ABSTRACT
Mammals have nine different homologues (ALKBH1-9) of the Escherichia coli DNA repair demethylase AlkB. ALKBH2 is a genuine DNA repair enzyme, but the in vivo function of the other ALKBH proteins has remained elusive. It was recently shown that ALKBH8 contains an additional transfer RNA (tRNA) methyltransferase domain, which generates the wobble nucleoside 5-methoxycarbonylmethyluridine (mcm(5)U) from its precursor 5-carboxymethyluridine (cm(5)U). In this study, we report that (R)- and 5-methoxycarbonylhydroxymethyluridine (mchm(5)U), hydroxylated forms of mcm(5)U, are present in mammalian tRNA-Arg(UCG), and tRNA-Gly(UCC), respectively, representing the first example of a diastereomeric pair of modified RNA nucleosides. Through in vitro and in vivo studies, we show that both diastereomers of mchm(5)U are generated from mcm(5)U, and that the AlkB domain of ALKBH8 specifically hydroxylates mcm(5)U into (S)-mchm(5)U in tRNA-Gly(UCC). These findings expand the function of the ALKBH oxygenases beyond nucleic acid repair and increase the current knowledge on mammalian wobble uridine modifications and their biogenesis.
Subject(s)
Codon/genetics , Protein Biosynthesis/physiology , RNA, Messenger/metabolism , RNA, Transfer, Gly/chemistry , Uridine/analogs & derivatives , tRNA Methyltransferases/genetics , AlkB Homolog 8, tRNA Methyltransferase , Animals , Cattle , Chromatography, Liquid , Computational Biology , Mice , Molecular Structure , Species Specificity , Tandem Mass Spectrometry , Uridine/chemistry , Uridine/metabolism , tRNA Methyltransferases/chemistry , tRNA Methyltransferases/metabolismABSTRACT
BACKGROUND: Escherichia coli AlkB is a 2-oxoglutarate- and iron-dependent dioxygenase that reverses alkylated DNA damage by oxidative demethylation. Mouse AlkB homolog 1 (Alkbh1) is one of eight members of the newly discovered family of mammalian dioxygenases. METHODS AND FINDINGS: In the present study we show non-Mendelian inheritance of the Alkbh1 targeted allele in mice. Both Alkbh1(-/-) and heterozygous Alkbh1(+/-) offspring are born at a greatly reduced frequency. Additionally, the sex-ratio is considerably skewed against female offspring, with one female born for every three to four males. Most mechanisms that cause segregation distortion, act in the male gametes and affect male fertility. The skewing of the sexes appears to be of paternal origin, and might be set in the pachythene stage of meiosis during spermatogenesis, in which Alkbh1 is upregulated more than 10-fold. In testes, apoptotic spermatids were revealed in 5-10% of the tubules in Alkbh1(-/-) adults. The deficiency of Alkbh1 also causes misexpression of Bmp2, 4 and 7 at E11.5 during embryonic development. This is consistent with the incompletely penetrant phenotypes observed, particularly recurrent unilateral eye defects and craniofacial malformations. CONCLUSIONS: Genetic and phenotypic assessment suggests that Alkbh1 mediates gene regulation in spermatogenesis, and that Alkbh1 is essential for normal sex-ratio distribution and embryonic development in mice.
Subject(s)
Dioxygenases/genetics , Embryo, Nonmammalian/metabolism , Eye Abnormalities/genetics , Sex Ratio , Transcription Factors/genetics , AlkB Homolog 1, Histone H2a Dioxygenase , Animals , Animals, Newborn , Anophthalmos/genetics , Bone Morphogenetic Protein 2/genetics , Bone Morphogenetic Protein 4/genetics , Bone Morphogenetic Protein 7/genetics , DNA-(Apurinic or Apyrimidinic Site) Lyase , Dioxygenases/deficiency , Embryo, Nonmammalian/abnormalities , Female , Gene Expression Profiling , Gene Expression Regulation, Developmental , In Situ Hybridization , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Microphthalmos/genetics , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , Spermatogenesis/genetics , Testis/embryology , Testis/growth & development , Testis/metabolism , Transcription Factors/deficiencyABSTRACT
Flap endonuclease 1 (FEN1) processes Okazaki fragments in lagging strand DNA synthesis, and FEN1 is involved in several DNA repair pathways. The interaction of FEN1 with the proliferating cell nuclear antigen (PCNA) processivity factor is central to the function of FEN1 in both DNA replication and repair. Here we present two gene-targeted mice with mutations in FEN1. The first mutant mouse carries a single amino acid point mutation in the active site of the nuclease domain of FEN1 (Fen1(E160D/E160D)), and the second mutant mouse contains two amino acid substitutions in the highly conserved PCNA interaction domain of FEN1 (Fen1(DeltaPCNA/DeltaPCNA)). Fen1(E160D/E160D) mice develop a considerably elevated incidence of B-cell lymphomas beginning at 6 months of age, particularly in females. By 16 months of age, more than 90% of the Fen1(E160D/E160D) females have tumors, primarily lymphomas. By contrast, Fen1(DeltaPCNA/DeltaPCNA) mouse embryos show extensive apoptosis in the forebrain and vertebrae area and die around stage E9.5 to E11.5.
Subject(s)
Apoptosis , Embryo, Mammalian/metabolism , Embryo, Mammalian/pathology , Flap Endonucleases/physiology , Lymphoma/etiology , Lymphoma/pathology , Proliferating Cell Nuclear Antigen/physiology , Animals , Animals, Newborn , Cell Cycle/physiology , Cell Proliferation , Cells, Cultured , Culture Media, Serum-Free , DNA Repair , DNA Replication , Embryo, Mammalian/cytology , Female , Genes, Immunoglobulin/genetics , Genes, Lethal , Insulin Resistance , Male , Mice , Mice, Mutant Strains , Mutation/genetics , Nucleic Acid Hybridization , Obesity/etiology , Protein Structure, Tertiary , Recombination, GeneticABSTRACT
Spinal cord long-term potentiation is often studied as a model for cellular memory of nociceptive information. In the present report, extracellular single-unit recordings and quantitative real-time reverse transcriptase polymerase chain reaction were used to examine whether the induction of spinal cord long-term potentiation involves changes in expression of Zif, c-fos and cyclooxygenase-2. The data demonstrated that induction of spinal cord long-term potentiation was associated with a transient increase in the expression of Zif at 120 min (p < 0.05, long-term potentiation group vs. control group). In contrast, a decrease or no changes were observed in the expression of c-fos and cyclooxygenase-2. The transient increase of the expression of Zif is consistent with an involvement in the transition from the early to the late-phase of spinal cord long-term potentiation.
