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BLM (the Causative Gene of Bloom Syndrome) Protein Translocation into the Nucleus by a Nuclear Localization Signal
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
240, 348–353 (1997)
RC977648
BLM (the Causative Gene of Bloom Syndrome) Protein Translocation into the Nucleus by a Nuclear Localization Signal Hideo Kaneko,* Koji O Orii,* Eiko Matsui,* Nobuyuki Shimozawa,* Toshiyuki Fukao,* Takehisa Matsumoto,† Akira Shimamoto,† Yasuhiro Furuichi,† Seiro Hayakawa,* Kimiko Kasahara,* and Naomi Kondo* *Department of Pediatrics, Gifu University School of Medicine, Tsukasa-machi 40, Gifu 500, Japan; and †AGENE Research Institute, 200 Kajiwara, Kamakura-shi, Kanagawa 247, Japan
Received October 13, 1997
gree of recombination between homologous chromosome is increased. An abnormally high number of sister chromatid exchanges are observed. In 1995 the causative gene for BS was identified using somatic crossover point mapping and termed BLM (7). BLM is a 4437 bp cDNA that encodes a 1417 amino acid peptide which is homologous to ATP-dependent DNA helicases. DNA helicases are the enzymes which catalyze the unwinding of double-stranded DNA to provide single-stranded templates for the processes of replication, repair, recombination and transcription. BLM is a member of the RecQ helicase family, consisting of human WRN, RECQL and yeast Sgs1 (8). WRN was identified as the causative gene for Werner’s syndrome (WS), a premature aging disorder (9). There is considerable structural and sequence divergence between these proteins outside of the helicase domains (10). Most strikingly, BLM protein and Sgs1 share a serine rich, highly charged amino-terminal domain of approximately 575 amino acids, which is completely absent from both RecQ and RECQL. Lu et al. reported that site-directed mutations that eliminate helicase activity of yeast Sgs1 can still complement certain Sgs1 mutants (11). These results suggest the region other than helicase domain has the unique function in a member of the helicase family. Nuclear proteins accumulate in the nucleus because they contain nuclear targeting signals (NLS) that allow selective entry through nuclear pore complex. NLS is essential for the transport of proteins into the nucleus. The seven-amino-acid nuclear targeting sequence of the SV40 large T antigen has been regarded as the model NLS (12,13). Digwall et al. reported that the Bloom syndrome (BS) is characterized by stunted NLS of nucleoplasmin consists of two essential dogrowth, sun sensitivity and immunodeficiency (1-4). So- mains of basic amino acids that function in an interdematic cells from subjects with BS accumulate excessive pendent manner and a 10 amino acid spacer region numbers of mutations, including those of both coding that separates the two basic domains and which when sequences and noncoding repetitive DNA (5,6). The de- mutated gives rise to null phenotypes (14). In a dataBloom syndrome (BS) is a rare genetic disorder characterized by small body size, sun sensitivity, immunodeficiency and a high predisposition to various types of cancer. BLM was identified as the causative gene for BS, and BLM protein is homologous to DNA helicase. There are two putative nuclear localization signals (NLSs) within amino acid residues 1334–1349 in the Cterminus of the BLM protein, which has the distinctive structure of two basic residue arms separated by a spacer. The entire coding or deleted BLM sequences of various sizes were ligated into an enhanced green fluorescent protein (EGFP) vector and transfected into HeLa cells. The EGFP vector harboring the entire BLM coding sequence was transported to the nucleus. The BLM protein truncated at 1341 amino acid, containing an intact helicase