Laboratory of Molecular Gerontology, National Institute on Aging, NIH 5600, Nathan Shock Drive, Baltimore, MD 21224, USA
* Author for correspondence (e-mail: vbohr{at}nih.gov)
Accepted 29 July 2002
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Summary |
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Key words: Werner syndrome, Nucleolar targeting sequence, In vivo localization
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Introduction |
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The nuclear import and export of macromolecules depends on energy and
receptor-associated processes (Izaurralde
et al., 1997). Molecules below the exclusion limit size (
50
kDa) can cross the nuclear pore complexes by simple diffusion. However, in the
case of large macromolecules, consensus sequence elements are required for
traffic in and out of the nucleus (nuclear export signal and NLS,
respectively) (for a review, see Gorlich
and Kutay, 1999
). The nucleolus has no known physical barrier
separating it from the nucleoplasm, and in principle, any soluble protein
should be able to diffuse in and out of the nucleoli. However, it has become
clear that many proteins contain sequences that are required for the entry
into the nucleolar region, but the biological functions of these regions are
not yet understood. Perhaps the nucleolar targeting of proteins is related to
direct or indirect interaction with the ribosomal DNA (rDNA) or rRNA
(Carmo-Fonseca et al.,
2000
).
To investigate the regulation of the nucleolar localization of WRN we have used a battery of different WRN regions as EGFP fusion proteins and analyzed their intracellular distribution in living cells by confocal microscopy. Here, we demonstrate the existence of a nuclear targeting sequence (NTS) in the WRN region containing amino acids 949-1092. As expected for a conserved intracellular targeting sequence, we can demonstrate that this NTS is functional in the different human cell lines that we have tested as well as in a mouse cell line. We also show that the nucleolar localization of this domain requires the WRN NLS, although the presence of the NLS alone does not provide the nucleolar targeting of WRN.
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Materials and Methods |
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Transfections, localization in living cells and immunofluorescence
assays
The U-2 OS and B16F10 cells were from ATCC, and the AG11395 cells were from
Coriell. Cells were grown in Dulbecco's modified Eagle's medium (Life
Technologies) and 10% FBS. For in vivo localization assays, the cells were
grown in glass-bottom microwell dishes (MatTek) and transfected with the
corresponding vectors using the Calphos mammalian transfection kit (Clontech).
After 15 hours, cells were viewed under a laser scan confocal microscope
(Zeiss 410) in the green (488 nm) channel (63x NA 1.4 lens). The images
were then overlaid and analyzed with Metamorp imaging system 4.1 (Universal
Imaging Corporation). For the colocalization assays, transfected cells growing
on coverslips (15 hours after transfection) were fixed with 4%
paraformaldehyde in 1x PBS (for 15 minutes at RT), and permeabilized
with 0.4% Triton X-100 in 1x PBS (for 10 minutes at RT). After the
blocking step (0.1% Tween-20, 2% BSA in 1x PBS, for 1 hour at RT),
coverslips were incubated with goat anti-B23 antibodies (Santa Cruz) (1:200
dilution in blocking buffer) for 2 hours at RT. Goat antibodies were then
detected with Texas Red-conjugated anti-goat antibodies (Jackson Laboratories;
1:200 in blocking buffer) for 1 hour at RT. After washing, the coverslips were
mounted on Vectashield (Vector Laboratories) and viewed under a laser scan
confocal microscope (Zeiss 410) in separate channels (green, 488 nm; red, 568
nm). The images were then processed as before.
Western blot analysis for protein expression levels of the different
GFP-WRN fragments
Approximately 15 hours after transfection, cells growing on 10 cm dishes
(80-90% confluent) were collected. The pellets were resuspended in 200
µl/pellet of SDS sample buffer, boiled and analyzed (8% of the sample)
by SDS-PAGE followed by western blot with anti-GFP monoclonal antibody
(Clontech).
