Isolation, characterization and developmental regulation of the human apobec-1 complementation factor (ACF) gene.
(49/1237)
Mammalian apolipoprotein B (apo B) mRNA undergoes site-specific C to U deamination which is mediated by a multicomponent enzyme complex containing a minimal core composed of apobec-1 and a complementation factor, ACF. We have isolated and characterized the human ACF gene and examined its tissue-specific and developmental expression. The ACF gene spans approximately 80 kb and contains 15 exons, three of which are non-coding. Multiple alternative splice acceptor sites were found, generating at least nine different transcripts. Of these, the majority (approximately 75-89%) encode functional protein. In order to examine the role of ACF mRNA expression in the regulation of apo B mRNA editing, we examined a panel of fetal intestinal and hepatic mRNAs as well as RNA from an intestinal cell line. A developmental increase in C to U RNA editing has been previously noted in the human intestine. In both instances, the pattern of alternative splicing and overall abundance of ACF mRNA was relatively constant during development in both liver and small intestine. Taken together, the data demonstrate a complex pattern of differential, tissue-specific splicing of ACF mRNA, but suggest that other mechanisms are responsible for the developmental increase noted in intestinal apo B mRNA editing in humans. (+info)
C-->U editing of neurofibromatosis 1 mRNA occurs in tumors that express both the type II transcript and apobec-1, the catalytic subunit of the apolipoprotein B mRNA-editing enzyme.
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C-->U RNA editing of neurofibromatosis 1 (NF1) mRNA changes an arginine (CGA) to a UGA translational stop codon, predicted to result in translational termination of the edited mRNA. Previous studies demonstrated varying degrees of C-->U RNA editing in peripheral nerve-sheath tumor samples (PNSTs) from patients with NF1, but the basis for this heterogeneity was unexplained. In addition, the role, if any, of apobec-1, the catalytic deaminase that mediates C-->U editing of mammalian apolipoprotein B (apoB) RNA, was unresolved. We have examined these questions in PNSTs from patients with NF1 and demonstrate that a subset (8/34) manifest C-->U editing of RNA. Two distinguishing characteristics were found in the PNSTs that demonstrated editing of NF1 RNA. First, these tumors express apobec-1 mRNA, the first demonstration, in humans, of its expression beyond the luminal gastrointestinal tract. Second, PNSTs with C-->U editing of RNA manifest increased proportions of an alternatively spliced exon, 23A, downstream of the edited base. C-->U editing of RNA in these PNSTs was observed preferentially in transcripts containing exon 23A. These findings were complemented by in vitro studies using synthetic RNA templates incubated in the presence of recombinant apobec-1, which again confirmed preferential editing of transcripts containing exon 23A. Finally, adenovirus-mediated transfection of HepG2 cells revealed induction of editing of apoB RNA, along with preferential editing of NF1 transcripts containing exon 23A. Taken together, the data support the hypothesis that C-->U RNA editing of the NF1 transcript occurs both in a subset of PNSTs and in an alternatively spliced form containing a downstream exon, presumably an optimal configuration for enzymatic deamination by apobec-1. (+info)
Cytidine deaminase from two extremophilic bacteria: cloning, expression and comparison of their structural stability.
(51/1237)
We cloned, purified and characterized two extremophilic cytidine deaminases: CDA(Bcald) and CDA(Bpsy), isolated from Bacillus caldolyticus (growth at 72 degrees C) and Bacillus psychrophilus (growth at 10 degrees C), respectively. We compared their thermostability also with the mesophilic counterpart, CDA(Bsubt), isolated from Bacillus subtilis (growth at 37 degrees C). The DNA fragments encoding CDA(Bcald) and CDA(Bpsy) were sequenced and the deduced amino acid sequences showed 70% identity. High sequence similarity was also found with the mesophilic CDA(Bsubt). Both enzymes were found to be homotetramers of approximately 58 kDa. CDA(Bcald) was found to be highly thermostable, as expected, up to 65 degrees C, whereas CDA(Bpsy) showed higher specific activity at lower temperatures and was considerably less thermostable than CDA(Bcald). After partial denaturation at 72 degrees C for 30 min, followed by renaturation on ice, CDA(Bcald) recovered 100% of its enzymatic activity, whereas CDA(Bpsy) as well as CDA(Bsubt) were irreversibly inactivated. Circular dichroism (CD) spectra of CDA(Bcald) and CDA(Bpsy) at temperatures ranging from 10 to 95 degrees C showed a markedly different thermostability of their secondary structures: at 10 and 25 degrees C the CD spectra were indistinguishable, suggesting a similar overall structure, but as temperature increases up to 50-70 degrees C, the alpha-helices of CDA(Bpsy) unfolded almost completely, whereas its beta-structure and the aromatic amino acids core remained pretty stable. No significant differences were seen in the secondary structures of CDA(Bcald) with increase in temperature. (+info)
Apolipoprotein B mRNA editing and the reduction in synthesis and secretion of the atherogenic risk factor, apolipoprotein B100 can be effectively targeted through TAT-mediated protein transduction.