domain and only one proximal arm, was not transported to the nucleus. The BLM protein truncated at 1357 amino acid, containing an intact helicase domain and two arms, was transported to the nucleus. The EGFP vector harboring DNA fragments encoding a protein having only the distal arms of basic amino acids in the C-terminus was also transported to the nucleus. The truncated BLM proteins corresponding to previously reported mutated BLM proteins were retained in the cytoplasm or both the cytoplasm and the nucleus as was the EGFP vector with no insert. These results show that the BLM protein translocates into the nucleus and that the distal arm of the bipartite basic residues in the C-terminus of the BLM protein is essential for targeting the nucleus. q 1997 Academic Press
0006-291X/97 $25.00
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base search of BLM amino acid sequences, we found amino acid sequences which exhibit a high degree of homology to bipartite NLS in its C-terminus. To clarify the precise function of the BLM protein, we investigated the subcellular localization and NLS of the BLM protein using EGFP fusion proteins (15,16). In this study we revealed that the BLM protein has a functional NLS in its C-terminal and is transported to the nucleus. MATERIALS AND METHODS Construction of EGFP fusion proteins. The EGFP fusion proteins were expressed transiently in HeLa cells that were transfected with expression vectors constructed from pEGFP-C3 (Clontech, Palo Alto, CA) and various length of BLM cDNA. Full-length BLM cDNA was cloned from mRNA obtained from the HeLa cells. Truncated BLM fragments were amplified from the full-length BLM cDNA using pfu DNA polymerase (Stratagene, La Jolla, CA) with 10 PCR cycles. Primer sets using in this experiments are listed as follows. Fragments P-1 to P-11 were amplified by combining X-S primer with P1 to P11 primers. Fragments of X-1 to X-3 were amplified by combining the X-1 to X-3 primers with the P-1 primer. The number in the primers is the position in cDNA of BLM. (forward) X-S 5*-CCG CTC GAG AGG ATT ATG GCT GCT GTT-3 * (69-86) X-1 5*-CCG CTC GAG GAA ATA CCC GTA TCT-3 * (4021-4040) X-2 5*-CCG CTC GAG GCC TCC CAA AGG TCT AAG-3 * (4095-4112) X-3 5*-CCG CTC GAG GCT TCC AGT GGT TCC AAG-3 * (4125-4142) (reverse) P-1 5*-AA CTG CAG TTA TGA GAA TGC ATA TGA AGG C-3* (4307-4328) P-2 5*-AA CTG CAG TGC CTT GGA ACC ACT GGA AGC-3* (4135-4155) P-3 5*-AA CTG CAG GGC TGG CAT CTT TTT CCT-3* (4079-4097) P-4 5*-AA CTG CAG GGT ATT TCC TCG TCA AGC TC-3* (4014-4034) P-5 5*-AA CTG CAG GGA ATT TTC TGT TTC CAT-3* (3641-3659) P-7 5*-AA CTG CAG GTC CTT CTG TAC CCT GGG-3* (2577-2593) P-8 5*-AA CTG CAG ATT AGT TCT AAA ATT ATG-3* (2069-2087) P-9 5*-AA CTG CAG TCC TAG TCT TGG TGT TTC-3* (1592-1610) P-10 5*-AA CTG CAG CTT TTC GCT ATT ATC TCT-3* (869-887) P-11 5*-AA CTG CAG TTT CTG AGC AGT GCT TAC-3* (611-629)
Amplified DNA fragments were digested by XhoI and PstI and were inserted in frame at the XhoI/PstI sites of the pEGFP plasmid downstream of the EGFP coding region, which was confirmed by dye-terminator sequencing (Applied Biosystemes, Chiba, Japan) using pEGFP specific primers. Plasmid DNA was prepared using the Qiagen kit (Qiagen, Hilden, Germany) and purified by CsCl ultracentrifugation. Electroporation. HeLa cells (5 1 106) were transfected with 10 mg of BLM fragments inserted into pEGFP plasmid by electroporation (Bio-Rad, Hercules, CA) and were seeded onto a cover glass. After 12 hrs, the cells were washed with PBS. After 24 hrs, the cells were fixed with 4% paraformaldehyde in PBS for 1 hour and washed with PBS. A cover glass was mounted with 0.2 mg/ ml of 4*,6-diamidino-2-phenylindole (Sigma, St. Louis, MO) in antifade solution. The cells were visualized by fluorescence microscopy.
