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Karen M. Frank - One of the best experts on this subject based on the ideXlab platform.
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The Rothmund-Thomson gene product RECQL4 localizes to the nucleolus in response to oxidative stress.
Experimental Cell Research, 2006Co-Authors: Kazunobu Futami, Akira Shimamoto, Yasuhiro Furuichi, Karen M. FrankAbstract:Mutations in the RECQL4 helicase gene have been linked to Rothmund-Thomson syndrome (RTS), which is characterized by poikiloderma, growth deficiency, and a predisposition to cancer. Examination of RECQL4 subcellular Localization in live cells demonstrated a nucleoplasmic pattern and, to a lesser degree, staining in nucleoli. Analysis of RECQL4-GFP deletion mutants revealed two nuclear Localization regions in the N-terminal region of RECQL4 and a Nucleolar Localization Signal at amino acids 376–386. RECQL4 Localization did not change after treatment with the DNA-damaging agents bleomycin, etoposide, UV irradiation and γ irradiation, in contrast to the Bloom and Werner syndrome helicases that relocate to distinct nuclear foci after damage. However, in a significant number of cells exposed to hydrogen peroxide or streptonigrin, RECQL4 accumulated in nucleoli. Using a T7 phage display screen, we determined that RECQL4 interacts with poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme that promotes genomic integrity through its involvement in DNA repair and Signaling pathways. The RECQL4 Nucleolar Localization was inhibited by pretreatment with a PARP-1 inhibitor. The C-terminal portion of RECQL4 was found to be an in vitro substrate for PARP-1. These results demonstrate changes in the intracellular Localization of RECQL4 in response to oxidative stress and identify an interaction between RECQL4 and PARP-1.
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The Rothmund-Thomson gene product RECQL4 localizes to the nucleolus in response to oxidative stress
Experimental Cell Research, 2006Co-Authors: Leslie L. Woo, Kazunobu Futami, Yoko Furuichi, Akira Shimamoto, Karen M. FrankAbstract:Mutations in the RECQL4 helicase gene have been linked to Rothmund-Thomson syndrome (RTS), which is characterized by poikiloderma, growth deficiency, and a predisposition to cancer. Examination of RECQL4 subcellular Localization in live cells demonstrated a nucleoplasmic pattern and, to a lesser degree, staining in nucleoli. Analysis of RECQL4-GFP deletion mutants revealed two nuclear Localization regions in the N-terminal region of RECQL4 and a Nucleolar Localization Signal at amino acids 376-386. RECQL4 Localization did not change after treatment with the DNA-damaging agents bleomycin, etoposide, UV irradiation and γ irradiation, in contrast to the Bloom and Werner syndrome helicases that relocate to distinct nuclear foci after damage. However, in a significant number of cells exposed to hydrogen peroxide or streptonigrin, RECQL4 accumulated in nucleoli. Using a T7 phage display screen, we determined that RECQL4 interacts with poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme that promotes genomic integrity through its involvement in DNA repair and Signaling pathways. The RECQL4 Nucleolar Localization was inhibited by pretreatment with a PARP-1 inhibitor. The C-terminal portion of RECQL4 was found to be an in vitro substrate for PARP-1. These results demonstrate changes in the intracellular Localization of RECQL4 in response to oxidative stress and identify an interaction between RECQL4 and PARP-1. © 2006 Elsevier Inc. All rights reserved.
Takehisa Matsumoto - One of the best experts on this subject based on the ideXlab platform.