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Results |
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After transfection in HeLa cells, EGFP-full length WRN (EGFP-WRN) (Fig. 1A, EGFP I) exhibited two main localization patterns: nucleolar-nuclear diffuse (60-80% of the analyzed cells) (Fig. 2Aa-b) and nuclear foci-nuclear diffuse staining (40-20%) (Fig. 2Ac-d). When the exonuclease and helicase domains together (aa 54-946 +NLS) (Fig. 1A, EGFP VII) were targeted to the nucleus, this construct showed (100% of the cells) a nuclear diffuse staining and interestingly, total nucleolar exclusion (Fig. 2Ae-h). This pattern was also seen when the exonuclease domain alone was targeted to the nucleus with the NLS sequence (data not shown). The C-terminal region of WRN (aa 949-1432) (Fig. 2Ai-l; Fig. 1A, EGFP II), exhibited nuclear diffuse staining and clear nucleolar staining. This result demonstrates that the C-terminal region of WRN contains a NTS. To further map the NTS region within the C-terminal domain, we constructed various deletion mutants in this region. Fig. 2B shows the localization pattern of three representative mutants. An EGFP protein fused to a region of WRN containing the NLS (aa 1358-1432) (Fig. 1A, EGFP VI) consistently showed a clear nuclear diffuse staining (Fig. 2Ba-d). The same nuclear diffuse pattern was seen when the cells were transfected with a longer C-terminal region of WRN that also included the NLS (aa 1072-1432) (Fig. 2Be-h; Fig. 1A, EGFP III). However, a WRN region encompassing amino acids 949-1092, when fused to the NLS (Fig. 1A, EGFP IV), showed clear nucleolar staining (100% of the analyzed cells) (Fig. 2Bi-l). This nucleolar signal was demonstrated by colocalization analysis, using a specific antibody recognizing a nucleolar protein (B23) in the EGFP-WRN (949-1092+NLS)-transfected cells (Fig. 2C). We found 100% colocalization of the EGFP-WRN (949-1092 +NLS) construct with B23, demonstrating that this WRN construct localized specifically to the nucleoli.
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To confirm that the EGFP-WRN fragments used for the mapping of WRN NTS were expressed and were of the expected size, we performed western blot analysis (Fig. 2D). The expression levels of all the fusion proteins were almost identical, except for EGFP-WRN (54-946 +NLS), which was expressed at a lower level (Fig. 2D, lane 8).
The NTS-containing WRN region, when not targeted to the nucleus (without the NLS) (EGFP-WRN 949-1092; Fig. 1A, EGFP V) showed cytoplasmic and nuclear diffuse staining, resembling the localization of EGFP alone (Fig. 3Aa-d). This construct (EGFP-WRN 949-1092) is small enough (<50 kDa) to cross the nuclear pore complex by simple diffusion However, once inside the nucleus, this WRN fragment was not targeted to the nucleoli, indicating that this sequence (aa 949-1092) does not contain nucleolar accumulation properties. Thus, the nucleolar staining of EGFP-WRN (949-1092 +NLS) is an active process, coupled to the nuclear import machinery (WRN-NLS-dependent process). The exonuclease and helicase domains together (aa 54-946) as an EGFP fusion protein without the NLS (Fig. 1A, EGFP VIII), localized completely to the cytoplasm (Fig. 3e-f), which was also expected based on its size (>50 kDa).
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The fragments EGFP V and VIII were expressed with the expected size, as demonstrated by western blot analysis (Fig. 3B).
