No abstract

A functional mooring sequence, known to be required for apolipoprotein B (apoB) mRNA editing, exists in the mRNA encoding the neurofibromatosis type I (NF1) tumor suppressor. Editing of NF1 mRNA modifies cytidine in an arginine codon (CGA) at nucleotide 2914 to a uridine (UGA), creating an in frame translation stop codon. NF1 editing occurs in normal tissue but was several-fold higher in tumors. In vitro editing and transfection assays demonstrated that apoB and NF1 RNA editing will take place in both neural tumor and hepatoma cells. Unlike apoB, NF1 editing did not demonstrate dependence on rate-limiting quantities of APOBEC-1 (the apoB editing catalytic subunit) suggesting that different trans-acting factors may be involved in the two editing processes.

Apolipoprotein B (apoB) RNA editing involves sitespecific deamination of a cytidine to a uridine. A mooring sequence, a spacer region, and a regulator region are components of the apoB RNA editing motif of which only the mooring sequence is both necessary and sufficient for editosome assembly and editing. The catalytic component of the editosome is APOBEC-1. In rat hepatoma, stable cell lines, overexpression of APOBEC-1 resulted in 3-6-fold stimulation of the editing efficiency on either rat endogenous apoB RNA or transiently expressed human apoB RNA. In these cell lines, cytidines in addition to the one at the wild type site were edited. The occurrence and efficiency of this "promiscuous" editing increased with increasing expression of APO-BEC-1. Promiscuous editing was restricted to cytidines 5 of the mooring sequence and only occurred on RNAs that had been edited at the wild type site. Moreover, RNAs with mutant editing motifs supported high efficiency but low fidelity editing in the presence of high levels of APOBEC-1. This study demonstrates that overexpression of APOBEC-1 can increase the efficiency of site-specific editing but can also result in promiscuous editing. Apolipoprotein B (apoB)1 RNA editing (1, 2) is a post-transcriptional (3), site-specific deamination of a cytidine residue to a uridine. This nucleotide transition (at nt 6666, C 6666 ) converts a glutamine codon (CAA) at amino acid position 2153 to an in-frame STOP codon (UAA), which results in the translation of a truncated apoB48 protein (reviewed in Ref. 4). An 11-nucleotide (UGAUCAGUAUA) mooring sequence (5-7) is both necessary and sufficient for site-specific editing in a variety of RNA backgrounds (6, 8 -10). A macromolecular complex or "editosome" specifically assembles upon the mooring sequence (11) and directs editing of cytidine residues appropriately located in only the 5Ј direction (10). Between the mooring sequence and C 6666 is a region of lax sequence specificity (12-14) but whose appropriate length is critical for efficient sitespecific editing (12). Immediately 5Ј of C 6666 exists a regulator element (UGAUA). Enhancement of RNA editing efficiency by this element does not require a specific sequence, although TA immediately adjacent to C 6666 is most effective (12). The deamination reaction involved in apoB RNA editing is a zinc-dependent process mediated by . This protein has extensive sequence homology to other cytidine deaminases from Escherichia coli and mammals (4,15,19), especially within the zinc coordination domain wherein mutations abolish editing activity (16,20,21). The enzyme stimulates editing activity in vitro when complemented with suitable cell extracts (16), provides editing activity to human liver cell lines (HepG2) in vitro (22), and enhances apoB mRNA editing in mice when expressed by adenovirus-mediated gene transfer (23). The efficiency of apoB RNA editing is an important determinant in the proportion of full-length (apoB100) or truncated (apoB48) protein variants assembled as triglyceride-rich lipoprotei...

Activation-induced deaminase (AID) uses base deamination for class-switch recombination and somatic hypermutation and is related to the mammalian RNA-editing enzyme apolipoprotein B editing catalytic subunit 1 (APOBEC-1). CDD1 is a yeast ortholog of APOBEC-1 that exhibits cytidine deaminase and RNA-editing activity. Here, we present the crystal structure of CDD1 at 2.0-Å resolution and its use in comparative modeling of APOBEC-1 and AID. The models explain dimerization and the need for trans-acting loops that contribute to active site formation. Substrate selectivity appears to be regulated by a central active site ''flap'' whose size and flexibility accommodate large substrates in contrast to deaminases of pyrimidine metabolism that bind only small nucleosides or free bases. Most importantly, the results suggested both AID and APOBEC-1 are equally likely to bind single-stranded DNA or RNA, which has implications for the identification of natural AID targets.A ntibody diversity is generated during B cell development through class-switch recombination (CSR) and somatic hypermutation (SHM) (1), and in many vertebrates by gene conversion (2). Expression of activation-induced deaminase (AID) is critical for all three processes (3-5) and ectopic AID expression was sufficient to activate SHM in B cell lines, hybridomas, and fibroblasts (6-9), gene conversion in B cells (4, 5), as well as CSR in fibroblast cell lines (10). Mutations in the AID protein have been detected in humans with hyper-IgM type 2 syndrome (11). Expression of AID in Escherichia coli caused deoxycytidine-todeoxyuridine deamination in actively transcribed host genes that was enhanced in strains deficient in the deoxyuridine repair enzyme DNA uracil N-glycosylase (12). DNA uracil N-glycosylase deficiency in mammals resulted in altered patterns of CSR and SHM (13,14). In vitro studies revealed that AID catalyzed deamination on single-stranded DNA at WRCY hot spots, but not on doublestranded DNA, RNA-DNA hybrids, or single-stranded RNA (15,16). Cytidine deamination on single-stranded DNA within transcription bubbles (17, 18) and the observation that transcription rates influenced SHM activity (17)(18)(19) suggested that the biological substrate for AID is DNA.In contrast, the homology of AID to the mRNA-editing enzyme APOBEC-1 (3), suggested AID might edit RNA (3). This idea was intriguing because edited mRNAs either encode novel proteins or lose the ability to express proteins (20,21). Consistent with expression of a novel protein from edited mRNA, de novo protein synthesis was required for CSR subsequent to AID activation (22).APOBEC-1 and AID have proven difficult to purify at levels sufficient for structural studies. CDD1 from Saccharomyces cerevisiae (ScCDD1) can be purified readily, which is relevant due to its orthology with APOBEC-1 at the level of both cytidine deaminase (CDA) sequence similarity (27%) and mRNA-editing activity on apolipoprotein B (apoB) substrates (23). We determined the crystal structure of ScCDD1 to 2.0 Å resolution, which ...

ROS-dependent acetylation of CyPA is required for the generation of extracellular CyPA. Acetylated extracellular CyPA regulates VSMC and EC activation, suggesting that inhibition of acetylation of CyPA may prevent the pathogenesis of oxidative stress-related cardiovascular diseases.