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WRN helicase accelerates the transcription of ribosomal RNA as a component of an RNA polymerase I-associated complex
Oncogene, 2002Co-Authors: Miwa Shiratori, Takahisa Suzuki, Chie Itoh, Makoto Goto, Yasuhiro Furuichi, Takehisa MatsumotoAbstract:Werner syndrome (WS) is a rare autosmomal recessive genetic disorder causing premature aging. The gene ( WRN ) responsible for WS encodes a protein homologous to the RecQ-type helicase. WRN has a Nucleolar Localization Signal and shows intranuclear trafficking between the nucleolus and the nucleoplasm. WRN is recruited into the nucleolus when rRNA transcription is reactivated in quiescent cells. Inhibition of mRNA transcription with α-amanitin has no effect on Nucleolar Localization of WRN whereas inhibition of rRNA transcription with actinomycin D releases WRN from nucleoli, suggesting that Nucleolar WRN is closely related to rRNA transcription by RNA polymerase I (RPI). A possible function of WRN on rRNA transcription through interaction with RPI is supported by the results described here showing that WRN is co-immunoprecipitated with an RPI subunit, RPA40. Here we show that WS fibroblasts are characterized by a decreased level of rRNA transcription compared with wild-type cells, and that the decreased level of rRNA transcription in WS fibroblasts recovers when wild-type WRN is exogenously expressed. By contrast, exogenously expressed mutant-type WRN lacking an ability to migrate into the nucleolus fails to stimulate rRNA transcription. These results suggest that WRN promotes rRNA transcription as a component of an RPI-associated complex in the nucleolus.
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Diverged nuclear Localization of Werner helicase in human and mouse cells
Oncogene, 2001Co-Authors: Takahisa Suzuki, Miwa Shiratori, Yasuhiro Furuichi, Takehisa MatsumotoAbstract:Werner syndrome (WS) is a rare autosomal recessive genetic disorder causing premature aging and rare cancers. A gene responsible for WS ( WRN ) encodes a protein with 1432 amino acids (a.a.) homologous to the E. coli RecQ -type DNA helicase. Transcriptional activation facilitated Nucleolar Localization of human WRN protein (hWRNp) and serum starvation induced translocation of hWRNp from the nucleoli to the nucleoplasm in human cultured cells, suggesting a Nucleolar-nucleoplasm trafficking of hWRNp depending on transcriptional state. Mutant hWRNp lacking the C-terminal 30 a.a. residues (Δ1403–1432) failed to localize in the nucleolus, whereas Δ1405–1432 can migrate into the nucleolus. Here we identify a region putative for Nucleolar Localization Signal (NoLS) containing a sequence of two positively charged amino acids (Arg^1403-Lys^1404) in the C-terminal area of hWRNp. By contrast, the mouse homolog (mWRNp) exists only in the nucleoplasm. We show that the inability of mWRNp to migrate into the nucleolus is due to a difference of a sequence in the region corresponding to the NoLS of hWRNp. In addition, mouse cells cannot recognize the NoLS of hWRNp. Our study suggests that defect in Nucleolar function of hWRNp may be linked to the premature aging which is not observed in mWRN ^−/− mice.
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Diverged nuclear Localization of Werner helicase in human and mouse cells.
Oncogene, 2001Co-Authors: Takahisa Suzuki, Miwa Shiratori, Yasuhiro Furuichi, Takehisa MatsumotoAbstract:: Werner syndrome (WS) is a rare autosomal recessive genetic disorder causing premature aging and rare cancers. A gene responsible for WS (WRN) encodes a protein with 1432 amino acids (a.a.) homologous to the E. coli RecQ-type DNA helicase. Transcriptional activation facilitated Nucleolar Localization of human WRN protein (hWRNp) and serum starvation induced translocation of hWRNp from the nucleoli to the nucleoplasm in human cultured cells, suggesting a Nucleolar-nucleoplasm trafficking of hWRNp depending on transcriptional state. Mutant hWRNp lacking the C-terminal 30 a.a. residues (Delta1403-1432) failed to localize in the nucleolus, whereas Delta1405-1432 can migrate into the nucleolus. Here we identify a region putative for Nucleolar Localization Signal (NoLS) containing a sequence of two positively charged amino acids (Arg(1403)-Lys(1404)) in the C-terminal area of hWRNp. By contrast, the mouse homolog (mWRNp) exists only in the nucleoplasm. We show that the inability of mWRNp to migrate into the nucleolus is due to a difference of a sequence in the region corresponding to the NoLS of hWRNp. In addition, mouse cells cannot recognize the NoLS of hWRNp. Our study suggests that defect in Nucleolar function of hWRNp may be linked to the premature aging which is not observed in mWRN(-/-) mice.
