Publications

• Comparison of RNA Editing Activity of APOBEC1-A1CF and APOBEC1-RBM47 Complexes Reconstituted in HEK293T Cells.

  Wolfe A.D., Arnold D.B., Chen X.S.

  RNA editing is an important form of regulating gene expression and activity. APOBEC1 cytosine deaminase was initially characterized as pairing with a cofactor, A1CF, to form an active RNA editing complex that specifically targets APOB RNA in regulating lipid metabolism. Recent studies revealed tha ... >> More

  RNA editing is an important form of regulating gene expression and activity. APOBEC1 cytosine deaminase was initially characterized as pairing with a cofactor, A1CF, to form an active RNA editing complex that specifically targets APOB RNA in regulating lipid metabolism. Recent studies revealed that APOBEC1 may be involved in editing other potential RNA targets in a tissue-specific manner, and another protein, RBM47, appears to instead be the main cofactor of APOBEC1 for editing APOB RNA. In this report, by expressing APOBEC1 with either A1CF or RBM47 from human or mouse in an HEK293T cell line with no intrinsic APOBEC1/A1CF/RBM47 expression, we have compared direct RNA editing activity on several known cellular target RNAs. By using a sensitive cell-based fluorescence assay that enables comparative quantification of RNA editing through subcellular localization changes of eGFP, the two APOBEC1 cofactors, A1CF and RBM47, showed clear differences for editing activity on APOB and several other tested RNAs, and clear differences were observed when mouse versus human genes were tested. In addition, we have determined the minimal domain requirement of RBM47 needed for activity. These results provide useful functional characterization of RBM47 and direct biochemical evidence for the differential editing selectivity on a number of RNA targets. << Less

  J. Mol. Biol. 431:1506-1517(2019) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Optimization of apolipoprotein B mRNA editing by APOBEC1 apoenzyme and the role of its auxiliary factor, ACF.

  Chester A., Weinreb V., Carter C.W. Jr., Navaratnam N.

  Expression and purification to homogeneity of the apolipoprotein B mRNA editing subunit, APOBEC1, has allowed the demonstration that this apoenzyme has considerable residual enzymatic activity on a minimal apoB mRNA substrate, even in the absence of any auxiliary factors. Assay of this activity as ... >> More

  Expression and purification to homogeneity of the apolipoprotein B mRNA editing subunit, APOBEC1, has allowed the demonstration that this apoenzyme has considerable residual enzymatic activity on a minimal apoB mRNA substrate, even in the absence of any auxiliary factors. Assay of this activity as a function of various experimental conditions has led to substantial optimization of assay conditions through the use of incomplete factorial and response surface experiments. Surprisingly, the apoenzyme is thermostable, and has a temperature optimum near 45 degrees C. We have used these optimized conditions, to assess steady-state kinetic parameters for APOBEC1 mRNA editing activity with and without the auxiliary factor, ACF. An important effect of the auxiliary factor is to broaden the temperature range of APOBEC1 activity, lowering the optimal temperature and enabling it to function optimally at lower temperatures. A model consistent with this observation is that at lower temperatures ACF promotes a conformational transition in the RNA substrate that occurs spontaneously at higher temperature. Notably, the substantial RNA editing activity of APOBEC1 alone may be responsible for the "hyperediting" observed upon overexpression of APOBEC1 in transgenic mice. << Less

  RNA 10:1399-1411(2004) [PubMed] [EuropePMC]

• Apolipoprotein B mRNA editing in vitro is a zinc-dependent process.

  Barnes C., Smith H.C.

  Apolipoprotein B mRNA editing involves the conversion of a cytidine at nucleotide 6666 to a uridine, thereby creating a translational stop codon from a glutamine codon. Recent evidence suggest that a cytidine deaminase is responsible for catalyzing the C to U conversion. All known nucleotide deami ... >> More

  Apolipoprotein B mRNA editing involves the conversion of a cytidine at nucleotide 6666 to a uridine, thereby creating a translational stop codon from a glutamine codon. Recent evidence suggest that a cytidine deaminase is responsible for catalyzing the C to U conversion. All known nucleotide deaminases require zinc as part of their catalytic domain. Utilizing the selective chelator for zinc, 1,10-penanthroline, we demonstrate that in vitro editing activity in rat liver extracts is zinc dependent. These data, taken together with recent reports, strongly support a role for a cytidine deaminase and nucleotide conversion as the catalytic mechanism for apoB mRNA editing. << Less

  Biochem Biophys Res Commun 197:1410-1414(1993) [PubMed] [EuropePMC]

• The APOBEC-2 crystal structure and functional implications for the deaminase AID.