RESULTS Putative NLS in the C-Terminus of BLM By investigating BLM protein amino acids sequences, we identified a putative bipartite NLS in the C-terminus. Close examination of the BLM protein re-
vealed that there are two arms of basic amino acids in the C-terminus, proximal arm: Arg (R)-Lys (K)-Arg (R)Lys (K)-Lys (K) (1334-1338) and distal arm: Arg (R)Ser (S)-Lys (K)-Arg (R)-Arg (R)-Lys (K) (1344-1349) (Fig. 1a). Figure 1a shows that these bipartite basic amino acids stretches are conserved in a number of known NLSs (14,17). Identification of NLS in BLM We prepared the primers for amplifying various lengths of BLM (Fig. 1b). Each amplified fragment corresponded to a putative NLS, or helicase domain or previously reported nonsense mutations. The fragments P-9, P-10 and P-11 were produced to correspond to the mutations previously reported in the Bloom’s Syndrome Registry, designated 97, 112 and 93, respectively (7). The predicted peptides of frameshift mutation observed in registry No. 15, 42, 107 and Nr2 were 739 amino acid residues long and were located between the P-7 and P-8 fragments. EGFP from the jellyfish Aequorea victoria yields a strong fluorescent signal in heterologous cell types and has been used as a marker of gene expression and for visualizing subcellular organelles and protein translocation in living cells. The EGFP vector harboring the entire coding sequence of BLM, P1, was transfected into HeLa cells, which express BLM well (data not shown), and EGFP was localized in the nucleus (Fig. 2a). Nuclear localization in P1 was confirmed by nuclear staining using 4*,6-diamidino-2-phenylindole (Fig. 2a*). The BLM protein truncated at 1341 amino acids, containing helicase domain and only one proximal arm of basic amino acids, P3, was not transported to the nucleus but retained in the cytoplasm (Fig. 2b and 2b*). The BLM protein truncated at 1357 amino acids, containing helicase domain and two arms, P2, was transported to the nucleus (Fig. 2c). The EGFP vector, harboring DNA fragments encoding BLM protein having only two arms of basic amino acid residues in the C-terminus, X-1, was also transported to the nucleus (Fig. 2d). The EGFP vector harboring fragments encoding BLM proteins with only a distal arm, X-2, was transported to the nucleus (Fig. 2e). These results suggest that the distal arm of the basic amino acids residues in the BLM protein is essential for nuclear localization (Fig. 1c). The EGFP vector with no insert was diffusely distributed in both the cytoplasm and nucleus (Fig. 2g). The truncated BLM protein, corresponding to previously reported BLM mutations, was retained in the cytoplasm or both the cytoplasm and the nucleus (Fig. 1b). The distribution of short BLM fragments, P-9 to P-11 and X-3 were observed in both the cytoplasm and the nucleus as well as EGFP vector with no insert. DISCUSSION We confirmed the nuclear localization of the BLM protein and identified the NLS within amino acid resi-
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FIG. 1. (a) Comparison of NLS of BLM with other known bipartite NLS (17). The NLS of BLM is aligned with sequences at the sites of other NLS. Capital letters reveal basic amino acids. Amino acids important in nuclear targeting are indicated by bold letters. (b) The summary of the localization of EGFP-BLM fusion proteins and the BLM fragments used in this experiment. The numbers showed the position of amino acid residues in the BLM protein. Shaded regions revealed untranslated regions, helicase domain and NLS. Arrowheads showed the position of nonsense mutations in a previously reported paper (7). nuc: nuclear localization of EGFP in all cells observed, cyt: cytoplasmic localization in all cells observed. In those cases where the subcellular localization was not exclusively nuclear or cytoplasmic, the results has been given by nucÅcyt, or nucõcyt to indicate the predominant pattern of staining observed in the transfected cells. (c) Amino acids of NLS in the C-terminus of BLM. Two basic amino acids stretch were underlined.
