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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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NOL7 subnuclear localization is dynamically regulated by changes in RNA composition. 293T cells were stably transfected with NOL7-GFP and treated with RNase A (100 μg/ml, 2 hours), DNase I (100 μg/ml, 2 hours), actinomycin D (0.05 μg/ml, 4 hours), or α-amanitin (50 μg/ml, 4 hours) to specifically deplete individual nucleic acid species. Treatment with DNase (total DNA), RNase (total RNA), ActD (rRNA), or α-amanitin (mRNA) was performed and localization of NOL7 was confirmed by fluorescent microscopy of the GFP tag.
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Figure 10: NOL7 subnuclear localization is dynamically regulated by changes in RNA composition. 293T cells were stably transfected with NOL7-GFP and treated with RNase A (100 μg/ml, 2 hours), DNase I (100 μg/ml, 2 hours), actinomycin D (0.05 μg/ml, 4 hours), or α-amanitin (50 μg/ml, 4 hours) to specifically deplete individual nucleic acid species. Treatment with DNase (total DNA), RNase (total RNA), ActD (rRNA), or α-amanitin (mRNA) was performed and localization of NOL7 was confirmed by fluorescent microscopy of the GFP tag.

Mentions: The shuttling of NOL7 between the nucleus and nucleolus suggested that specific interactions within these compartments may regulate the nucleolar occupancy of NOL7. Due to the highly basic nature of the protein, it was hypothesized that subnuclear localization of NOL7 may be due to interactions with nucleic acids. To investigate, various cell treatments were employed to change the abundance of different nucleic acid species (Figure 10). Cells overexpressing GFP-tagged NOL7 were treated with RNase, DNase, actinomycin D (ActD), or α-amanitin and visualized by fluorescence microscopy for changes in subcellular localization. RNase treatment resulted in nucleolar loss and nucleoplasmic accumulation of NOL7, while cells treated with DNase did not show any significant change. Culture of mammalian cells in low doses of ActD selectively inhibit rRNA synthesis while having no effect on tRNA, 5S rRNA, nuclear RNA and mRNA synthesis [73,74]. Similarly, treatment with low doses of α-amanitin inhibits RNAPII and subsequent mRNA synthesis without affecting the abundances of other RNA species. Loss of these specific RNA species has been shown to selectively deplete their RNA-binding protein counterparts from different cellular compartments, enabling visualization of binding activities that may participate in protein localization [75-77]. Upon treatment with ActD, NOL7 was found to translocate to the nucleoplasm. Upon treatment with α-amanitin, no change in the nucleolar localization of NOL7 was observed. However, the nucleoplasmic localization of NOL7 previously observed was absent. This data suggests that targeting of NOL7 to both the nucleus and nucleolus results in multiple RNA-dependent interactions.


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

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

NOL7 subnuclear localization is dynamically regulated by changes in RNA composition. 293T cells were stably transfected with NOL7-GFP and treated with RNase A (100 μg/ml, 2 hours), DNase I (100 μg/ml, 2 hours), actinomycin D (0.05 μg/ml, 4 hours), or α-amanitin (50 μg/ml, 4 hours) to specifically deplete individual nucleic acid species. Treatment with DNase (total DNA), RNase (total RNA), ActD (rRNA), or α-amanitin (mRNA) was performed and localization of NOL7 was confirmed by fluorescent microscopy of the GFP tag.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 10: NOL7 subnuclear localization is dynamically regulated by changes in RNA composition. 293T cells were stably transfected with NOL7-GFP and treated with RNase A (100 μg/ml, 2 hours), DNase I (100 μg/ml, 2 hours), actinomycin D (0.05 μg/ml, 4 hours), or α-amanitin (50 μg/ml, 4 hours) to specifically deplete individual nucleic acid species. Treatment with DNase (total DNA), RNase (total RNA), ActD (rRNA), or α-amanitin (mRNA) was performed and localization of NOL7 was confirmed by fluorescent microscopy of the GFP tag.
Mentions: The shuttling of NOL7 between the nucleus and nucleolus suggested that specific interactions within these compartments may regulate the nucleolar occupancy of NOL7. Due to the highly basic nature of the protein, it was hypothesized that subnuclear localization of NOL7 may be due to interactions with nucleic acids. To investigate, various cell treatments were employed to change the abundance of different nucleic acid species (Figure 10). Cells overexpressing GFP-tagged NOL7 were treated with RNase, DNase, actinomycin D (ActD), or α-amanitin and visualized by fluorescence microscopy for changes in subcellular localization. RNase treatment resulted in nucleolar loss and nucleoplasmic accumulation of NOL7, while cells treated with DNase did not show any significant change. Culture of mammalian cells in low doses of ActD selectively inhibit rRNA synthesis while having no effect on tRNA, 5S rRNA, nuclear RNA and mRNA synthesis [73,74]. Similarly, treatment with low doses of α-amanitin inhibits RNAPII and subsequent mRNA synthesis without affecting the abundances of other RNA species. Loss of these specific RNA species has been shown to selectively deplete their RNA-binding protein counterparts from different cellular compartments, enabling visualization of binding activities that may participate in protein localization [75-77]. Upon treatment with ActD, NOL7 was found to translocate to the nucleoplasm. Upon treatment with α-amanitin, no change in the nucleolar localization of NOL7 was observed. However, the nucleoplasmic localization of NOL7 previously observed was absent. This data suggests that targeting of NOL7 to both the nucleus and nucleolus results in multiple RNA-dependent interactions.

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