  Prochnow C., Bransteitter R., Klein M.G., Goodman M.F., Chen X.S.

  APOBEC-2 (APO2) belongs to the family of apolipoprotein B messenger RNA-editing enzyme catalytic (APOBEC) polypeptides, which deaminates mRNA and single-stranded DNA. Different APOBEC members use the same deamination activity to achieve diverse human biological functions. Deamination by an APOBEC ... >> More

  APOBEC-2 (APO2) belongs to the family of apolipoprotein B messenger RNA-editing enzyme catalytic (APOBEC) polypeptides, which deaminates mRNA and single-stranded DNA. Different APOBEC members use the same deamination activity to achieve diverse human biological functions. Deamination by an APOBEC protein called activation-induced cytidine deaminase (AID) is critical for generating high-affinity antibodies, and deamination by APOBEC-3 proteins can inhibit retrotransposons and the replication of retroviruses such as human immunodeficiency virus and hepatitis B virus. Here we report the crystal structure of APO2. APO2 forms a rod-shaped tetramer that differs markedly from the square-shaped tetramer of the free nucleotide cytidine deaminase, with which APOBEC proteins share considerable sequence homology. In APO2, two long alpha-helices of a monomer structure prevent the formation of a square-shaped tetramer and facilitate formation of the rod-shaped tetramer via head-to-head interactions of two APO2 dimers. Extensive sequence homology among APOBEC family members allows us to test APO2 structure-based predictions using AID. We show that AID deamination activity is impaired by mutations predicted to interfere with oligomerization and substrate access. The structure suggests how mutations in patients with hyper-IgM-2 syndrome inactivate AID, resulting in defective antibody maturation. << Less

  Nature 445:447-451(2007) [PubMed] [EuropePMC]

• C-->U editing of apolipoprotein B mRNA in marsupials: identification and characterisation of APOBEC-1 from the American opossum Monodelphus domestica.

  Fujino T., Navaratnam N., Jarmuz A., von Haeselaer A., Scott J.

  The C->U editing of RNA is widely found in plant and animal species. In mammals it is a discrete process confined to the editing of apolipoprotein B (apoB) mRNA in eutherians and the editing of the mitochondrial tRNA for glycine in marsupials. Here we have identified and characterised apoB mRNA ed ... >> More

  The C->U editing of RNA is widely found in plant and animal species. In mammals it is a discrete process confined to the editing of apolipoprotein B (apoB) mRNA in eutherians and the editing of the mitochondrial tRNA for glycine in marsupials. Here we have identified and characterised apoB mRNA editing in the American opossum Monodelphus domestica. The apoB mRNA editing site is highly conserved in the opossum and undergoes complete editing in the small intestine, but not in the liver or other tissues. Opossum APOBEC-1 cDNA was cloned, sequenced and expressed. The encoded protein is similar to APOBEC-1 of eutherians. Motifs previously identified as involved in zinc binding, RNA binding and catalysis, nuclear localisation and a C-terminal leucine-rich domain are all conserved. Opossum APOBEC-1 contains a seven amino acid C-terminal extension also found in humans and rabbits, but not present in rodents. The opossum APOBEC-1 gene has the same intron/exon organisation in the coding sequence as the eutherian gene. Northern blot and RT-PCR analyses and an editing assay indicate that no APOBEC-1 was expressed in the liver. Thus the far upstream promoter responsible for hepatic expression in rodents does not operate in the opossum. An APOBEC-1-like enzyme such as might be involved in C->U RNA editing of tRNA in marsupial mitochondria was not demonstrated. The activity of opossum APOBEC-1 in the presence of both chicken and rodent auxiliary editing proteins was comparable to that of other mammals. These studies extend the origins of APOBEC-1 back 170 000 000 years to marsupials and help bridge the gap in the origins of this RNA editing process between birds and eutherian mammals. << Less

  Nucleic Acids Res. 27:2662-2671(1999) [PubMed] [EuropePMC]

• The apolipoprotein B mRNA editing complex performs a multifunctional cycle and suppresses nonsense-mediated decay.

