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Expression and sub-cellular localization of human ABH family molecules.

Tsujikawa K, Koike K, Kitae K, Shinkawa A, Arima H, Suzuki T, Tsuchiya M, Makino Y, Furukawa T, Konishi N, Yamamoto H - J. Cell. Mol. Med. (2007 Sep-Oct)

Bottom Line: Interestingly, the EmGFP-hABH2 splice variant localized in nucleoplasm with a dot-like pattern.In some HeLa cells transfected with EmGFP-hABH5, dot-like fluorescence was also detected in the cytoplasm.These observations provide important information for the future annotation of the hABH family of molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan. tujikawa@phs.osaka-u.ac.jp

ABSTRACT
AlkB is an Escherichia coli protein that catalyses the oxidative demethylation of 1-methyladenine and 3-methylcytosine in DNA and RNA. The enzyme activity of AlkB is dependent on a 2-oxoglutarate- and Fe(II)-dependent (2OG-Fe[II]) oxygenase domain. Human AlkB homologues (hABH), hABH1, hABH2 and hABH3, which also possess the 2OG-Fe(II) oxygenase domain, have previously been identified. Recent bioinformatics analysis suggests the existence of an additional five ABH genes in humans. In this study, we identified the hABH4-hABH7 mRNAs and determined their expression in human tissues. Moreover, an hABH2 splice variant lacking the 2OG-Fe(II) oxygenase domain and a new gene, hABH8, were cloned from testis cDNA. hABH8 possesses not only the 2OG-Fe(II) oxygenase domain but both an RNA-binding motif and a methyl-transferase domain. mRNA of the eight hABH molecules was detected in the 16 normal human tissues examined. The sub-cellular localization of EmGFP-hABH8 was restricted to the cytoplasm. EmGFP-hABH1, 3, 4, 6 and 7 were localized in both the cytoplasm and nuclei. Interestingly, the EmGFP-hABH2 splice variant localized in nucleoplasm with a dot-like pattern. In some HeLa cells transfected with EmGFP-hABH5, dot-like fluorescence was also detected in the cytoplasm. These observations provide important information for the future annotation of the hABH family of molecules.

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Immunoblot analysis of the EmGFP-hABH family of molecules in transfected HeLa cells. HeLa cells were transfected with pcDNA6.2/N-EmGFP (Mock), pcDNA6.2/N -EmGFP-hABH1–hABH8, and pcDNA6.2/N-EmGFP-hABH2 variant 1 (▵hABH2) family expression constructs. After incubation for 24 hrs, the transfected cells were lysed, electrophoresed on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was immunoblotted with rat anti-GFP antibody followed by HRP-conjugated antirat IgG. lane 1, EmGFP; lane 2, EmGFP-hABH1; lane 3, EmGFP-hABH2; lane 4, EmGFP-hABH3; lane 5, EmGFP-hABH4; lane 6, EmGFP-hABH5; lane 7, EmGFP-hABH6; lane 8, EmGFP-hABH7; lane 9, EmGFP-hABH8;and lane 10, EmGFP- hABH2.
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fig05: Immunoblot analysis of the EmGFP-hABH family of molecules in transfected HeLa cells. HeLa cells were transfected with pcDNA6.2/N-EmGFP (Mock), pcDNA6.2/N -EmGFP-hABH1–hABH8, and pcDNA6.2/N-EmGFP-hABH2 variant 1 (▵hABH2) family expression constructs. After incubation for 24 hrs, the transfected cells were lysed, electrophoresed on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was immunoblotted with rat anti-GFP antibody followed by HRP-conjugated antirat IgG. lane 1, EmGFP; lane 2, EmGFP-hABH1; lane 3, EmGFP-hABH2; lane 4, EmGFP-hABH3; lane 5, EmGFP-hABH4; lane 6, EmGFP-hABH5; lane 7, EmGFP-hABH6; lane 8, EmGFP-hABH7; lane 9, EmGFP-hABH8;and lane 10, EmGFP- hABH2.

Mentions: In order to examine the sub-cellular localization of the hABH family molecules, we generated hABH expression vectors in which EmGFP was fused at the N terminus of each hABH1-hABH8 coding region. HeLa cells were transiently transfected with the constructs encoding the EmGFP-hABH fusion proteins as well as EmGFP alone as a control. The cell lysates were prepared 24 hrs after transfection, resolved on an SDS-PAGE gel, and then transferred to a PVDF membrane. The membrane was immunoblotted with anti-GFP antiserum. As shown in Figure 5, discrete bands with the expected molecular masses were detected in each lysate of the EmGFP-hABH-transfected cells, indicating successful construction and transfection of each EmGFP-hABH expression vector. The sub-cellular localization of the EmGFP-hABH fusion proteins was examined with fluorescence microscopy 24 hrs after transfection. As shown in Figure 6A, the control EmGFP was diffusely distributed in the cell and did not exhibit any particular localization pattern. As previously reported, hABH2 accumulates in the nuclei with particularly bright nucleolar staining and hABH3 localizes in both the nuclei and cytoplasm. hABH1, hABH4, hABH5, hABH6 and hABH7 were observed throughout the cells but hABH8 was detected only in the cytoplasm. Interestingly, many bright EmGFP foci were detected in the cytoplasm of some EmGFP-hABH5-expressing HeLa cells after approximately 24 hrs of transfection (Fig. 6B). However, we could not detect the specific localization of EmGFP-hABH5 in the cell organelles.