dues 1334-1349 in the C-terminus of the BLM protein. Amino acid residues 1334-1349 in the C-terminus of the BLM protein have the consensus amino acid sequence of bipartite NLS, which are two arms of basic amino acids and a 10 amino acid spacer region that separates the two basic domains. A series of fusion proteins with EGFP and a fraction of the C-terminal amino acids that were truncated from the N-terminal region showed that the distal arm of the basic amino acid residues in the BLM protein is essential for nuclear localization. However, we could not eliminate the possibility that the proximal arm has a cooperative or additive effect on the distal arm. Full-length BLM and WRN proteins differ in length by only 15 residues and share a highly conserved helicase domain. Both the N-terminal and C-terminal domains are unique to the BLM and WRN proteins. Recently, it was revealed that the WRN protein also has a functional NLS in its C-terminus (18). It is reasonable
that the BLM protein as well as the WRN protein is transported to the nucleus, since the helicases catalyze the unwinding of double-stranded DNA to provide single-stranded templates mainly in the nucleus. Based on previous reports, although many BLM mutations truncate the protein upstream of the helicase domain, WRN mutations truncate the protein downstream of the helicase domain. The products of some mutated WRN genes probably retain some DNA helicase activity. Most of the mutations in WRN result in impaired nuclear import of the WRN protein and this defect is probably a major contributing factor in the molecular pathology of WS (18). In contrast to WRN mutations, previously reported BLM mutations are assumed to result in a protein which has lost its helicase activity (7). Seven of them result in truncated proteins which do not contain the full length of the helicase domain, other three are missense mutation in the helicase domain. We analyzed
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FIG. 1—Continued
all the mutations leading to truncation. Therefore, the impact of these missense mutation on nuclear localization remains to be elucidated. Short fragments, P-9 to P11 and X-3, were retained in both the cytoplasm and the nucleus. These short fragments might pass through the nuclear pores freely and distributed in both the cytoplasm and the nucleus as does the EGFP vector with no insert, because these fragments have a low molecular weight. In these cases, even if such short
BLM fragments were transported to the nucleus, they could not function because of the defective helicase domain. To date, mutations in BLM in subjects with BS have been identified in only 10 cases. If the number of mutations identified in BLM increases, it can be clarified whether mutations in the gene coding NLS resulting in the impaired nuclear transport but intact helicase activity of the BLM protein cause BS. Since all the mutations identified in BLM gene affect
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FIG. 2. Subcellular distributions of EGFP–BLM fusion proteins. The transfected HeLa cells cultured for 24 hr were photographed (1400). EGFP signal is shown in panels of a) to g), P-1 (a), P-3 (b), P-2 (c), X-1 (d), X-2 (e), X-3, (f) EGFP with no insert (g). Nuclear staining by 4*,6-diamidino-2-phenylindole was shown in panels of a*) and b*).
the helicase domain, we must reserve the significance of the NLS for the pathogenesis of BS at the moment. However, on the basis of our results, it is assumed that the correct functioning of the BLM protein requires both intact helicase activity and an intact NLS in the C-terminus. Further studies will reveal the exact mechanism by which the loss of the function of the BLM protein causes the various symptoms of BS, including a high predisposition to cancer.
REFERENCES 1. German, J. (1983) (German, J., ed), pp. 97–134, Alan R. Liss, New York. 2. German, J., and Passarge, E. (1989) Clin. Genet. 35, 57–69. 3. German, J. (1993) Medicine 72, 393–406. 4. German, J. (1995) Dermatol. Clin. 13, 7–18. 5. Groden, J., and German, J. (1992) Hum. Genet. 90, 360–367. 6. Kaneko, H., Inoue, R., Yamada, Y., Sukegawa, K., Fukao, T.,
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7.
8. 9.
10. 11.
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Tashita, H., Teramoto, T., Kasahara, K., Takami, T., and Kondo, N. (1996) Int. J. Canc. 69, 480–483. Ellis, N. A., Groden, J., Ye, TZ., Straughen, J., Lennon, D. J., Ciocci, S., Proytcheva, M., and German, J. (1995) Cell 83, 655– 666. Watt, P. M., Hickson, I. D., Borts, R. H., and Louis, E. J. (1996) Genetics 144, 935–945. Yu, C-E., Oshima, J., Fu, Y-H., Wijsman, E. M., Hisama, F., Alisch, R., Matthews, S., Nakura, J., Miki, T., Ouais, S., Martin, G. M., Mulligan, J., and Schenllenberg, G. D. (1996) Science 272, 258–262. Watt, P. M., and Hickson, I. D. (1996) Curr. Biol. 6, 265–267. Lu, J., Mullen, J. R., Brill, S. J., Kleff, S., Romeo, A. M., and Sternglanz, R. (1996) Nature 383, 678–679.
