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First published online December 11, 2006
doi: 10.1242/10.1242/jcs.03292

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Multiple controls regulate nucleostemin partitioning between nucleolus and nucleoplasm

¹ Center for Cancer and Stem Cell Biology, Alkek Institute of Biosciences and Technology, Texas A&M Health Science Center, 2121 W Holcombe Blvd, Houston, TX 77030-3303, USA

View larger version (42K): [in this window] [in a new window]   Fig. 1. Nucleostemin containes two distinct nucleolus-targeting regions with different nucleolar retention time. (A) Diagram of nucleostemin (NS) protein structure and mutant constructs used to determine the nucleolus-targeting domains of nucleostemin. An SV40 nuclear localization signal (NLS, black circle) was introduced in mutants that lack an endogenous NLS (black boxes). Numbers indicate the aa positions. B, basic; C, coiled-coil; G, GTP-binding; I, intermediate; A, acidic. (B) Subcellular distributions of mutant proteins in U2OS cells were revealed by a C-terminal GFP-tag; counterstaining with anti-B23 antibody is shown in the right upper quadrants in all panels on a 50% scale. Both the B- and the G-domains (nlsG) displayed a nucleolar distribution pattern. Mutation of the conserved GTP-binding residue G256, yielding nlsG(256), did not affect the nucleolar localization of the G-domain alone. In the presence of the I-domain, such a mutation [nlsGI(256)] abolished its nucleolar localization. A cytoplasmic hydrolase protein (Hd3) was tagged with the SV40 NLS to demonstrate that this sequence was not sufficient to confer nucleolar localization. Bar, 10 µm. (C1) FRAP recovery times (x-axis, in seconds) of the B-domain (trace 1), the full-length nucleostemin (trace 2) and the GI-domain (trace 3) were determined in CHO cells transiently transfected with the GFP-fusion constructs. The y-axis represents the percentage of fluorescence intensity in the bleached area relative to the prebleached intensity. (C2) The FRAP recovery time of the GI-domain (nlsGI) and the A-domain deletion mutant (NSdA) was measured as described in the Materials and Methods. (C3) Statistical analyses at 5, 10, 20 and 30 seconds (mean ± standard error of mean (s.e.m.), n=20).

View larger version (85K): [in this window] [in a new window]   Fig. 2. The nucleolar localization of nucleostemin is gated by a nucleoplasmic-retaining mechanism independent of its nucleolus-targeting domains. (A1-E2) When fused to the N-terminus (A1) or the C-terminus (B1) of B23, the I-domain of nucleostemin significantly increased the amount of B23 in the nucleoplasm compared with the epitope-tagged (A2, B2) or the GFP-tagged proteins (A3, B3) at their respective ends. This nucleoplasmic-retaining activity of the I-domain could also be transferred to three ribosomal proteins, L5, L11 and L23. Unlike the nucleolar distributions of their original proteins (C2, D2, E2), the I-domain fusions of these proteins (C1, D1, E1) localized almost exclusively in the nucleoplasm. Anti-fibrillarin or anti-B23 immunostainings of the same cells are shown in the bottom panels. Fusion constructs are depicted at the bottom of each panel. Bars, 10 µm.

View larger version (39K): [in this window] [in a new window]   Fig. 3. Nucleostemin interacts with RSL1D1. (A) Protein sequences of mouse RSL1D1 (GenBank accession no. NM_025546) and two closely related genes, L10A (GenBank accession no. NM_011287), and L10 (GenBank accession no. XM_138143), were aligned by the Clustal W (1.81) program. Shaded and underlined areas represent the ribosomal L1 and the coiled-coil domain, respectively. Three putative NLS are marked in bold. *, fully conserved residues;:, conservation of strong groups;., conservation of weak groups. Biochemical interaction between nucleostemin and RSL1D1 was shown by affinity binding assays using a GST-RSL1D1 fusion protein to pull down HA-tagged nucleostemin (B1) or a GST-nucleostemin fusion protein to pull down HA-tagged RSL1D1 (B2). (C) The nucleostemin-RSL1D1 interaction was confirmed by coimmunoprecipitation in both directions. HEK293 cells were co-transfected with HA-tagged nucleostemin and Myc-tagged RSL1D1 and immunoprecipitated with anti-Myc antibody (rows 1 and 2, left column, -myc), anti-HA antibody (rows 3 and 4, rows, left column, -HA), or mouse IgG (rows 1-4, right column). The co-purified proteins (rows 1 and 3) and the immunoprecipitates (rows 2 and 4) were detected by immunoblotting with the indicated polyclonal antibodies. (D) Left panel: Myc-tagged RSL1D1 was co-purified with endogenous nucleostemin in HEK293 cells by nucleostemin antiserum (-NS), but not by preimmune serum (Cntrl). Right panel: endogenous nucleostemin was also be co-purified with Myc-tagged RSL1D1 by anti-Myc antibody (-myc) but not by mouse IgG.