Yasuhiro Furuichi - One of the best experts on this subject based on the ideXlab platform.
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The Rothmund-Thomson gene product RECQL4 localizes to the nucleolus in response to oxidative stress.
Experimental Cell Research, 2006Co-Authors: Kazunobu Futami, Akira Shimamoto, Yasuhiro Furuichi, Karen M. FrankAbstract:Mutations in the RECQL4 helicase gene have been linked to Rothmund-Thomson syndrome (RTS), which is characterized by poikiloderma, growth deficiency, and a predisposition to cancer. Examination of RECQL4 subcellular Localization in live cells demonstrated a nucleoplasmic pattern and, to a lesser degree, staining in nucleoli. Analysis of RECQL4-GFP deletion mutants revealed two nuclear Localization regions in the N-terminal region of RECQL4 and a Nucleolar Localization Signal at amino acids 376–386. RECQL4 Localization did not change after treatment with the DNA-damaging agents bleomycin, etoposide, UV irradiation and γ irradiation, in contrast to the Bloom and Werner syndrome helicases that relocate to distinct nuclear foci after damage. However, in a significant number of cells exposed to hydrogen peroxide or streptonigrin, RECQL4 accumulated in nucleoli. Using a T7 phage display screen, we determined that RECQL4 interacts with poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme that promotes genomic integrity through its involvement in DNA repair and Signaling pathways. The RECQL4 Nucleolar Localization was inhibited by pretreatment with a PARP-1 inhibitor. The C-terminal portion of RECQL4 was found to be an in vitro substrate for PARP-1. These results demonstrate changes in the intracellular Localization of RECQL4 in response to oxidative stress and identify an interaction between RECQL4 and PARP-1.
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WRN helicase accelerates the transcription of ribosomal RNA as a component of an RNA polymerase I-associated complex
Oncogene, 2002Co-Authors: Miwa Shiratori, Takahisa Suzuki, Chie Itoh, Makoto Goto, Yasuhiro Furuichi, Takehisa MatsumotoAbstract:Werner syndrome (WS) is a rare autosmomal recessive genetic disorder causing premature aging. The gene ( WRN ) responsible for WS encodes a protein homologous to the RecQ-type helicase. WRN has a Nucleolar Localization Signal and shows intranuclear trafficking between the nucleolus and the nucleoplasm. WRN is recruited into the nucleolus when rRNA transcription is reactivated in quiescent cells. Inhibition of mRNA transcription with α-amanitin has no effect on Nucleolar Localization of WRN whereas inhibition of rRNA transcription with actinomycin D releases WRN from nucleoli, suggesting that Nucleolar WRN is closely related to rRNA transcription by RNA polymerase I (RPI). A possible function of WRN on rRNA transcription through interaction with RPI is supported by the results described here showing that WRN is co-immunoprecipitated with an RPI subunit, RPA40. Here we show that WS fibroblasts are characterized by a decreased level of rRNA transcription compared with wild-type cells, and that the decreased level of rRNA transcription in WS fibroblasts recovers when wild-type WRN is exogenously expressed. By contrast, exogenously expressed mutant-type WRN lacking an ability to migrate into the nucleolus fails to stimulate rRNA transcription. These results suggest that WRN promotes rRNA transcription as a component of an RPI-associated complex in the nucleolus.
