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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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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.
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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.

Mentions: Protein occupancy and complex assembly in subnuclear bodies has been shown to relate to function for a majority of proteins [68-70]. Therefore, the nucleolar occupancy of NOL7 was evaluated by FRAP. The occupancy was described by the recovery half life (t1/2) and mobile fraction (Mf) of GFP-fusion constructs. In order to define an upper and lower limit for nucleolar protein mobility, NOL7 was evaluated with the controls NCL, a freely diffusing nuclear/nucleolar shuttle with functions in both the nucleus and nucleolus, and RPS5, a low-mobility resident nucleolar protein (Figure 9A). These proteins represent typical controls within the literature and allow for comparison to other dynamic studies [71]. The t1/2 of NOL7 was found to be most similar to a shuttling protein such as NCL, suggesting that NOL7 can freely exchange with the nucleoplasm (Figure 9B). Conversely, the immobile fraction (Mf) of NOL7 was found to be most similar to an immobile, complexed nucleolar protein like RPS5 (Figure 9B). This is consistent with previous reports describing the nucleolar occupation of a number of nucleolar proteins, including NPM, NCL, and RPS5 [71,72]. This suggests that a large pool of nucleolar NOL7 is functionally occupied in a nucleolar complex, while the free protein is able to shuttle rapidly between subnuclear compartments. Compared to literature reports, this data indicates that NOL7 is most similar to proteins with multiple nuclear and nucleolar roles like NPM, which is both a nucleolar shuttle and associates in functional nucleolar complexes, than either NCL or RPS5. Further, these data suggest that NOL7 shuttles between the nucleolus and nucleoplasm and may play a functional role in both compartments.


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

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

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2957388&req=5

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.
Mentions: Protein occupancy and complex assembly in subnuclear bodies has been shown to relate to function for a majority of proteins [68-70]. Therefore, the nucleolar occupancy of NOL7 was evaluated by FRAP. The occupancy was described by the recovery half life (t1/2) and mobile fraction (Mf) of GFP-fusion constructs. In order to define an upper and lower limit for nucleolar protein mobility, NOL7 was evaluated with the controls NCL, a freely diffusing nuclear/nucleolar shuttle with functions in both the nucleus and nucleolus, and RPS5, a low-mobility resident nucleolar protein (Figure 9A). These proteins represent typical controls within the literature and allow for comparison to other dynamic studies [71]. The t1/2 of NOL7 was found to be most similar to a shuttling protein such as NCL, suggesting that NOL7 can freely exchange with the nucleoplasm (Figure 9B). Conversely, the immobile fraction (Mf) of NOL7 was found to be most similar to an immobile, complexed nucleolar protein like RPS5 (Figure 9B). This is consistent with previous reports describing the nucleolar occupation of a number of nucleolar proteins, including NPM, NCL, and RPS5 [71,72]. This suggests that a large pool of nucleolar NOL7 is functionally occupied in a nucleolar complex, while the free protein is able to shuttle rapidly between subnuclear compartments. Compared to literature reports, this data indicates that NOL7 is most similar to proteins with multiple nuclear and nucleolar roles like NPM, which is both a nucleolar shuttle and associates in functional nucleolar complexes, than either NCL or RPS5. Further, these data suggest that NOL7 shuttles between the nucleolus and nucleoplasm and may play a functional role in both compartments.

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