An auxiliary factor containing a 240-kDa protein complex is involved in apolipoprotein B RNA editing.

Abstract

A protein complex involved in apolipoprotein B (apoB) RNA editing, referred to as AUX240 (auxiliary factor containing p240), has been identified through the production of monoclonal antibodies against in vitro assembled 27S editosomes. The 240-kDa protein antigen of AUX240 colocalized with editosome complexes on immunoblots of native gels. Immunoadsorbed extracts were impaired in their ability to assemble editosomes beyond early intermediates and in their ability to edit apoB RNA efficiently. Supplementation of adsorbed extract with AUX240 restored both editosome assembly and editing activities. Several proteins, in addition to p240, ranging in molecular mass from 150 to 45 kDa coimmunopurify as AUX240 under stringent wash conditions. The activity of the catalytic subunit of the editosome APOBEC-1 and mooring sequence RNA binding proteins of 66 and 44 kDa could not be demonstrated in AUX240. The data suggest that p240 and associated proteins constitute an auxiliary factor required for efficient apoB RNA editing. We propose that the role of AUX240 may be regulatory and involve mediation or stabilization of interactions between APOBEC-1 subunits and editing site recognition proteins leading the assembly of the rat liver C/U editosome.Editing of apolipoprotein B (apoB) mRNA involves deamination of cytidine to uridine at nucleotide 6666, thereby converting a glutamine codon to a translation stop codon (1, 2). The amount of apoB mRNA edited is an important determinant in the proportion of triglyceride-rich serum lipoprotein particles containing either full-length (apoBloo) or truncated (apoB48) proteins (3-5). The cytidine deaminase involved in this process, APOBEC-1, has extensive sequence homology with other cytidine deaminases from Escherichia coli and mammals (6-11). Characteristic of deamination reactions, apoB mRNA editing is a zinc-dependent process (6,10,12) and mutations within APOBEC-1 predicted to disrupt its zinc-coordination domain abolished editing activity (10,13,14). The abundance of APOBEC-1 in tissues and transfected human and rat hepatoma cell lines correlated with the proportion of edited apoB mRNA (6-8, 10, 13-15).The mooring sequence model predicts that site-specific apoB mRNA editing requires the assembly of an editosome containing multiple proteins (16,17). In support of this model, current data have demonstrated that APOBEC-1 requires an auxiliary protein factor(s) for site-specific RNA editing (4-8, 13, 18). In the absence of complementing extracts, APOBEC-1 displayed only cytidine deaminase activity (6, 14) and a low but apparently nonselective affinity for binding RNA (14,19,20 In rat liver extracts, the 66-and 44-kDa auxiliary factors, APOBEC-1 and presumably other auxiliary factors essential for high specific activity apoB RNA editing and editosome assembly uniquely resided within 60S preeditosomal complexes (21). In the presence of apoB RNA substrate and under the conditions of the in vitro editing reaction, 60S complexes disaggregated to form 27S complexes containing 66-and 44-...

A Sequence-Specific RNA-Binding Protein Complements Apobec-1 To Edit Apolipoprotein B mRNA

“…In support of this hypothesis, an increasing number of proteins have been shown to interact with apo-B mRNA or apobec-1 (34,35). In addition to the complementing activity, these include p60 and p40, which cross-link to apo-B mRNA; ABBP-1, which interacts with apobec-1 (21); and a 240-kDa protein that is associated with the editosome and may regulate the efficiency of editing (28). However, the role of these proteins remains ambiguous.…”

Apolipoprotein B mRNA and lipoprotein secretion are increased in McArdle RH-7777 cells by expression of betaine-homocysteine S-methyltransferase

“…Therefore, identification of factors that regulate hepatic apo B gene expression and metabolism is important for understanding control of VLDL and LDL synthesis. After the apo B gene is transcribed, apo B secretion is regulated through post-transcriptional mechanisms that may include changes in apo B-mRNA stability [3], apo BmRNA editing [4] and post-translational apo B degradation [5]. Utilizing a monoclonal-antibody strategy that identified an auxiliary factor involved in apo B-mRNA editing [4], we now identify a protein that is the rat homologue of human betainehomocysteine S-methyltransferase (BHMT, EC 2.1.1.5).…”

ApoB mRNA editing is mediated by a coordinated modulation of multiple apoB mRNA editing enzyme components

“…The minimal enzyme complex that can mediate C to U editing of apoB RNA in vitro comprises two proteins: apoB mRNA editing enzyme, catalytic polypeptide 1 (apobec-1), the catalytic subunit, and apobec-1 complementation factor (ACF), a novel RNA-binding protein that serves as the RNA recognition component of the editing enzyme (26,28,38). Other proteins including CUG binding protein 2 (CUGBP2), glycine-arginine-tyrosine-rich RNA binding protein (GRY-RBP), heterogenous nuclear ribonucleoprotein (hnRNP)-C1, apobec-1 binding protein (ABBP)1, ABBP2, KH-type splicing regulatory binding protein (KSRP), Bcl-2-associated anthogene 4 (BAG4), and auxiliary factor (AUX)240 have been identified by using detection assays that primarily reflect their ability to interact with apobec-1, ACF, or apoB RNA (3,7,19,(21)(22)(23)(24)(25)(26)32). However, the specific functional role that each candidate auxiliary protein might have in the editing complex remains to be clarified, and the precise composition of the holoenzyme in vivo is yet to be elucidated.…”