12. Kalderon, D., Richardson, W. D., Markham, A. T., and Smith, A. E. (1984) Nature 311, 33–38. 13. Kalderon, D., Roberts, B. L., Richardson, W. D., and Smith, A. E. (1984) Cell 39, 499–509. 14. Robbins, R., Dilworth, S. M., Laskey, R. A., and Dingwall, C. (1991) Cell 64, 615–623. 15. Chalfile, M., Tu, Y., Euskirchen, G., Ward, W. W., and Prasher, D. C. (1994) Science 263, 802–805. 16. Monosov, E. Z., Wenzel, T. J., Luers, G. H., Heyman, J. A., and Subramani, S. (1996) J. Histochem. Cytochem. 44, 581–589. 17. Dingwall, C., and Laskey, R. A. (1991) Trends Biochem. Sci. 16, 478–481. 18. Matsumoto, T., Shimamoto, A., Goto, M., and Furuichi, Y. (1997) Nature Genet. 16, 335.
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BLM (the Causative Gene of Bloom Syndrome) Protein Translocation into the Nucleus by a Nuclear Localization Signal Hideo Kaneko,* Koji O Orii,* Eiko Matsui,* Nobuyuki Shimozawa,* Toshiyuki Fukao,* Takehisa Matsumoto,† Akira Shimamoto,† Yasuhiro Furuichi,† Seiro Hayakawa,* Kimiko Kasahara,* and Naomi Kondo* *Department of Pediatrics, Gifu University School of Medicine, Tsukasa-machi 40, Gifu 500, Japan; and †AGENE Research Institute, 200 Kajiwara, Kamakura-shi, Kanagawa 247, Japan
Received October 13, 1997
gree of recombination between homologous chromosome is increased. An abnormally high number of sister chromatid exchanges are observed. In 1995 the causative gene for BS was identified using somatic crossover point mapping and termed BLM (7). BLM is a 4437 bp cDNA that encodes a 1417 amino acid peptide which is homologous to ATP-dependent DNA helicases. DNA helicases are the enzymes which catalyze the unwinding of double-stranded DNA to provide single-stranded templates for the processes of replication, repair, recombination and transcription. BLM is a member of the RecQ helicase family, consisting of human WRN, RECQL and yeast Sgs1 (8). WRN was identified as the causative gene for Werner’s syndrome (WS), a premature aging disorder (9). There is considerable structural and sequence divergence between these proteins outside of the helicase domains (10). Most strikingly, BLM protein and Sgs1 share a serine rich, highly charged amino-terminal domain of approximately 575 amino acids, which is completely absent from both RecQ and RECQL. Lu et al. reported that site-directed mutations that eliminate helicase activity of yeast Sgs1 can still complement certain Sgs1 mutants (11). These results suggest the region other than helicase domain has the unique function in a member of the helicase family. Nuclear proteins accumulate in the nucleus because they contain nuclear targeting signals (NLS) that allow selective entry through nuclear pore complex. NLS is essential for the transport of proteins into the nucleus. The seven-amino-acid nuclear targeting sequence of the SV40 large T antigen has been regarded as the model NLS (12,13). Digwall et al. reported that the Bloom syndrome (BS) is characterized by stunted NLS of nucleoplasmin consists of two essential dogrowth, sun sensitivity and immunodeficiency (1-4). So- mains of basic amino acids that function in an interdematic cells from subjects with BS accumulate excessive pendent manner and a 10 amino acid spacer region numbers of mutations, including those of both coding that separates the two basic domains and which when sequences and noncoding repetitive DNA (5,6). The de- mutated gives rise to null phenotypes (14). In a dataBloom syndrome (BS) is a rare genetic disorder characterized by small body size, sun sensitivity, immunodeficiency and a high predisposition to various types of cancer. BLM was identified as the causative gene for BS, and BLM protein is homologous to DNA helicase. There are two putative nuclear localization signals (NLSs) within amino acid residues 1334–1349 in the Cterminus of the BLM protein, which has the distinctive structure of two basic residue arms separated by a spacer. The entire coding or deleted BLM sequences of various sizes were ligated into an enhanced green fluorescent protein (EGFP) vector and transfected into HeLa cells. The EGFP vector harboring the entire BLM coding sequence was transported to the nucleus. The BLM protein truncated at 1341 amino acid, containing an intact helicase domain and only one proximal arm, was not transported to the nucleus. The BLM protein truncated at 1357 amino acid, containing an intact helicase domain and two arms, was transported to the nucleus. The EGFP vector harboring DNA fragments encoding a protein having only the distal arms of basic amino acids in the C-terminus was also transported to the nucleus. The truncated BLM proteins corresponding to previously reported mutated BLM proteins were retained in the cytoplasm or both the cytoplasm and the nucleus as was the EGFP vector with no insert. These results show that the BLM protein translocates into the nucleus and that the distal arm of the bipartite basic residues in the C-terminus of the BLM protein is essential for targeting the nucleus. q 1997 Academic Press
0006-291X/97 $25.00
348
Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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base search of BLM amino acid sequences, we found amino acid sequences which exhibit a high degree of homology to bipartite NLS in its C-terminus. To clarify the precise function of the BLM protein, we investigated the subcellular localization and NLS of the BLM protein using EGFP fusion proteins (15,16). In this study we revealed that the BLM protein has a functional NLS in its C-terminal and is transported to the nucleus. MATERIALS AND METHODS Construction of EGFP fusion proteins. The EGFP fusion proteins were expressed transiently in HeLa cells that were transfected with expression vectors constructed from pEGFP-C3 (Clontech, Palo Alto, CA) and various length of BLM cDNA. Full-length BLM cDNA was cloned from mRNA obtained from the HeLa cells. Truncated BLM fragments were amplified from the full-length BLM cDNA using pfu DNA polymerase (Stratagene, La Jolla, CA) with 10 PCR cycles. Primer sets using in this experiments are listed as follows. Fragments P-1 to P-11 were amplified by combining X-S primer with P1 to P11 primers. Fragments of X-1 to X-3 were amplified by combining the X-1 to X-3 primers with the P-1 primer. The number in the primers is the position in cDNA of BLM. (forward) X-S 5*-CCG CTC GAG AGG ATT ATG GCT GCT GTT-3 * (69-86) X-1 5*-CCG CTC GAG GAA ATA CCC GTA TCT-3 * (4021-4040) X-2 5*-CCG CTC GAG GCC TCC CAA AGG TCT AAG-3 * (4095-4112) X-3 5*-CCG CTC GAG GCT TCC AGT GGT TCC AAG-3 * (4125-4142) (reverse) P-1 5*-AA CTG CAG TTA TGA GAA TGC ATA TGA AGG C-3* (4307-4328) P-2 5*-AA CTG CAG TGC CTT GGA ACC ACT GGA AGC-3* (4135-4155) P-3 5*-AA CTG CAG GGC TGG CAT CTT TTT CCT-3* (4079-4097) P-4 5*-AA CTG CAG GGT ATT TCC TCG TCA AGC TC-3* (4014-4034) P-5 5*-AA CTG CAG GGA ATT TTC TGT TTC CAT-3* (3641-3659) P-7 5*-AA CTG CAG GTC CTT CTG TAC CCT GGG-3* (2577-2593) P-8 5*-AA CTG CAG ATT AGT TCT AAA ATT ATG-3* (2069-2087) P-9 5*-AA CTG CAG TCC TAG TCT TGG TGT TTC-3* (1592-1610) P-10 5*-AA CTG CAG CTT TTC GCT ATT ATC TCT-3* (869-887) P-11 5*-AA CTG CAG TTT CTG AGC AGT GCT TAC-3* (611-629)
Amplified DNA fragments were digested by XhoI and PstI and were inserted in frame at the XhoI/PstI sites of the pEGFP plasmid downstream of the EGFP coding region, which was confirmed by dye-terminator sequencing (Applied Biosystemes, Chiba, Japan) using pEGFP specific primers. Plasmid DNA was prepared using the Qiagen kit (Qiagen, Hilden, Germany) and purified by CsCl ultracentrifugation. Electroporation. HeLa cells (5 1 106) were transfected with 10 mg of BLM fragments inserted into pEGFP plasmid by electroporation (Bio-Rad, Hercules, CA) and were seeded onto a cover glass. After 12 hrs, the cells were washed with PBS. After 24 hrs, the cells were fixed with 4% paraformaldehyde in PBS for 1 hour and washed with PBS. A cover glass was mounted with 0.2 mg/ ml of 4*,6-diamidino-2-phenylindole (Sigma, St. Louis, MO) in antifade solution. The cells were visualized by fluorescence microscopy.