The same WRN NTS region localizes to nucleoli in other human cell
lines and in mouse cells
We next transfected other cell lines with the WRN constructs to determine
whether our observations were specific to HeLa cells or were more general. We
tested U-2 OS, a telomerase negative cell line, and an SV40-transformed WS
AG11395 cell line, which is telomerase negative and lacks endogenous nuclear
WRN. These cell lines were transfected with EGFP-WRN, EGFP-WRN (1072-1432) and
EGFP-WRN (949-1092 +NLS) constructs. As shown in
Fig. 4, the C-terminal fragment
of WRN (aa 1072-1432) showed the same nuclear diffuse staining (100% of the
analyzed cells) in both the U-2 OS and WS cells
(Fig. 4e-h) as was observed in
the HeLa cells. Full length WRN localized mainly to nuclear foci
(Fig. 4a-d) in the U-2 OS and
WS cells (80% of the analyzed cells). However, we cannot rule out that
some of the nuclear foci are actually within the nucleoli. Importantly, the
EGFP-WRN (949-1092 +NLS) construct, as seen in HeLa cells, accumulated in the
nucleoli of both cell lines (100% of the cells)
(Fig. 4i-l). Similar results
were obtained using the SV40-transformed WS AG07066B cell line (data not
shown). Thus, the nucleolar targeting of this WRN fragment (aa 949-1092) is
independent of the presence of endogenous WRN and of telomerase expression
levels.
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To investigate the species conservation of the WRN NTS, we examined cells from another species. Mouse B16F10 cells were transfected with either EGFP-WRN, EGFP-WRN (1072-1432) or EGFP-WRN (949-1092 +NLS) constructs. After transfection we analyzed the intracellular distribution of each construct as before (living cells). As shown in Fig. 5, full length WRN (EGFP-WRN) showed nuclear diffuse and nuclear foci staining (Fig. 5a-d). Interestingly, full length WRN also showed a clear nucleolar exclusion pattern. The NLS-containing C-terminal fragment of WRN (aa 1072-1432) was distributed throughout the nucleus (80-90% of the analyzed cells), with some nucleolar exclusion (20-10%) (Fig. 5e-h). In contrast, the NTS-containing region (aa 949-1092 +NLS) was efficiently targeted to the nucleoli in all the analyzed cells (Fig. 5i-l). Thus, this domain of the human WRN protein is as active as an NTS in a mouse cell line.
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Nucleolar exclusion pattern of a WRN RQC (RecQ conserved) domain
deletion mutant
To further demonstrate that the WRN region comprising amino acids 949-1092
contains a NTS, we generated an EGFP-tagged RQC domain deletion mutant of WRN
(EGFP-WRN853-1089) and transfected it into HeLa cells
(Fig. 6A,B). After
transfection, 80% of the analyzed cells showed nuclear diffuse staining, and
more interestingly, total nucleolar exclusion of this construct (100% of the
analyzed cells). Thus, the presence of an active NLS in the C-terminal domain
of WRN does not alone provide the targeting of the protein to the nucleoli.
The nucleolar targeting is dependent on the WRN RQC domain (aa 853-1089),
specifically on amino acids 949-1092, as shown before.
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Discussion |
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The human WRN sequence contains a NTS (aa 949-1092) that directs the protein to the nucleoli in a number of human cells and that is recognized by the nucleolar targeting machinery in rodent cells. However, when this sequence is expressed in the context of the full length protein (EGFP-WRN), it is unable to target the protein to the nucleolus in the mouse cell line we studied. This situation resembles the differences in the WRN localization that we observed between the human cell lines. The full length protein (EGFP-WRN) showed predominantly a nucleolar diffuse staining in HeLa cells, whereas in ALT cells (U-2 OS and AG11395 cell lines) it was principally in nuclear foci (compare Fig. 2Aa-d with Fig. 4a-d). In contrast, the EGFP-WRN (949-1092 +NLS) construct was efficiently targeted to the nucleoli in all cell lines tested. In agreement with these data, a WRN RQC deletion mutant (containing an active NLS) showed a nuclear diffuse and nucleolar exclusion pattern (Fig. 6A,B). This result clearly demonstrates that the RQC domain of WRN contains a NTS. The NLS also needs to be present for the protein to reach the nucleolus, but the NLS alone will not target WRN to the nucleolus (Fig. 2B).
Conformational changes (post-translational modifications) may render some
regions of WRN more accessible and this may be a determining factor in
targeting of WRN to different intranuclear structures or may affect the
binding of WRN to interacting proteins. Support for this hypothesis is found
in a previous study (Gray et al.,
1998), which suggests that tyrosine phosphorylation could modulate
the intranuclear trafficking of WRN by either direct (WRN phosphorylation) or
indirect (phosphorylation of a WRN interacting protein) modification.
