Multiple protein domains determine the cell type-specific nuclear distribution of the catalytic subunit required for apolipoprotein B mRNA editing

Abstract

Apolipoprotein B (apoB) mRNA editing catalyzed by apoB mRNA editing catalytic subunit 1 (APOBEC-1) has been proposed to be a nuclear process. To test this hypothesis, the subcellular distribution of hemagglutinin-(HA) tagged APOBEC-1 expressed in transiently transfected hepatoma cells was determined by indirect immunof luorescence microscopy. HA-APOBEC-1 was detected in both the nucleus and cytoplasm of rat and human hepatoma cells. Mutagenesis of APOBEC-1 demonstrated that the N-terminal 56 amino acids (1-56) were necessary for the nuclear distribution of APOBEC-1, but this region did not contain a functional nuclear localization signal (NLS). However, we identified a 24-amino acid domain in the C terminus of APOBEC-1 with characteristics of a cytoplasmic retention signal (CRS) or a nuclear export signal (NES). These data suggest, therefore, that the nuclear import of APOBEC-1 may not be mediated by a positive NLS; rather, it may be achieved by overcoming the effect of a CRS͞NES. We also demonstrated that the nuclear distribution of APOBEC-1 occurred only in cell lines that were capable of editing apoB RNA. We propose that the cellular distribution of APOBEC-1 is determined by multiple domains within this protein, and a nuclear localization of the enzyme may be regulated by cell type-specific factors that render these cells uniquely editing competent.Apolipoprotein B (apoB) mRNA undergoes posttranscriptional deamination editing of a cytidine at nucleotide 6666, converting a CAA glutamine codon to a UAA in-frame translation stop codon (1, 2). Translation of unedited and edited variants of apoB mRNA generates two isoforms of apoB proteins, apoB 100 and apoB 48 , which play distinct roles in lipid metabolism pathways (3). A tripartite RNA sequence motif consisting of a mooring sequence, a spacer, and a regulatory element is required for site-specific RNA editing (4-8).apoB mRNA editing catalytic subunit 1 (APOBEC-1) is the catalytic subunit of the editosome, a multiprotein complex assembled to edit apoB RNA (9-11). It is a cytidine deaminase and shares amino acid homology in its catalytic domain with a variety of cytidine and adenosine deaminases (12-14). In the absence of editosomal proteins, APOBEC-1 cannot edit apoB RNA (11,12,15). Proteins that complement APOBEC-1 in apoB mRNA editing activity (auxiliary proteins) have been identified in diverse tissues and cell types (11,12,(16)(17)(18)) and seem to play important but uncharacterized roles in regulating editing activity during tissue development as well as in response to nutritional stress and hormone stimulation (17)(18)(19).Compelling evidence for a nuclear localization of the editing activity has been provided through the demonstration that a portion of cellular apoB RNA molecules is edited before polyadenylation and splicing (20,21). APOBEC-1 has two short stretches of basic amino acids separated by 12 residues in its N terminus, which closely resemble the consensus bipartite nuclear localization signal (NLS) (18,22).Indirect immunofluorescence mi...

Active nuclear import and cytoplasmic retention of activation-induced deaminase

“…AID dimerization through the β2 strand has been proposed based on the structure of A2 33 . We tested the effect of perturbing the predicted AID β2, residues [40][41][42][43][44][45][46][47][48][49][50][51][52][53] in our model, on oligomerization by introducing one (F46A), two (F46A/Y48A) 33 or four (F46A/Y48A/R50G/N51A, named AID FYRN) mutations. The ability of each of these mutants to interact with wt AID was monitored by comparing their efficiency in coimmunoprecipitating with AID-Flag.…”

“…Similarly to AID, the N-terminal domain of APOBEC1 is necessary but not sufficient for nuclear import 45,46 suggesting that APOBEC1 might also have a conformational NLS.…”

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

“…Several other auxiliary protein candidates have also been described that had binding affinities for APOBEC-1 and\or apoB mRNA and that demonstrated the ability to modulate editing efficiency [23,24,26,31,33,34,37,38]. Although, under biological conditions, editing occurs only in the nucleus [8,9], nuclear and cytoplasmic distributions have been described for both APOBEC-1 and ACF [9,52,53]. Nuclear editing has been characterized as occurring coincident with, or immediately after, pre-mRNA splicing [8][9][10].…”

“…Current evidence suggests that editing of all RNAs occurred in the nucleus ; however, we cannot rule out the possibility that editing of the unspliced apoB chimaeric RNA occured after it was exported to the cytoplasm. Although we have reported cytoplasmic localization of APOBEC-1 and ACF [9,52], there is no precedent for them to form active editosomes in the cytoplasm in i o or for there to be cytoplasmic editing when APOBEC-1 expression was under biological regulation [9]. Specifically, cytoplasmic APOBEC-1 and ACF existed within an inactive 60 S complex that only became an activated 27 S editosome in the nucleus [11,22].…”

Phylogenetic Analysis of the Apolipoprotein B mRNA-editing Region

“…The apoB mRNA editing complex locates the apoB pre-mRNA among tens of thousands pre-mRNAs (12,13). No other mRNAs have been identified that are edited by this mechanism, and with the exception of a minor site in the human apoB mRNA, no other cytidines in the apoB mRNA are edited (14).…”