View larger version (88K): [in this window] [in a new window]   Fig. 4. Tissue and subcellular distributions of RSL1D1 overlap with those of nucleostemin. (A,B) Northern blot analyses of expression patterns of RSL1D1 and nucleostemin in the developing whole embryos from E10.5 to E16.5 (A) and in the adult mice (B). (C1-E3) Double-labeled immunofluorescence and confocal analyses showing colocalization of endogenous nucleostemin (NS, red) and Myc-tagged RSL1D1 (green) (C), NS (red) and B23 (green) (D), NS (red) and fibrillarin (Fib, green) (E). High magnifications of the indicated areas (squares) are shown in (C2, D2 and E2). Dashed lines delineate the nucleo-cytoplasmic boundaries. Bars, 5 µm for C1, D1, E1; 2 µm for C2, D2 and E2. Quantification of colocalization is shown in C3, D3 and E3. All pixels were plotted on the basis of their red (y-axis) and green (x-axis) fluorescence intensities, and pseudocolored on the basis of the event frequency, with red representing the highest and blue the lowest event frequency.

View larger version (28K): [in this window] [in a new window]   Fig. 5. RSL1D1 has a longer nucleolar retention time than nucleostemin. (A) Time-sequenced FRAP images of nucleostemin and RSL1D1 in the nucleolus. A circle of 1 µm in diameter within the nucleolus (arrows) was bleached. Of note, low intensity spots in the upper panels (*) existed before photobleaching. Numbers indicate the time in seconds after the bleaching event. Bars, 1 µm. (B) The FRAP recovery curves of RSL1D1 and nucleostemin depict the average of the fluorescence recovery level (y-axis; n=20) relative to the prebleached intensity (set as 1) over a 31.6-second period following photobleaching (x-axis) in seconds. Error bars represent ± standard deviations (±s.d.) and are omitted on the top and bottom side of the RSL1D1 and nucleostemin recovery curves for clarity. (C) t-test analyses of the FRAP results were conducted at 5, 10, 20 and 30 seconds after photobleaching (mean ± s.e.m.; n=20).

View larger version (28K): [in this window] [in a new window]   Fig. 6. RSL1D1 interacts with the B- and G-domains of nucleostemin. (A) Schematic diagrams of truncated nucleostemin mutants used to determine RSL1D1-interacting domain(s). (B) Affinity binding assays showing that GST fusions of RSL1D1 are able to pull down all single-domain deletion mutants of nucleostemin, suggesting involvement of multiple regions. (C) The use of truncation mutants showed that RSL1D1 binds both the BC- and the GI-domains, but not the G- and IA-domains or a GI-domain containing a G256V mutation (GI(256)). (D) Nucleostemin RSL1D1-binding domains were further narrowed down to the B-domain of the BC mutant (D1) and the GI1-domain of the GI mutant (D2). Double-deletion mutants (NSdBG and NSdCI) confirmed the importance of the B- and G-domains, but not of the C- and I-domains, in mediating the interaction of nucleostemin and RSL1D1 (D2).

