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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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Related in: MedlinePlus

Nucleotide and predicted amino acid sequences of hABH8. The conserved 2OG-Fe(II) oxy-genase domain is boxed, the RNA-binding motif is underlined and the methyltrans-ferase domain is shaded. Numbering of the nucleotide and amino acid positions is indicated on the left.
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fig03: Nucleotide and predicted amino acid sequences of hABH8. The conserved 2OG-Fe(II) oxy-genase domain is boxed, the RNA-binding motif is underlined and the methyltrans-ferase domain is shaded. Numbering of the nucleotide and amino acid positions is indicated on the left.

Mentions: The hABH8 PCR product was approximately 2000 bp in length (Fig. 2), which is considerably longer than the nucleotide number of the predicted hABH8 ORF (717 bp) in the database (accession no. NM_138775). The nucleotide sequence of the amplified RT-PCR product suggested that the hABH8 registered in the NCBI database would result from the alternative splicing of our identified product. Therefore, we cloned the hABH8 cDNA encoding a product of 2144 nucleotides in length–including an ORF of 664 amino acids–based on a combination of M. mulatta (XM_001102947) cDNA and information obtained from the NCBI human genome resources database (Fig. 3). We submitted the sequence to the DDBJ database as hABH8 (accession no. AB218768). The NCBI human genome resources database indicated that the cloned hABH8 gene is located in chromosome region 11q23.1 and comprises at least 12 exons. An EMBL-EBI InterProScan sequence search (http://www. ebi. ac. uk/interpro/) indicated that the cloned hABH8 is a member of the 2OG-Fe(II) oxygenase superfamily. Moreover, the existence of an RNA recognition motif and a methyltransferase domain are recognized in hABH8 (Fig. 1A), suggesting that hABH8 might be a unique molecule that uses RNA as a substrate and expresses both methylation and demethylation enzymatic activities.


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)

Nucleotide and predicted amino acid sequences of hABH8. The conserved 2OG-Fe(II) oxy-genase domain is boxed, the RNA-binding motif is underlined and the methyltrans-ferase domain is shaded. Numbering of the nucleotide and amino acid positions is indicated on the left.
© Copyright Policy
Related In: Results  -  Collection

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

fig03: Nucleotide and predicted amino acid sequences of hABH8. The conserved 2OG-Fe(II) oxy-genase domain is boxed, the RNA-binding motif is underlined and the methyltrans-ferase domain is shaded. Numbering of the nucleotide and amino acid positions is indicated on the left.
Mentions: The hABH8 PCR product was approximately 2000 bp in length (Fig. 2), which is considerably longer than the nucleotide number of the predicted hABH8 ORF (717 bp) in the database (accession no. NM_138775). The nucleotide sequence of the amplified RT-PCR product suggested that the hABH8 registered in the NCBI database would result from the alternative splicing of our identified product. Therefore, we cloned the hABH8 cDNA encoding a product of 2144 nucleotides in length–including an ORF of 664 amino acids–based on a combination of M. mulatta (XM_001102947) cDNA and information obtained from the NCBI human genome resources database (Fig. 3). We submitted the sequence to the DDBJ database as hABH8 (accession no. AB218768). The NCBI human genome resources database indicated that the cloned hABH8 gene is located in chromosome region 11q23.1 and comprises at least 12 exons. An EMBL-EBI InterProScan sequence search (http://www. ebi. ac. uk/interpro/) indicated that the cloned hABH8 is a member of the 2OG-Fe(II) oxygenase superfamily. Moreover, the existence of an RNA recognition motif and a methyltransferase domain are recognized in hABH8 (Fig. 1A), suggesting that hABH8 might be a unique molecule that uses RNA as a substrate and expresses both methylation and demethylation enzymatic activities.

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