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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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Schematic diagram of the hABH family molecules. (A) The conserved 2OG-Fe(II) oxygenase domain, the methyltransferase domain and the RNA-binding motif are drawn as a solid box, a hatched box and a shaded box, respectively. In hABH7, the AlkB homologous region is drawn as a solid box. (B) Relationship of the exon structure and protein domains of hABH2 and ▵hABH2. The conserved 2OG-Fe(II) oxygenase domain is drawn as a solid box. A dotted box indicates a completely different amino acid sequence from hABH2 due to a frameshift.
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fig01: Schematic diagram of the hABH family molecules. (A) The conserved 2OG-Fe(II) oxygenase domain, the methyltransferase domain and the RNA-binding motif are drawn as a solid box, a hatched box and a shaded box, respectively. In hABH7, the AlkB homologous region is drawn as a solid box. (B) Relationship of the exon structure and protein domains of hABH2 and ▵hABH2. The conserved 2OG-Fe(II) oxygenase domain is drawn as a solid box. A dotted box indicates a completely different amino acid sequence from hABH2 due to a frameshift.

Mentions: In our previous paper, we described the cloning of hABH3 as a highly expressed gene in human prostate cancer (Fig. 1A) [18]. Recently, based on in silico analysis, the existence of five members of the hABH family (hABH4 through hABH8) was predicted. Therefore, we first examined the existence of these experimentally un-characterized genes. Since we have observed the highest expression of hABH3 mRNA in human testis, the mRNA expression of the other hABH family members was also examined using human testis cDNA. The RT-PCR primer pairs used were designed to amplify the full-length of the ORF of each hABH mRNA (Table 1). As shown in Figure 2, with the exception of hABH8, the products amplified using each of the hABH primer pairs corresponded closely to the expected sizes. The nucleotide sequences of the amplified products of hABH1 through hABH7 were almost completely identical to those in the National Center for Biotechnology Information (NCBI) database, indicating that these six hABH family members, including hABH3, are at least expressed in the testis.


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)

Schematic diagram of the hABH family molecules. (A) The conserved 2OG-Fe(II) oxygenase domain, the methyltransferase domain and the RNA-binding motif are drawn as a solid box, a hatched box and a shaded box, respectively. In hABH7, the AlkB homologous region is drawn as a solid box. (B) Relationship of the exon structure and protein domains of hABH2 and ▵hABH2. The conserved 2OG-Fe(II) oxygenase domain is drawn as a solid box. A dotted box indicates a completely different amino acid sequence from hABH2 due to a frameshift.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4401260&req=5

fig01: Schematic diagram of the hABH family molecules. (A) The conserved 2OG-Fe(II) oxygenase domain, the methyltransferase domain and the RNA-binding motif are drawn as a solid box, a hatched box and a shaded box, respectively. In hABH7, the AlkB homologous region is drawn as a solid box. (B) Relationship of the exon structure and protein domains of hABH2 and ▵hABH2. The conserved 2OG-Fe(II) oxygenase domain is drawn as a solid box. A dotted box indicates a completely different amino acid sequence from hABH2 due to a frameshift.
Mentions: In our previous paper, we described the cloning of hABH3 as a highly expressed gene in human prostate cancer (Fig. 1A) [18]. Recently, based on in silico analysis, the existence of five members of the hABH family (hABH4 through hABH8) was predicted. Therefore, we first examined the existence of these experimentally un-characterized genes. Since we have observed the highest expression of hABH3 mRNA in human testis, the mRNA expression of the other hABH family members was also examined using human testis cDNA. The RT-PCR primer pairs used were designed to amplify the full-length of the ORF of each hABH mRNA (Table 1). As shown in Figure 2, with the exception of hABH8, the products amplified using each of the hABH primer pairs corresponded closely to the expected sizes. The nucleotide sequences of the amplified products of hABH1 through hABH7 were almost completely identical to those in the National Center for Biotechnology Information (NCBI) database, indicating that these six hABH family members, including hABH3, are at least expressed in the testis.

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