Apobec-1 Complementation Factor Research Articles

Mutagenesis of Apobec-1 Complementation Factor Reveals Distinct Domains That Modulate RNA Binding, Protein-Protein Interaction with Apobec-1, and Complementation of C to U RNA-editing Activity

C to U editing of apolipoprotein B (apoB) RNA requires a multicomponent holoenzyme complex in which minimal constituents include apobec-1 and apobec-1 complementation factor (ACF). We have examined the predicted functional domains in ACF in binding apoB RNA, interaction with apobec-1, and complementation of RNA editing. We demonstrate that apoB RNA binding and apobec-1-interacting domains are defined by two partially overlapping regions containing the NH(2)-terminal RNA recognition motifs of ACF. Both apoB RNA binding and apobec-1 interaction are required for editing complementation activity. ACF is a nuclear protein that upon cotransfection with apobec-1 results in nuclear colocalization and redistribution of apobec-1 from the cytoplasm. ACF constructs with deletions or mutations in the putative nuclear localization signal (NLS) still localize in the nucleus of transfected cells but do not colocalize with apobec-1, the latter remaining predominantly cytoplasmic. These observations suggest that the putative NLS motif in ACF is not responsible for its nucleo-cytoplasmic trafficking. By contrast, protein-protein interaction is important for the nuclear import of apobec-1. Taken together, these data suggest that functional complementation of C to U RNA editing by apobec-1 involves the NH(2)-terminal 380 residues of ACF.

ARCD-1, an apobec-1-related cytidine deaminase, exerts a dominant negative effect on C to U RNA editing.

Mammalian apolipoprotein B (apoB) C to U RNA editing is catalyzed by a multicomponent holoenzyme containing a single catalytic subunit, apobec-1. We have characterized an apobec-1 homologue, ARCD-1, located on chromosome 6p21.1, and determined its role in apoB mRNA editing. ARCD-1 mRNA is ubiquitously expressed; phylogenetic analysis reveals it to be a distant member of the RNA editing family. Recombinant ARCD-1 demonstrates cytidine deaminase and apoB RNA binding activity but does not catalyze C to U RNA editing, either in vitro or in vivo. Although not competent itself to mediate deamination of apoB mRNA, ARCD-1 inhibits apobec-1-mediated C to U RNA editing. ARCD-1 interacts and heterodimerizes with both apobec-1 and apobec-1 complementation factor (ACF) and localizes to both the nucleus and cytoplasm of transfected cells. Together, the data suggest that ARCD-1 is a novel cytidine deaminase that interacts with apobec-1 and ACF to inhibit apoB mRNA editing, possibly through interaction with other protein components of the apoB RNA editing holoenzyme.

Isolation, characterization and developmental regulation of the human apobec-1 complementation factor (ACF) gene

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 ∼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 (∼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.

Identification of GRY-RBP as an apolipoprotein B RNA binding protein that interacts with both apobec-1 and with apobec-1 complementation factor (ACF) to modulate C-to-U editing

Identification of GRY-RBP as an apolipoprotein B RNA binding protein that interacts with both apobec-1 and with apobec-1 complementation factor (ACF) to modulate C-to-U editing

Identification of GRY-RBP as an apolipoprotein B RNA binding protein that interacts with both apobec-1 and with apobec-1 complementation factor (ACF) to modulate C-to-U editing

Identification of GRY-RBP as an apolipoprotein B RNA binding protein that interacts with both apobec-1 and with apobec-1 complementation factor (ACF) to modulate C-to-U editing

Two-Hybrid Cloning Identifies an RNA-Binding Protein, GRY-RBP, as a Component of apobec-1 Editosome

ApoB mRNA editing is mediated by an editosome complex with apobec-1 as its catalytic component. By yeast two-hybrid cloning using apobec-1 as bait we identified a 69.6-kDa RNA binding protein, GRY-RBP, that contains 3 RNA-recognition motifs (RRMs) as a novel apobec-1 associating protein. GRY-RBP may be an alternatively spliced species of NASP1, a protein of known function. GRY-RBP was shown to bind to apobec-1, the catalytic component of apoB mRNA editosome, in vivo and in vitro. Immunodepletion using a monospecific rabbit antibody abolished editing in apobec-1 expressing HepG2 S-100 extracts. GRY-RBD interacted with apobec-1 through its C-terminus. It contains three RRM (RNA recognition motifs) domains that are homologous to those found in human ACF (apobec-1 complementation factor). Phylogeny analysis of the RRM domain-containing proteins indicates that GRY-RBP clusters with hnRNP-R, ACF, and ABBP-1 (another apobec-1 binding protein). In addition to its involvement with apobec-1 editosome, the suggested cellular functions of GRY-RBD and its structural homologues include RNA transport and RNA secondary structure stabilization.