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Diverged nuclear Localization of Werner helicase in human and mouse cells
Oncogene, 2001Co-Authors: Takahisa Suzuki, Miwa Shiratori, Yasuhiro Furuichi, Takehisa MatsumotoAbstract:Werner syndrome (WS) is a rare autosomal recessive genetic disorder causing premature aging and rare cancers. A gene responsible for WS ( WRN ) encodes a protein with 1432 amino acids (a.a.) homologous to the E. coli RecQ -type DNA helicase. Transcriptional activation facilitated Nucleolar Localization of human WRN protein (hWRNp) and serum starvation induced translocation of hWRNp from the nucleoli to the nucleoplasm in human cultured cells, suggesting a Nucleolar-nucleoplasm trafficking of hWRNp depending on transcriptional state. Mutant hWRNp lacking the C-terminal 30 a.a. residues (Δ1403–1432) failed to localize in the nucleolus, whereas Δ1405–1432 can migrate into the nucleolus. Here we identify a region putative for Nucleolar Localization Signal (NoLS) containing a sequence of two positively charged amino acids (Arg^1403-Lys^1404) in the C-terminal area of hWRNp. By contrast, the mouse homolog (mWRNp) exists only in the nucleoplasm. We show that the inability of mWRNp to migrate into the nucleolus is due to a difference of a sequence in the region corresponding to the NoLS of hWRNp. In addition, mouse cells cannot recognize the NoLS of hWRNp. Our study suggests that defect in Nucleolar function of hWRNp may be linked to the premature aging which is not observed in mWRN ^−/− mice.
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Diverged nuclear Localization of Werner helicase in human and mouse cells.
Oncogene, 2001Co-Authors: Takahisa Suzuki, Miwa Shiratori, Yasuhiro Furuichi, Takehisa MatsumotoAbstract:: Werner syndrome (WS) is a rare autosomal recessive genetic disorder causing premature aging and rare cancers. A gene responsible for WS (WRN) encodes a protein with 1432 amino acids (a.a.) homologous to the E. coli RecQ-type DNA helicase. Transcriptional activation facilitated Nucleolar Localization of human WRN protein (hWRNp) and serum starvation induced translocation of hWRNp from the nucleoli to the nucleoplasm in human cultured cells, suggesting a Nucleolar-nucleoplasm trafficking of hWRNp depending on transcriptional state. Mutant hWRNp lacking the C-terminal 30 a.a. residues (Delta1403-1432) failed to localize in the nucleolus, whereas Delta1405-1432 can migrate into the nucleolus. Here we identify a region putative for Nucleolar Localization Signal (NoLS) containing a sequence of two positively charged amino acids (Arg(1403)-Lys(1404)) in the C-terminal area of hWRNp. By contrast, the mouse homolog (mWRNp) exists only in the nucleoplasm. We show that the inability of mWRNp to migrate into the nucleolus is due to a difference of a sequence in the region corresponding to the NoLS of hWRNp. In addition, mouse cells cannot recognize the NoLS of hWRNp. Our study suggests that defect in Nucleolar function of hWRNp may be linked to the premature aging which is not observed in mWRN(-/-) mice.
Leslie L. Woo - One of the best experts on this subject based on the ideXlab platform.
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The Rothmund-Thomson gene product RECQL4 localizes to the nucleolus in response to oxidative stress
Experimental Cell Research, 2006Co-Authors: Leslie L. Woo, Kazunobu Futami, Yoko Furuichi, Akira Shimamoto, Karen M. FrankAbstract:Mutations in the RECQL4 helicase gene have been linked to Rothmund-Thomson syndrome (RTS), which is characterized by poikiloderma, growth deficiency, and a predisposition to cancer. Examination of RECQL4 subcellular Localization in live cells demonstrated a nucleoplasmic pattern and, to a lesser degree, staining in nucleoli. Analysis of RECQL4-GFP deletion mutants revealed two nuclear Localization regions in the N-terminal region of RECQL4 and a Nucleolar Localization Signal at amino acids 376-386. RECQL4 Localization did not change after treatment with the DNA-damaging agents bleomycin, etoposide, UV irradiation and γ irradiation, in contrast to the Bloom and Werner syndrome helicases that relocate to distinct nuclear foci after damage. However, in a significant number of cells exposed to hydrogen peroxide or streptonigrin, RECQL4 accumulated in nucleoli. Using a T7 phage display screen, we determined that RECQL4 interacts with poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme that promotes genomic integrity through its involvement in DNA repair and Signaling pathways. The RECQL4 Nucleolar Localization was inhibited by pretreatment with a PARP-1 inhibitor. The C-terminal portion of RECQL4 was found to be an in vitro substrate for PARP-1. These results demonstrate changes in the intracellular Localization of RECQL4 in response to oxidative stress and identify an interaction between RECQL4 and PARP-1. © 2006 Elsevier Inc. All rights reserved.
