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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 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.
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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.

Mentions: Database analysis of the full length sequence of human NOL7, utilizing the protein domain prediction programs PSORT [55], TargetP [56], SAPS [57] and NetNES [58], identified five distinct biochemical domains and four putative NLSs within NOL7 (Figure 2). SAPS analysis revealed the existence of four highly basic regions and one acidic domain which correlates with its high pI of 9.7. Stretches of highly basic residues have been shown to participate in nucleic acid binding, nuclear transport, and may contribute to the tumor suppressive function and localization of NOL7 (Figure 2A). While NOL7 lacks homology to other known proteins and domains, the significant sequence conservation among its orthologs suggests a consistent evolutionary role. In particular, four long stretches of basic amino acids are particularly conserved throughout evolution (Figure 2B). Not coincidently, the prediction programs also identified potential NLSs within these basic biochemical domains. However, NOL7 was not predicted to contain any NESs. Putative Sequence (PS) 1, amino acids 1-10 appeared to be a monopartite NLS, while PS2, amino acids 88-112 was predicted to be an example of the recently described tripartite NLS. PS3, amino acids 144-162 appeared to be bipartite NLS and PS4, amino acids 242-257, was predicted to be a bipartite NLS (Figure 2B).


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

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

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

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

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.
Mentions: Database analysis of the full length sequence of human NOL7, utilizing the protein domain prediction programs PSORT [55], TargetP [56], SAPS [57] and NetNES [58], identified five distinct biochemical domains and four putative NLSs within NOL7 (Figure 2). SAPS analysis revealed the existence of four highly basic regions and one acidic domain which correlates with its high pI of 9.7. Stretches of highly basic residues have been shown to participate in nucleic acid binding, nuclear transport, and may contribute to the tumor suppressive function and localization of NOL7 (Figure 2A). While NOL7 lacks homology to other known proteins and domains, the significant sequence conservation among its orthologs suggests a consistent evolutionary role. In particular, four long stretches of basic amino acids are particularly conserved throughout evolution (Figure 2B). Not coincidently, the prediction programs also identified potential NLSs within these basic biochemical domains. However, NOL7 was not predicted to contain any NESs. Putative Sequence (PS) 1, amino acids 1-10 appeared to be a monopartite NLS, while PS2, amino acids 88-112 was predicted to be an example of the recently described tripartite NLS. PS3, amino acids 144-162 appeared to be bipartite NLS and PS4, amino acids 242-257, was predicted to be a bipartite NLS (Figure 2B).

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