View larger version (104K): [in this window] [in a new window]   Fig. 7. Nucleolar distribution and the nucleostemin-interaction of RSL1D1 are controlled by separate domains. (A) RSL1D1 contains an L1 domain (aa 150-254), a coiled-coil domain (C) and three putative NLS (black boxes). Myc-tagged truncated RSL1D1 mutants were generated to map nucleostemin-interacting and nucleolus-targeting regions. (B) Affinity binding assays show that GST fusions of both the BC- and GI-domains bind the (aa 317-452) part of RSL1D1 that does not contain the L1- or C-domain. (C-L) Anti-Myc and anti-nucleostemin double-labeled immunofluorescence demonstrate that the 150-316 aa region of RSL1D1 is localized in the nucleolus (C). The N-terminal 1-149 aa region is cytoplasmic by itself (D), and becomes partially nucleolar when tagged with an SV40 NLS (E). Distribution of the C-terminal 317-452 aa region is diffuse in the nucleus (F), with some cells showing more signals in the nucleolus than in the nucleoplasm (G). Within the aa 150-316 segment, the L1 domain (150-254) by itself is primarily cytoplasmic (H), but becomes mostly nucleolar when fused to an SV40 NLS (I,J, nlsL1). The coiled-coil domain (255-316) is diffusely localized in the nucleus (K). The nucleostemin signal is diminished or absent from the nucleolus of many cells expressing nlsL1 (J). Bars, 10 µm.

View larger version (60K): [in this window] [in a new window]   Fig. 8. Overexpression of a nucleolar form of the L1-domain (nlsL1) disperses nucleostemin from the nucleolus. (A1) The intensities of nucleostemin signals in the nucleolus are diminished or disappear in many cells that express the nlsL1 mutant. (A2) High magnification of the nlsL1-expressing cell show that its remaining nucleostemin signals display a reticular pattern of distribution, similar to the nucleostemin distribution in wild-type cells. (B) In some cells, nucleostemin is scattered around the nlsL1 signals. Overexpression of the nlsL1 mutant does not affect the signal intensities or the distribution patterns of fibrillarin (C) or B23 (D). Bars, 10 µm for A1; 5 µm for A2, B, C, D.

View larger version (37K): [in this window] [in a new window]   Fig. 9. Loss of RSL1D1 expression decreases the compartmental size and protein amount of nucleostemin and B23 in the nucleolus. (A1) The knockdown efficiency of the RSL1D1-specific siRNA duplex (siRSL1D1) was examined at the RNA and protein levels. Compared with samples treated with the control siRNA duplex (siNEG), siRSL1D1 can reduce the endogenous RSL1D1 mRNA by 73% (top panel), and the exogenously expressed Myc-tagged RSL1D1 protein by 43% in HEK293 cells (bottom panel). The siRSL1D1 treatment does not affect the total amount of nucleostemin protein (A2). Tub, ß-tubulin for northern blots (NB), and -tubulin for western blots (WB). The effect of a partial loss of RSL1D1 expression on the nucleolar distribution of nucleostemin was measured in U2OS cells double-labeled with anti-nucleostemin (Ab2438) and anti-B23 antibody. Quantitative analyses show that a partial knockdown of RSL1D1 expression decreased the total nucleolar area (No) occupied by nucleostemin (B1) (P<0.001, n=130). A similar effect was seen in the B23-containing regions (B2). On the y-axis the percentage of cells at or below the size indicated on the x-axis is shown. On the x-axis the nucleoluar area in units of 100 pixels (equals 0.89 µm2) is shown. (C) Immunofluorescence images representative of the average of each group are shown. Dashed lines delineate the nucleo-cytoplasmic boundaries. Bars, 5 µm.

View larger version (50K): [in this window] [in a new window]   Fig. 10. Diagram of the mechanism controlling nucleostemin distribution between the nucleolar and nucleoplasmic compartments, and the effects of RSL1D1 knockdown and nlsL1 overexpression. Nucleostemin in the GTP-unbound state is blocked from entering the nucleolus by a nucleoplasmic-retaining mechanism that acts on the I-domain. GTP binding releases this lock and allows nucleostemin to move into the nucleolus. Nucleostemin interacts with nucleolar protein RSL1D1 through the nucleolus-targeting B- and G-domains. When not bound by GTP, the GI-domain fails to interact with RSL1D1, suggesting a link between the nucleolar exit of nucleostemin and GTP hydrolysis. RSL1D1 co-resides with nucleostemin in subnucleolar domains surrounding fibrillarin. A partial knockdown of RSL1D1 expression reduces the compartmental size and, to a lesser extent, the protein amount of nucleostemin in the nucleolus, supporting the idea that RSL1D1 provides the nucleolar binding site for nucleostemin. Overexpression of nlsL1 disperses nucleostemin signals from the nucleolus by occupying the nucleolar binding sites for the endogenous RSL1D1 capable of interacting with nucleostemin. NoLS, nucleolar localization sequence(s); NOR, nucleolar organization region; Fib, fibrillarin.

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