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Hepatic very-low-density lipoprotein particles (VLDL) containing full-length apolipoprotein B100 are metabolized in the blood stream to low-density lipoprotein (LDL) particles, whose elevated levels increase the risk of atherosclerosis. Statins and bile-acid sequestrants are effective LDL-lowering therapies for many patients. Development of alternative therapies remains important for patients with adverse reactions to conventional therapy, with defects in the LDL receptor-dependent lipoprotein uptake pathway and for intervention in children. Editing of apoB mRNA by the enzyme APOBEC-1 changes a glutamine codon to a stop codon, leading to the synthesis and secretion of apoB48-containing VLDL, which are rapidly cleared before they can be metabolized to LDL. Human liver does not edit apoB mRNA because it does not express APOBEC-1. Although initially promising, enthusiasm for apobec-1 gene therapy for hypercholesterolemia was blunted by the finding that uncontrolled transgenic expression of APOBEC-1 led to nonspecific editing of mRNAs and pathology. We demonstrate that APOBEC-1 fused to TAT entered primary hepatocytes, where it induced a transient increase in mRNA editing activity and enhanced synthesis and secretion of VLDL containing apoB48. Protein transduction of APOBEC-1 transiently stimulated high levels of apoB mRNA editing in a dose-dependent manner without loss of fidelity. These results suggested that apoB mRNA editing should be re-evaluated as a LDL-lowering therapeutic target in the new context of protein transduction therapy. (+info)
Two proteins essential for apolipoprotein B mRNA editing are expressed from a single gene through alternative splicing.
(53/1237)
Apolipoprotein B (apoB) mRNA editing involves site-specific deamination of cytidine to form uridine, resulting in the production of an in-frame stop codon. Protein translated from edited mRNA is associated with a reduced risk of atherosclerosis, and hence the protein factors that regulate hepatic apoB mRNA editing are of interest. A human protein essential for apoB mRNA editing and an eight-amino acid-longer variant of no known function have been recently cloned. We report that both proteins, henceforth referred to as ACF64 and ACF65, supported APOBEC-1 (the catalytic subunit of the editosome) equivalently in editing of apoB mRNA. They are encoded by a single 82-kb gene on chromosome 10. The transcripts are encoded by 15 exons that are expressed from a tissue-specific promoter minimally contained within the -0.33-kb DNA sequence. ACF64 and ACF65 mRNAs are expressed in both liver and intestinal cells in an approximate 1:4 ratio. Exon 11 is alternatively spliced to include or exclude 24 nucleotides of exon 12, thereby encoding ACF65 and ACF64, respectively. Recognition motifs for the serine/arginine-rich (SR) proteins SC35, SRp40, SRp55, and SF2/ASF involved in alternative RNA splicing were predicted in exon 12. Overexpression of these SR proteins in liver cells demonstrated that alternative splicing of a minigene-derived transcript to express ACF65 was enhanced 6-fold by SRp40. The data account for the expression of two editing factors and provide a possible explanation for their different levels of expression. (+info)
Requirement of the activation-induced deaminase (AID) gene for immunoglobulin gene conversion.
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Three phenotypically distinct processes-somatic hypermutation, gene conversion, and switch recombination-remodel the functionally rearranged immunoglobulin (Ig) loci in B cells. Somatic hypermutation and switch recombination have recently been shown to depend on the activation-induced deaminase (AID) gene product. Here, we show that the disruption of the AID gene in the chicken B cell line DT40 completely blocks Ig gene conversion and that this block can be complemented by reintroduction of the AID complementary DNA. This demonstrates that the AID master gene controls all B cell-specific modifications of vertebrate Ig genes. (+info)
Activation-induced deaminase (AID)-directed hypermutation in the immunoglobulin Smu region: implication of AID involvement in a common step of class switch recombination and somatic hypermutation.
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Somatic hypermutation (SHM) and class switch recombination (CSR) cause distinct genetic alterations at different regions of immunoglobulin genes in B lymphocytes: point mutations in variable regions and large deletions in S regions, respectively. Yet both depend on activation-induced deaminase (AID), the function of which in the two reactions has been an enigma. Here we report that B cell stimulation which induces CSR but not SHM, leads to AID-dependent accumulation of SHM-like point mutations in the switch mu region, uncoupled with CSR. These findings strongly suggest that AID itself or a single molecule generated by RNA editing function of AID may mediate a common step of SHM and CSR, which is likely to be involved in DNA cleavage. (+info)
The editosome for cytidine to uridine mRNA editing has a native complexity of 27S: identification of intracellular domains containing active and inactive editing factors.
(56/1237)
Apolipoprotein B mRNA cytidine to uridine editing requires the assembly of a multiprotein editosome comprised minimally of the catalytic subunit, apolipoprotein B mRNA editing catalytic subunit 1 (APOBEC-1), and an RNA-binding protein, APOBEC-1 complementation factor (ACF). A rat homolog has been cloned with 93.5% identity to human ACF (huACF). Peptide-specific antibodies prepared against huACF immunoprecipitated a rat protein of similar mass as huACF bound to apolipoprotein B (apoB) RNA in UV cross-linking reactions, thereby providing evidence that the p66, mooring sequence-selective, RNA-binding protein identified previously in rat liver by UV cross-linking and implicated in editosome assembly is a functional homolog of huACF. The rat protein (p66/ACF) was distributed in both the nucleus and cytoplasm of rat primary hepatocytes. Within a thin section, a significant amount of total cellular p66/ACF was cytoplasmic, with a concentration at the outer surface of the endoplasmic reticulum. Native APOBEC-1 co-fractionated with p66/ACF in the cytoplasm as 60S complexes. In the nucleus, the biological site of apoB mRNA editing, native p66/ACF, was localized to heterochromatin and fractionated with APOBEC-1 as 27S editosomes. When apoB mRNA editing was stimulated in rat primary hepatocytes with ethanol or insulin, the abundance of p66/ACF in the nucleus markedly increased. It is proposed that the heterogeneity in size of complexes containing editing factors is functionally significant and reflects functionally engaged editosomes in the nucleus and an inactive cytoplasmic pool of factors. (+info)