Gene structure and expression of the human apobec-1 complementation factor gene, ACF

Gene structure and expression of the human apobec-1 complementation factor gene, ACF

Gene structure and expression of the human apobec-1 complementation factor gene, ACF

Gene structure and expression of the human apobec-1 complementation factor gene, ACF

Ô‖Identification of GRY-RBP as an Apolipoprotein B RNA-binding Protein That Interacts with Both Apobec-1 and Apobec-1 Complementation Factor to Modulate C to U Editing

C to U editing of apolipoprotein B (apoB) mRNA involves the interaction of a multicomponent editing enzyme complex with a requisite RNA sequence embedded within an AU-rich context. This enzyme complex includes apobec-1, an RNA-specific cytidine deaminase, and apobec-1 complementation factor (ACF), a novel 65-kDa RNA-binding protein, that together represent the minimal core of the editing enzyme complex. The precise composition of the holo-enzyme, however, remains unknown. We have previously isolated an enriched fraction of S100 extracts, prepared from chicken intestinal cells, that displays apoB RNA binding and which, following supplementation with apobec-1, permits efficient C to U editing. Peptide sequencing of this most active fraction reveals the presence of ACF as well as GRY-RBP, an RNA-binding protein with approximately 50% homology to ACF. GRY-RBP was independently isolated from a two-hybrid screen of chicken intestinal cDNA. GRY-RBP binds to ACF, to apobec-1, and also binds apoB RNA. Experiments using recombinant proteins demonstrate that GRY-RBP binds to ACF and inhibits both the binding of ACF to apoB RNA and C to U RNA editing. This competitive inhibition is rescued by addition of ACF, suggesting that GRY-RBP binds to and sequesters ACF. As further evidence of the role of GRY-RBP, rat hepatoma cells treated with an antisense oligonucleotide to GRY-RBP demonstrated an increase in C to U editing of endogenous apoB RNA. ACF and GRY-RBP colocalize in the nucleus of transfected cells and, in cotransfection experiments with apobec-1, each appears to colocalize in a predominantly nuclear distribution. Taken together, the results indicate that GRY-RBP is a member of the ACF gene family that may function to modulate C to U RNA editing through binding either to ACF or to apobec-1 or, alternatively, to the target RNA itself.

Molecular cloning of apobec-1 complementation factor, a novel RNA-binding protein involved in the editing of apolipoprotein B mRNA.

The C-to-U editing of apolipoprotein B (apo-B) mRNA is catalyzed by a multiprotein complex that recognizes an 11-nucleotide mooring sequence downstream of the editing site. The catalytic subunit of the editing enzyme, apobec-1, has cytidine deaminase activity but requires additional unidentified proteins to edit apo-B mRNA. We purified a 65-kDa protein that functionally complements apobec-1 and obtained peptide sequence information which was used in molecular cloning experiments. The apobec-1 complementation factor (ACF) cDNA encodes a novel 64.3-kDa protein that contains three nonidentical RNA recognition motifs. ACF and apobec-1 comprise the minimal protein requirements for apo-B mRNA editing in vitro. By UV cross-linking and immunoprecipitation, we show that ACF binds to apo-B mRNA in vitro and in vivo. Cross-linking of ACF is not competed by RNAs with mutations in the mooring sequence. Coimmunoprecipitation experiments identified an ACF-apobec-1 complex in transfected cells. Immunodepletion of ACF from rat liver extracts abolished editing activity. The immunoprecipitated complexes contained a functional holoenzyme. Our results support a model of the editing enzyme in which ACF binds to the mooring sequence in apo-B mRNA and docks apobec-1 to deaminate its target cytidine. The fact that ACF is widely expressed in human tissues that lack apobec-1 and apo-B mRNA suggests that ACF may be involved in other RNA editing or RNA processing events.