Basic components of the intracellular protein trafficking machinery are
conserved among different species (i.e. NLS and nuclear export signals)
(Dimaano et al., 2001;
Schmitt et al., 1999
;
Jans et al., 2000
). Thus,
specific intracellular targeting sequences would be expected to function in
different species and cell lines, as is the case of the WRN NTS presented
here.
Our studies were conducted in living cells. A previous study
(Suzuki et al., 2001) was
performed with fixed cells and reported that two amino acids (1403-1404) in
the C-terminal region of WRN contains the NTS, but no other WRN regions were
analyzed. A high concentration of formaldehyde (10%) was used in the fixation
protocol in that study (Suzuki et al.,
2001
). In our hands, the fixation protocol greatly affects the
nucleolar localization of the proteins, and we therefore argue that it is of
importance to perform this type of analysis in live cells.
The role of WRN in the nucleoli is unclear. WRN may participate in
nucleolar processes such as transcription or it may be located there for
temporal storage (sequestration), a phenomenon that has been observed for
other proteins (Visintin and Amon,
2000). This sequestration could prevent proteins from reaching
their targets in other cellular compartments. We have previously shown that
WRN is involved in RNA-polymerase-II-directed transcription, but not in RNA
polymerase I transcription in vitro
(Balajee et al., 1999
). While
the role of WRN in the nucleoli may not involve rDNA transcription, WRN may
function in other rDNA/rRNA metabolic processes.
It has been suggested previously that the nucleolar localization of
proteins depends upon the presence of an active NLS
(Creancier et al., 1993), thus
linking the processes of nuclear import and nucleolar targeting; our results
support this notion. It is likely that eukaryotic cells have evolved a
mechanism that discriminates between simple nonspecific diffusion and active
targeting of proteins to different subnuclear structures.
In summary, using four different cell lines and a battery of different EGFP-WRN fragments, we have demonstrated that amino acids 949-1092 of WRN contain an NTS that is responsible for the targeting and also requires the presence of a NLS. Supporting this data, we also showed that a WRN RQC deletion mutant (lacking amino acids 853-1089) exhibited a complete nucleolar exclusion pattern. The experiments were carried out in living cells. The 949-1092 WRN region not only targets the WRN protein to the nucleolus, but binds (and modulates) some of the WRN-interacting proteins, playing a central role(s) in the regulation and intracellular trafficking of WRN. It is possible that one of the protein binders to this WRN region is specifically involved in the transport to the nucleoli.
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Acknowledgments |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Balajee, A. S., Machwe, A., May, A., Gray, M. D., Oshima, J.,
Martin, G. M., Nehlin, J. O., Brosh, R., Orren, D. K. and Bohr, V. A.
(1999). The Werner syndrome protein is involved in RNA polymerase
II transcription. Mol. Biol. Cell
10,2655
-2668.
Brosh, Jr, R. M., von Kobbe, C., Sommers, J. A., Karmakar, P.,
Opresko, P. L., Piotrowski, J., Dianova, I., Dianov, G. L. and Bohr, V. A.
(2001). Werner syndrome protein interacts with human flap
endonuclease 1 and stimulates its cleavage activity. EMBO
J. 20,5791
-5801.
Carmo-Fonseca, M., Mendes-Soares, L. and Campos, I. (2000). To be or not to be in the nucleolus. Nat. Cell Biol. 2,E107 -E112.[CrossRef][Medline]
Creancier, L., Prats, H., Zanibellato, C., Amalric, F. and Bugler, B. (1993). Determination of the functional domains involved in nucleolar targeting of nucleolin. Mol. Biol. Cell 4,1239 -1250.[Abstract]
Dimaano, C., Ball, J. R., Prunuske, A. J. and Ullman, K. S.
(2001). RNA association defines a functionally conserved domain
in the nuclear pore protein nup153. J. Biol. Chem.
