title: 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.
abstract: 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.
authors.affiliation: Department of Internal Medicine and Department of Pharmacology and Molecular Biology, Washington University School of Medicine, St. Louis, Missouri 63110 , USA.
authors.forename: V
authors.initials: V
authors.lastname: Blanc
authors.forename: N
authors.initials: N
authors.lastname: Navaratnam
authors.forename: J O
authors.initials: JO
authors.lastname: Henderson
authors.forename: S
authors.initials: S
authors.lastname: Anant
authors.forename: S
authors.initials: S
authors.lastname: Kennedy
authors.forename: A
authors.initials: A
authors.lastname: Jarmuz
authors.forename: J
authors.initials: J
authors.lastname: Scott
authors.forename: N O
authors.initials: NO
authors.lastname: Davidson
mesh_terms: D000071479:APOBEC-1 Deaminase; D000595:Amino Acid Sequence; D000818:Animals; D001055:Apolipoproteins B / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D019556:COS Cells; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002460:Cell Line; D002645:Chickens; D003001:Cloning, Molecular; D003564:Cytidine Deaminase / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010802:Phylogeny; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms; D017393:RNA Editing; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques; D014466:Ultraviolet Rays; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
chemical_list: D001055:Apolipoproteins B; D018076:DNA, Complementary; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D016376:Oligonucleotides, Antisense; D010455:Peptides; D020033:Protein Isoforms; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C425312:SYNCRIP protein, human; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D000071479:APOBEC-1 Deaminase; C000605089:APOBEC1 protein, human; C000605091:Apobec1 protein, rat; D003564:Cytidine Deaminase