeLa cells. (A) Twenty hours after transfection, mutants were imaged by immunofluorescence against the HA tags and costained with DAPI and WGA to delineate the nucleus and cytoplasm. Using ImageJ, the nuclear-to-total cell fluorescence ratio was calculated for twenty cells per construct. Error bars represent standard error. (B) Cells were transfected with the different NOL7 NLS constructs and imaged at 5, 6, 7, and 8 hours post-transfection. The nuclear accumulation was measured by α-HA immunofluorescence and the rates were calculated as the change in nuclear signal over time. Bars represent the average rate for ten cells and error bars are representative of the standard error.

Mentions: For the efficiency experiments, data was collected twenty hours after transfection, when import had reached steady-state equilibrium (Figure 7A). It was found that WT NOL7 was most efficiently localized to the nucleus, and the strictly nucleoplasmic mutant N23(-), was least efficiently targeted. The single mutants demonstrated nearly 10% more efficient nuclear targeting than the double mutants, with a p-value of 2.13×10-7. The most dramatic loss in targeting efficiency was observed upon the combined loss of NLS2 and NLS3, with over a 15% decrease in efficiency for N23(-) compared to WT NOL7 and over 10% decrease compared to all other mutants. This decrease was highly significant, with a p-value of 9.45×10-9 compared to WT NOL7 and 2.68×10-5 and 1.24×10-4 compared to the other double mutants N12(-) and N13(-), respectively. Together, these observations suggest that NLS2 and NLS3 are the major sequences involved in the efficient targeting of NOL7 to the nucleus.


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

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

Each NLS contributes differently to the rate and efficiency of NOL7 nuclear localization. The steady-state efficiency and rate of import for NLS mutants was evaluated to determine their relative contribution to the subcellular localization of NOL7 in HeLa cells. (A) Twenty hours after transfection, mutants were imaged by immunofluorescence against the HA tags and costained with DAPI and WGA to delineate the nucleus and cytoplasm. Using ImageJ, the nuclear-to-total cell fluorescence ratio was calculated for twenty cells per construct. Error bars represent standard error. (B) Cells were transfected with the different NOL7 NLS constructs and imaged at 5, 6, 7, and 8 hours post-transfection. The nuclear accumulation was measured by α-HA immunofluorescence and the rates were calculated as the change in nuclear signal over time. Bars represent the average rate for ten cells and error bars are representative of the standard error.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Each NLS contributes differently to the rate and efficiency of NOL7 nuclear localization. The steady-state efficiency and rate of import for NLS mutants was evaluated to determine their relative contribution to the subcellular localization of NOL7 in HeLa cells. (A) Twenty hours after transfection, mutants were imaged by immunofluorescence against the HA tags and costained with DAPI and WGA to delineate the nucleus and cytoplasm. Using ImageJ, the nuclear-to-total cell fluorescence ratio was calculated for twenty cells per construct. Error bars represent standard error. (B) Cells were transfected with the different NOL7 NLS constructs and imaged at 5, 6, 7, and 8 hours post-transfection. The nuclear accumulation was measured by α-HA immunofluorescence and the rates were calculated as the change in nuclear signal over time. Bars represent the average rate for ten cells and error bars are representative of the standard error.
Mentions: For the efficiency experiments, data was collected twenty hours after transfection, when import had reached steady-state equilibrium (Figure 7A). It was found that WT NOL7 was most efficiently localized to the nucleus, and the strictly nucleoplasmic mutant N23(-), was least efficiently targeted. The single mutants demonstrated nearly 10% more efficient nuclear targeting than the double mutants, with a p-value of 2.13×10-7. The most dramatic loss in targeting efficiency was observed upon the combined loss of NLS2 and NLS3, with over a 15% decrease in efficiency for N23(-) compared to WT NOL7 and over 10% decrease compared to all other mutants. This decrease was highly significant, with a p-value of 9.45×10-9 compared to WT NOL7 and 2.68×10-5 and 1.24×10-4 compared to the other double mutants N12(-) and N13(-), respectively. Together, these observations suggest that NLS2 and NLS3 are the major sequences involved in the efficient targeting of NOL7 to the nucleus.

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