RESULTS Putative NLS in the C-Terminus of BLM By investigating BLM protein amino acids sequences, we identified a putative bipartite NLS in the C-terminus. Close examination of the BLM protein re-
vealed that there are two arms of basic amino acids in the C-terminus, proximal arm: Arg (R)-Lys (K)-Arg (R)Lys (K)-Lys (K) (1334-1338) and distal arm: Arg (R)Ser (S)-Lys (K)-Arg (R)-Arg (R)-Lys (K) (1344-1349) (Fig. 1a). Figure 1a shows that these bipartite basic amino acids stretches are conserved in a number of known NLSs (14,17). Identification of NLS in BLM We prepared the primers for amplifying various lengths of BLM (Fig. 1b). Each amplified fragment corresponded to a putative NLS, or helicase domain or previously reported nonsense mutations. The fragments P-9, P-10 and P-11 were produced to correspond to the mutations previously reported in the Bloom’s Syndrome Registry, designated 97, 112 and 93, respectively (7). The predicted peptides of frameshift mutation observed in registry No. 15, 42, 107 and Nr2 were 739 amino acid residues long and were located between the P-7 and P-8 fragments. EGFP from the jellyfish Aequorea victoria yields a strong fluorescent signal in heterologous cell types and has been used as a marker of gene expression and for visualizing subcellular organelles and protein translocation in living cells. The EGFP vector harboring the entire coding sequence of BLM, P1, was transfected into HeLa cells, which express BLM well (data not shown), and EGFP was localized in the nucleus (Fig. 2a). Nuclear localization in P1 was confirmed by nuclear staining using 4*,6-diamidino-2-phenylindole (Fig. 2a*). The BLM protein truncated at 1341 amino acids, containing helicase domain and only one proximal arm of basic amino acids, P3, was not transported to the nucleus but retained in the cytoplasm (Fig. 2b and 2b*). The BLM protein truncated at 1357 amino acids, containing helicase domain and two arms, P2, was transported to the nucleus (Fig. 2c). The EGFP vector, harboring DNA fragments encoding BLM protein having only two arms of basic amino acid residues in the C-terminus, X-1, was also transported to the nucleus (Fig. 2d). The EGFP vector harboring fragments encoding BLM proteins with only a distal arm, X-2, was transported to the nucleus (Fig. 2e). These results suggest that the distal arm of the basic amino acids residues in the BLM protein is essential for nuclear localization (Fig. 1c). The EGFP vector with no insert was diffusely distributed in both the cytoplasm and nucleus (Fig. 2g). The truncated BLM protein, corresponding to previously reported BLM mutations, was retained in the cytoplasm or both the cytoplasm and the nucleus (Fig. 1b). The distribution of short BLM fragments, P-9 to P-11 and X-3 were observed in both the cytoplasm and the nucleus as well as EGFP vector with no insert. DISCUSSION We confirmed the nuclear localization of the BLM protein and identified the NLS within amino acid resi-
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FIG. 1. (a) Comparison of NLS of BLM with other known bipartite NLS (17). The NLS of BLM is aligned with sequences at the sites of other NLS. Capital letters reveal basic amino acids. Amino acids important in nuclear targeting are indicated by bold letters. (b) The summary of the localization of EGFP-BLM fusion proteins and the BLM fragments used in this experiment. The numbers showed the position of amino acid residues in the BLM protein. Shaded regions revealed untranslated regions, helicase domain and NLS. Arrowheads showed the position of nonsense mutations in a previously reported paper (7). nuc: nuclear localization of EGFP in all cells observed, cyt: cytoplasmic localization in all cells observed. In those cases where the subcellular localization was not exclusively nuclear or cytoplasmic, the results has been given by nucÅcyt, or nucõcyt to indicate the predominant pattern of staining observed in the transfected cells. (c) Amino acids of NLS in the C-terminus of BLM. Two basic amino acids stretch were underlined.
