Induction of Cytidine to Uridine Editing on Cytoplasmic Apolipoprotein B mRNA by Overexpressing APOBEC-1

Abstract

Post-transcriptional editing of apolipoprotein B (apoB) mRNA is regulated in hepatic cells to achieve a steady state proportion of edited and unedited RNA molecules. This activity is catalyzed by APOBEC-1 (apoB mRNA editing catalytic subunit 1) in what has been widely accepted as nuclear event occurring during or after mRNA splicing. Introns impair the efficiency of editing within an adjacent exon in a distance-dependent manner in reporter RNAs. We show here that this inhibition can be overcome by overexpressing APOBEC-1 and that the enhanced editing efficiency on these reporter RNAs occurred after splicing on cytoplasmic transcripts. Given the absolute requirement of auxiliary proteins in apoB mRNA editing, the data suggested that auxiliary proteins were distributed with APOBEC-1 in both the nucleus and cytoplasm of McArdle cells. In fact, immunolocalization of one such auxiliary protein, APO-BEC-1 complementation factor (ACF) demonstrated a nuclear and cytoplasmic distribution. We also demonstrate that in the absence of alterations in APOBEC-1 expression, changes in edited apoB RNA induced by ethanol arise through the stimulation of nuclear editing activity. The finding that apoB mRNA editing can occur in the cytoplasm but normally does not suggests that under biological conditions, restricting editing activity to the nucleus must be an important step in regulating the proportion of the edited apoB mRNAs. Apolipoprotein B (apoB)1 mRNA editing involves a site-specific deamination reaction wherein a cytidine at nucleotide 6666 is changed to a uridine thereby placing an in frame UAA stop codon within the 14-kilobase apoB mRNA (1, 2). Translation of the unedited and edited variants of apoB mRNA generates two isoforms of apoB proteins, apoB100 and apoB48, which behave differently in terms of lipoprotein secretion and uptake by peripheral cells (3). A tripartite RNA sequence motif, consisting of an 11-nucleotide mooring sequence, a spacer, and a regulatory element, is required for site-specific RNA editing (4 -8). Recently, a stem-loop model of secondary structure involving the essential sequence required for editing has been proposed (9, 10). These cis-acting elements are required for the assembly of an editing complex, the C/U editosome (5, 11), and site-specific editing activity. APOBEC-1, the cytidine deaminase responsible for editing (12), is the catalytic subunit of the editosome (9) and may function in this capacity as a dimer (13,14).APOBEC-1 alone cannot edit apoB mRNA and requires multiple yet-to-be characterized proteins referred to collectively as the auxiliary proteins (15-20). Auxiliary proteins are broadly expressed in mammalian cell lines and tissues, independent of the expression of APOBEC-1 and apoB mRNA (12, 16 -18, 21). Recently, APOBEC-1 complementation factor (ACF) has been cloned (40). ACF is 64.3-kDa RNA binding protein, it binds to apoB mRNA in vitro and in vivo. ACF and APOBEC-1 comprise the minimal protein requirements for specific and efficient editing of apoB mRNA in vitro. Other candida...

Commitment of apolipoprotein B RNA to the splicing pathway regulates cytidine-to-uridine editing-site utilization

“…Nor does it occur when rat hepatic editing efficiencies are stimulated by metabolic or hormonal manipulations [49,50]. Promiscuous editing appears to be unique to cells in which APOBEC-1 has been artificially overexpressed [10,40,47] and is observed under these conditions on both nuclear and cytoplasmic transcripts [9]. The results presented in Figures 3 and 4 are therefore the first demonstration of promiscuous editing in the nucleus without the exogenous overexpression of APOBEC-1.…”

“…However, the editing efficiency was enhanced 5-fold when the Rev protein was coexpressed. Given that editing in the cytoplasm has never been demonstrated in wild-type McArdle cells [9], nor would it be driven by an increase in apoB RNA abundance in the cytoplasm [10], we propose that the enhanced editing occurred in the nucleus as a consequence of pre-mRNAs by-passing commitment to the spliceosome assembly and\or RNA export pathways. We cannot formally exclude the alternative explanation that unspliced CAT-apoB chimaeric RNAs were edited in the cytoplasm.…”

“…These results corroborate previous findings suggesting that there is a window of opportunity for editing apoB mRNA in the nucleus and that no further editing occurs in the cytoplasm of wild-type hepatic cells. Specifically, recently published subcellular-fractionation studies have shown that the low level of editing measured on this transcript as mRNA (1 %) occurred while the RNA was still in the nucleus [9].…”

“…Under physiological conditions, apoB RNA editing is a nuclear event [8,9]. It was first evident at the time pre-mRNAs were polyadenylated and spliced and was completed by the time the pre-mRNAs had been fully processed [8,10].…”

Flow-cytometric visualization of C>U mRNA editing reveals the dynamics of the process in live cells

“…17,30,61 Nonetheless, it has been shown that overexpression of APOBEC1 can induce editing also in the cytoplasm. 62 Considering that both APOBEC1 and ACF appear localized in different compartments depending on the cells in which they are expressed, 30,59,62-64 we wanted to assess the weight of the nuclear and of the cytoplasmic component of RNA editing in our model system.…”