  Chester A., Somasekaram A., Tzimina M., Jarmuz A., Gisbourne J., O'Keefe R., Scott J., Navaratnam N.

  The C to U editing of apolipoprotein B (apoB) mRNA is mediated by a minimal complex composed of an RNA-binding cytidine deaminase (APOBEC1) and a complementing specificity factor (ACF). This editing generates a premature termination codon and a truncated open reading frame. We demonstrate that the ... >> More

  The C to U editing of apolipoprotein B (apoB) mRNA is mediated by a minimal complex composed of an RNA-binding cytidine deaminase (APOBEC1) and a complementing specificity factor (ACF). This editing generates a premature termination codon and a truncated open reading frame. We demonstrate that the APOBEC1-ACF holoenzyme mediates a multifunctional cycle. The atypical APOBEC1 nuclear localization signal is involved in RNA binding and is used to import ACF into the nucleus as cargo. APOBEC1 alone induces nonsense-mediated decay (NMD). The APOBEC1-ACF complex edits and remains associated with the edited RNA to protect it from NMD. The APOBEC1 nuclear export signal is involved in the export of ACF and the edited apoB mRNA together, to the site of translation. << Less

  EMBO J. 22:3971-3982(2003) [PubMed] [EuropePMC]

• Cloning and mutagenesis of the rabbit ApoB mRNA editing protein. A zinc motif is essential for catalytic activity, and noncatalytic auxiliary factor(s) of the editing complex are widely distributed.

  Yamanaka S., Poksay K.S., Balestra M.E., Zeng G.-Q., Innerarity T.L.

  Apolipoprotein (apo) B mRNA editing is the specific deamination of cytidine (nucleotide 6666) to uridine in apoB mRNA. We isolated a full-length cDNA clone encoding the rabbit apoB mRNA editing protein (REPR), a subunit of the editing complex. Rabbit REPR is analogous to a rat enterocyte 27-kDa pr ... >> More

  Apolipoprotein (apo) B mRNA editing is the specific deamination of cytidine (nucleotide 6666) to uridine in apoB mRNA. We isolated a full-length cDNA clone encoding the rabbit apoB mRNA editing protein (REPR), a subunit of the editing complex. Rabbit REPR is analogous to a rat enterocyte 27-kDa protein that has been shown to have cytidine deaminase activity. Like rat REPR, rabbit REPR edited synthetic apoB RNA when mixed with chicken enterocyte extract. Surprisingly, the REPR also acquired editing activity when mixed with extracts from various organs of the rabbit (liver, gallbladder, stomach, intestine, adrenals, thyroid, testes, spleen, kidney, and lung) or the chicken (kidney and liver). In contrast, the rabbit REPR mRNA was found only in the small and large intestine. Thus, the auxiliary protein(s) of the apoB mRNA editing complex, which are essential for editing activity, exist in organs devoid of significant apoB mRNA editing or apoB synthesis. REPR requires zinc for its catalytic activity. We mutated putative zinc-coordinating residues (His61, Cys93, Cys96) and 2 additional residues (Glu63, Pro92) of the rabbit REPR that are conserved in other cytidine or deoxycytidylate deaminases and in rat REPR. The wild-type and mutant REPR cDNAs each produced 28-kDa proteins when transcribed and translated in vitro. Compared with the wild-type editing activity, the mutations of His61-->Ala, Glu63-->Ala, Cys93-->Ala, and Cys96-->Ala abolished or greatly reduced editing activity, whereas the mutations of His61-->Cys (which also can coordinate zinc) and Pro92-->Ala had a lesser effect. These results indicate that His61, Cys93, and Cys96 are essential for editing activity, probably because they coordinate zinc, whereas Glu63 also is essential, because it may be involved in the deaminase reaction. In addition, the widespread distribution of the auxiliary factor(s) portends their involvement in other RNA editing reactions. << Less

  J. Biol. Chem. 269:21725-21734(1994) [PubMed] [EuropePMC]