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. 2010 Sep 27:11:74.
doi: 10.1186/1471-2121-11-74.

Identification and functional analysis of NOL7 nuclear and nucleolar localization signals

Guolin Zhou  1 , Colleen L Doçi, Mark W Lingen
Affiliations

Identification and functional analysis of NOL7 nuclear and nucleolar localization signals

Guolin Zhou et al. BMC Cell Biol. .

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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Figures

Figure 1
Figure 1
NOL7 requires cytosolic factors for efficient nuclear localization. HeLa cells permeabilized with digitonin were incubated at 4°C or 37°C as indicated with (+) or without (-) full length NOL7 expressing a C-terminal GFP tag, cytosol, heat-inactivated cytosol, ATP, or WGA. Localization of NOL7 was confirmed by visualization of GFP.
Figure 2
Figure 2
NOL7 is composed of distinct biochemical domains and multiple putative NLSs that show evolutionary conservation. (A) Multiple sequential analysis programs confirmed the existence of four basic (blue) and one acidic (green) region in the full-length sequence of NOL7. Putative NLSs identified in sequence analysis programs are shown in red. (B) Sequence conservation between human NOL7 and its putative orthologs was analyzed for each of the putative NLSs and the alignment is shown. Black shaded boxes indicate identical amino acid conservation, while grey boxes signify similar amino acids to Homo sapiens. Numbers correspond to residues within the RefSeq sequences listed in the Materials and Methods 2.7.
Figure 3
Figure 3
NOL7 contains three separate NLSs that are necessary for nuclear localization. (A) Schematic representation of deletion constructs of NOL7 used to determine which regions of NOL7 are required for nuclear localization. Results as demonstrated in (B) are summarized in the column to the right, where "No" designates nucleolar localization, "Np" designates nucleoplasmic localization, and "C" designates cytoplasmic localization. (B) Localization of the constructs was confirmed in HeLa cells by immunofluorescence using an α-HA primary and FITC-coupled secondary antibody.
Figure 4
Figure 4
NOL7 contains three NLSs that are sufficient for nuclear localization. (A) Schematic representing the three different NLSs cloned in-frame with the cytoplasmic protein PK bearing a c-myc tag. Results demonstrated in (B) are summarized in the column to the right, where "Np" designates nucleoplasmic localization and "C" designates cytoplasmic localization. (B) Localization of the constructs in Hela cells was confirmed by immunofluorescence using an α-myc primary and Cy-3 conjugated secondary antibody. Costaining of the nucleus with DAPI is shown in blue.
Figure 5
Figure 5
Basic residues within each of the NLSs are required for nuclear localization of PK. (A) Schematic representing the three different NLSs bearing neutralizing mutations in the basic residues were cloned in-frame with the cytoplasmic protein PK bearing a c-Myc tag. Results demonstrated in (B-D) are summarized in the column to the right, where "Np" designates nucleoplasmic localization and "C" designates cytoplasmic localization. (BD) Subcellular localization was determined by immunofluorescence in HeLa cells using an α-myc primary and either a HRP or Cy-3 conjugated secondary antibody. Costaining of the nucleus with DAPI is shown in blue. (B) Mutations in either the first (NLS1-sub1), second (NLS1-sub2), or third (NLS1-sub3) basic cluster of NLS1 were mutated in alanine and visualized for nuclear localization. (C) Expression and localization of NLS2 mutants lacking either the first (NLS2-sub1), second (NLS2-sub2), or third (NLS2-sub3) basic cluster. (D) Expression and localization of the constructs bearing mutations in the first (NLS3-sub1) or second (NLS3-sub2) basic cluster of NLS3.
Figure 6
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.
Figure 7
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.
Figure 8
Figure 8
Basic residues within NLS2 and NLS3 are required for nucleolar localization of NOL7. Basic residues within each of the NLSs are required for nucleolar localization of full length NOL7. A) Schematic representation of the different mutant constructs used to evaluate nucleolar localization. Results demonstrated in (B) are summarized in the column on the right, where "No" designates nucleolar localization and "Np" designates nucleoplasmic localization. B) Localization of the constructs in HeLa cells was confirmed by GFP visualization. Costaining of the nucleus with DAPI is shown in blue.
Figure 9
Figure 9
FRAP analysis of NOL7 nucleolar occupancy demonstrates rapid recovery but low mobility within the nucleolus. (A) The fluorescence recovery within the nucleolus was measured over time for HeLa cells transfected with GFP-tagged NOL7, the high mobility shuttle NCL, and the low mobility resident protein RPS5. Measurements represent thirteen different cells per protein. The curves were fit to the line curve F(t) = F∞(1-eτ·t). (B) The nucleolar occupancy was plotted as a function of recovery versus mobility. The mobile fraction was used as a measurement of free versus complexed protein within the nucleolus and calculated from the regression values in (A) using the formula Mf = (F∞-F0)/(Fi-F0). The half-time to maximal recovery was calculated using the formula t1/2 = ln(0.5)/τ and used as a measurement of shuttling between the nucleolus and nucleoplasm. All error bars represent the standard error of the measurement.
Figure 10
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.
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