Gayatri Ramakrishna - One of the best experts on this subject based on the ideXlab platform.
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intracellular distribution of human sirt7 and mapping of the nuclear Nucleolar Localization Signal
FEBS Journal, 2013Co-Authors: Shashi Kiran, Nirupama Chatterjee, Sapna Singh, Sunil C Kaul, Renu Wadhwa, Gayatri RamakrishnaAbstract:: Sirtuins belong to a class of NAD-dependent deacetylases, and include seven distinct isoforms, of which SIRT7 is the least studied member. In the present study, the subcellular expression of SIRT7 in primary fibroblasts undergoing senescence was evaluated by immunocytochemistry and immunoblot assay. Expression of Nucleolar SIRT7 in young fibroblast was very prominent, decreased in pre-senescent cells, and became undetectable in the senescent cells. Interestingly, we observed previously unreported staining for cytoplasmic SIRT7 in fibroblasts, and report the existence of a steady-state level of SIRT7 in cytoplasm. Selective Localization of the high-molecular-mass (47.5 kDa) SIRT7 in the cytoplasmic fraction and the low-molecular-mass (45 kDa) SIRT7 in the nuclear fraction was observed in the immunoblot analysis of various cell types. The specificity of the N-terminal antibodies for detection of cytoplasmic SIRT7 was confirmed by RNAi and peptide competition assays. The two forms of SIRT7 showed reciprocal expression following serum starvation, nocodazole and okadaic acid treatments, and also during senescence. Using a combination of deletion constructs and site-directed mutagenesis, we defined the role of two distinct SIRT7 sequences in the N-terminal region (amino acids 61-76, LQGRSRRREGLKRRQE) and the C-terminal region (amino acids 392-400, KRTKRKKVT) for nuclear and Nucleolar import, respectively. In conclusion, we report for the first time the existence of a cytoplasmic pool of SIRT7 in addition to its well-known Nucleolar form, identify distinct Localization Signals for its nuclear/Nucleolar targeting, and describe an association between loss of Nucleolar SIRT7 and replicative senescence.
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Intracellular distribution of human SIRT7 and mapping of the nuclear/Nucleolar Localization Signal.
FEBS Journal, 2013Co-Authors: Shashi Kiran, Nirupama Chatterjee, Sapna Singh, Sunil C Kaul, Renu Wadhwa, Gayatri RamakrishnaAbstract:: Sirtuins belong to a class of NAD-dependent deacetylases, and include seven distinct isoforms, of which SIRT7 is the least studied member. In the present study, the subcellular expression of SIRT7 in primary fibroblasts undergoing senescence was evaluated by immunocytochemistry and immunoblot assay. Expression of Nucleolar SIRT7 in young fibroblast was very prominent, decreased in pre-senescent cells, and became undetectable in the senescent cells. Interestingly, we observed previously unreported staining for cytoplasmic SIRT7 in fibroblasts, and report the existence of a steady-state level of SIRT7 in cytoplasm. Selective Localization of the high-molecular-mass (47.5 kDa) SIRT7 in the cytoplasmic fraction and the low-molecular-mass (45 kDa) SIRT7 in the nuclear fraction was observed in the immunoblot analysis of various cell types. The specificity of the N-terminal antibodies for detection of cytoplasmic SIRT7 was confirmed by RNAi and peptide competition assays. The two forms of SIRT7 showed reciprocal expression following serum starvation, nocodazole and okadaic acid treatments, and also during senescence. Using a combination of deletion constructs and site-directed mutagenesis, we defined the role of two distinct SIRT7 sequences in the N-terminal region (amino acids 61-76, LQGRSRRREGLKRRQE) and the C-terminal region (amino acids 392-400, KRTKRKKVT) for nuclear and Nucleolar import, respectively. In conclusion, we report for the first time the existence of a cytoplasmic pool of SIRT7 in addition to its well-known Nucleolar form, identify distinct Localization Signals for its nuclear/Nucleolar targeting, and describe an association between loss of Nucleolar SIRT7 and replicative senescence.