Expression and sub-cellular localization of human ABH family molecules.

Tsujikawa K, Koike K, Kitae K, Shinkawa A, Arima H, Suzuki T, Tsuchiya M, Makino Y, Furukawa T, Konishi N, Yamamoto H - J. Cell. Mol. Med. (2007 Sep-Oct)

Immunoblot analysis of the EmGFP-hABH family of molecules in transfected HeLa cells. HeLa cells were transfected with pcDNA6.2/N-EmGFP (Mock), pcDNA6.2/N -EmGFP-hABH1–hABH8, and pcDNA6.2/N-EmGFP-hABH2 variant 1 (▵hABH2) family expression constructs. After incubation for 24 hrs, the transfected cells were lysed, electrophoresed on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was immunoblotted with rat anti-GFP antibody followed by HRP-conjugated antirat IgG. lane 1, EmGFP; lane 2, EmGFP-hABH1; lane 3, EmGFP-hABH2; lane 4, EmGFP-hABH3; lane 5, EmGFP-hABH4; lane 6, EmGFP-hABH5; lane 7, EmGFP-hABH6; lane 8, EmGFP-hABH7; lane 9, EmGFP-hABH8;and lane 10, EmGFP- hABH2.
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fig05: Immunoblot analysis of the EmGFP-hABH family of molecules in transfected HeLa cells. HeLa cells were transfected with pcDNA6.2/N-EmGFP (Mock), pcDNA6.2/N -EmGFP-hABH1–hABH8, and pcDNA6.2/N-EmGFP-hABH2 variant 1 (▵hABH2) family expression constructs. After incubation for 24 hrs, the transfected cells were lysed, electrophoresed on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was immunoblotted with rat anti-GFP antibody followed by HRP-conjugated antirat IgG. lane 1, EmGFP; lane 2, EmGFP-hABH1; lane 3, EmGFP-hABH2; lane 4, EmGFP-hABH3; lane 5, EmGFP-hABH4; lane 6, EmGFP-hABH5; lane 7, EmGFP-hABH6; lane 8, EmGFP-hABH7; lane 9, EmGFP-hABH8;and lane 10, EmGFP- hABH2.
Mentions: In order to examine the sub-cellular localization of the hABH family molecules, we generated hABH expression vectors in which EmGFP was fused at the N terminus of each hABH1-hABH8 coding region. HeLa cells were transiently transfected with the constructs encoding the EmGFP-hABH fusion proteins as well as EmGFP alone as a control. The cell lysates were prepared 24 hrs after transfection, resolved on an SDS-PAGE gel, and then transferred to a PVDF membrane. The membrane was immunoblotted with anti-GFP antiserum. As shown in Figure 5, discrete bands with the expected molecular masses were detected in each lysate of the EmGFP-hABH-transfected cells, indicating successful construction and transfection of each EmGFP-hABH expression vector. The sub-cellular localization of the EmGFP-hABH fusion proteins was examined with fluorescence microscopy 24 hrs after transfection. As shown in Figure 6A, the control EmGFP was diffusely distributed in the cell and did not exhibit any particular localization pattern. As previously reported, hABH2 accumulates in the nuclei with particularly bright nucleolar staining and hABH3 localizes in both the nuclei and cytoplasm. hABH1, hABH4, hABH5, hABH6 and hABH7 were observed throughout the cells but hABH8 was detected only in the cytoplasm. Interestingly, many bright EmGFP foci were detected in the cytoplasm of some EmGFP-hABH5-expressing HeLa cells after approximately 24 hrs of transfection (Fig. 6B). However, we could not detect the specific localization of EmGFP-hABH5 in the cell organelles.

Bottom Line: Interestingly, the EmGFP-hABH2 splice variant localized in nucleoplasm with a dot-like pattern.In some HeLa cells transfected with EmGFP-hABH5, dot-like fluorescence was also detected in the cytoplasm.These observations provide important information for the future annotation of the hABH family of molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan. tujikawa@phs.osaka-u.ac.jp

ABSTRACT
AlkB is an Escherichia coli protein that catalyses the oxidative demethylation of 1-methyladenine and 3-methylcytosine in DNA and RNA. The enzyme activity of AlkB is dependent on a 2-oxoglutarate- and Fe(II)-dependent (2OG-Fe[II]) oxygenase domain. Human AlkB homologues (hABH), hABH1, hABH2 and hABH3, which also possess the 2OG-Fe(II) oxygenase domain, have previously been identified. Recent bioinformatics analysis suggests the existence of an additional five ABH genes in humans. In this study, we identified the hABH4-hABH7 mRNAs and determined their expression in human tissues. Moreover, an hABH2 splice variant lacking the 2OG-Fe(II) oxygenase domain and a new gene, hABH8, were cloned from testis cDNA. hABH8 possesses not only the 2OG-Fe(II) oxygenase domain but both an RNA-binding motif and a methyl-transferase domain. mRNA of the eight hABH molecules was detected in the 16 normal human tissues examined. The sub-cellular localization of EmGFP-hABH8 was restricted to the cytoplasm. EmGFP-hABH1, 3, 4, 6 and 7 were localized in both the cytoplasm and nuclei. Interestingly, the EmGFP-hABH2 splice variant localized in nucleoplasm with a dot-like pattern. In some HeLa cells transfected with EmGFP-hABH5, dot-like fluorescence was also detected in the cytoplasm. These observations provide important information for the future annotation of the hABH family of molecules.

Show MeSH
Related in: MedlinePlus