dues 1334-1349 in the C-terminus of the BLM protein. Amino acid residues 1334-1349 in the C-terminus of the BLM protein have the consensus amino acid sequence of bipartite NLS, which are two arms of basic amino acids and a 10 amino acid spacer region that separates the two basic domains. A series of fusion proteins with EGFP and a fraction of the C-terminal amino acids that were truncated from the N-terminal region showed that the distal arm of the basic amino acid residues in the BLM protein is essential for nuclear localization. However, we could not eliminate the possibility that the proximal arm has a cooperative or additive effect on the distal arm. Full-length BLM and WRN proteins differ in length by only 15 residues and share a highly conserved helicase domain. Both the N-terminal and C-terminal domains are unique to the BLM and WRN proteins. Recently, it was revealed that the WRN protein also has a functional NLS in its C-terminus (18). It is reasonable
that the BLM protein as well as the WRN protein is transported to the nucleus, since the helicases catalyze the unwinding of double-stranded DNA to provide single-stranded templates mainly in the nucleus. Based on previous reports, although many BLM mutations truncate the protein upstream of the helicase domain, WRN mutations truncate the protein downstream of the helicase domain. The products of some mutated WRN genes probably retain some DNA helicase activity. Most of the mutations in WRN result in impaired nuclear import of the WRN protein and this defect is probably a major contributing factor in the molecular pathology of WS (18). In contrast to WRN mutations, previously reported BLM mutations are assumed to result in a protein which has lost its helicase activity (7). Seven of them result in truncated proteins which do not contain the full length of the helicase domain, other three are missense mutation in the helicase domain. We analyzed
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FIG. 1—Continued
all the mutations leading to truncation. Therefore, the impact of these missense mutation on nuclear localization remains to be elucidated. Short fragments, P-9 to P11 and X-3, were retained in both the cytoplasm and the nucleus. These short fragments might pass through the nuclear pores freely and distributed in both the cytoplasm and the nucleus as does the EGFP vector with no insert, because these fragments have a low molecular weight. In these cases, even if such short
BLM fragments were transported to the nucleus, they could not function because of the defective helicase domain. To date, mutations in BLM in subjects with BS have been identified in only 10 cases. If the number of mutations identified in BLM increases, it can be clarified whether mutations in the gene coding NLS resulting in the impaired nuclear transport but intact helicase activity of the BLM protein cause BS. Since all the mutations identified in BLM gene affect
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FIG. 2. Subcellular distributions of EGFP–BLM fusion proteins. The transfected HeLa cells cultured for 24 hr were photographed (1400). EGFP signal is shown in panels of a) to g), P-1 (a), P-3 (b), P-2 (c), X-1 (d), X-2 (e), X-3, (f) EGFP with no insert (g). Nuclear staining by 4*,6-diamidino-2-phenylindole was shown in panels of a*) and b*).
the helicase domain, we must reserve the significance of the NLS for the pathogenesis of BS at the moment. However, on the basis of our results, it is assumed that the correct functioning of the BLM protein requires both intact helicase activity and an intact NLS in the C-terminus. Further studies will reveal the exact mechanism by which the loss of the function of the BLM protein causes the various symptoms of BS, including a high predisposition to cancer.
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