276,45349
-45357.
Gorlich, D. and Kutay, U. (1999). Transport between the cell nucleus and the cytoplasm. Annu. Rev. Cell Dev. Biol. 15,607 -660.[CrossRef][Medline]
Gray, M. D., Shen, J. C., Kamath-Loeb, A. S., Blank, A., Sopher, B. L., Martin, G. M., Oshima, J. and Loeb, L. A. (1997). The Werner syndrome protein is a DNA helicase. Nat. Genet. 17,100 -103.[Medline]
Gray, M. D., Wang, L., Youssoufian, H., Martin, G. M. and Oshima, J. (1998). Werner helicase is localized to transcriptionally active nucleoli of cycling cells. Exp. Cell Res. 242,487 -494.[CrossRef][Medline]
Huang, S., Li, B., Gray, M. D., Oshima, J., Mian, I. S. and
Campisi, J. (1998). The premature ageing syndrome protein,
WRN, is a 3'5' exonuclease. Nat.
Genet. 20,114
-116.[CrossRef][Medline]
Izaurralde, E., Kutay, U., von Kobbe, C., Mattaj, I. W. and
Gorlich, D. (1997). The asymmetric distribution of the
constituents of the Ran system is essential for transport into and out of the
nucleus. EMBO J. 16,6535
-6547.
Jans, D. A., Xiao, C. Y. and Lam, M. H. (2000). Nuclear targeting signal recognition: a key control point in nuclear transport? Bioessays 22,532 -544.[CrossRef][Medline]
Marciniak, R. A., Lombard, D. B., Johnson, F. B. and Guarente,
L. (1998). Nucleolar localization of the Werner syndrome
protein in human cells. Proc. Natl. Acad. Sci. USA
95,6687
-6892.
Matsumoto, T., Shimamoto, A., Goto, M. and Furuichi, Y. (1997). Impaired nuclear localization of defective DNA helicases in Werner's syndrome. Nat. Genet. 16,335 -336.[Medline]
Sakamoto, S., Nishikawa, K., Heo, S. J., Goto, M., Furuichi, Y.
and Shimamoto, A. (2001). Werner helicase relocates into
nuclear foci in response to DNA damaging agents and co-localizes with RPA and
Rad51. Genes Cells 6,421
-430.
Schmitt, C., von Kobbe, C., Bachi, A., Pante, N., Rodrigues, J.
P., Boscheron, C., Rigaut, G., Wilm, M., Seraphin, B., Carmo-Fonseca, M. and
Izaurralde, E. (1999). Dbp5, a DEAD-box protein required for
mRNA export, is recruited to the cytoplasmic fibrils of nuclear pore complex
via a conserved interaction with CAN/Nup 159p. EMBO J.
18,4332
-4347.
Suzuki, N., Shimamoto, A., Imamura, O., Kuromitsu, J., Kitao,
S., Goto, M. and Furuichi, Y. (1997). DNA helicase activity
in Werner's syndrome gene product synthesized in a baculovirus system.
Nucleic Acids Res. 25,2973
-2978.
Suzuki, T., Shiratori, M., Furuichi, Y. and Matsumoto, T. (2001). Diverged nuclear localization of Werner helicase in human and mouse cells. Oncogene 20,2551 -2558.[CrossRef][Medline]
Visintin, R. and Amon, A. (2000). The nucleolus: the magician's hat for cell cycle tricks. Curr. Opin. Cell Biol. 12,752 .[CrossRef][Medline]
Yankiwski, V., Marciniak, R. A., Guarente, L. and Neff, N.
F. (2000). Nuclear structure in normal and Bloom syndrome
cells. Proc. Natl. Acad. Sci. USA
97,5214
-5219.
Yu, C. E., Oshima, J., Fu, Y. H., Wijsman, E. M., Hisama, F., Alisch, R., Matthews, S., Nakura, J., Miki, T., Ouais, S. et al. (1996). Positional cloning of the Werner's syndrome gene. Science 272,258 -262.[Abstract]