Ethanol stimulates apolipoprotein B mRNA editing in the absence of de novo RNA or protein synthesis.

Apolipoprotein B (apoB) mRNA editing involves a site-specific modification of cytidine to form uridine. The reaction is catalyzed in the nucleus by a multi-protein editosome. Rat hepatic editing is regulated during development, metabolically and in response to ethanol. Ethanol stimulated editing in hepatocytes within minutes of exposure. In the present study, we show that ethanol stimulated apoB mRNA synthesis and apoB mRNA editing. Significantly, the proportion of edited apoB mRNA also increased following ethanol treatment of transcription or translation arrested cells. These data suggested that ethanol could regulate editing activity using pre-existing editosomal proteins. In addition, the presence of a suppressor of apoB mRNA editing activity was suggested by the finding that inhibition of mRNA or protein synthesis alone was sufficient to increase the proportion of edited RNA. It is proposed that the level of editing activity observed in hepatocytes may be the end result of positive and negative regulatory proteins.

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

A tripartite motif located in the centre of the 7.5 kb exon 26 of apolipoprotein B (apoB) mRNA directs editosome assembly and site-specific cytidine-to-uridine editing at nucleotide 6666. apoB mRNA editing is a post-transcriptional event, occurring primarily at the time exon 26 is spliced or at a time after splicing, but before nuclear export. We show, through reporter RNA constructs, that RNA splice sites suppress editing of precursor RNAs when placed proximal or distal to the editing site. Processed RNAs were edited more efficiently than precursor RNAs. Mutation of both the splice donor and acceptor sites was necessary for RNAs to be edited efficiently. The results suggested that commitment of pre-mRNA to the splicing and/or nuclear-export pathways may play a role in regulating editing-site utilization. The HIV-1 Rev-Rev response element ('RRE') interaction was utilized to uncouple the commitment of precursor RNAs to the spliceosome assembly pathway and associated nuclear-export pathway. Under these conditions, unspliced reporter RNAs were edited efficiently. We propose that pre-mRNA passage through the temporal or spatial restriction point where they become committed to spliceosome assembly contributes regulatory information for subsequent editosome activity.

Intracellular trafficking determinants in APOBEC-1, the catalytic subunit for cytidine to uridine editing of apolipoprotein B mRNA.

The posttranscriptional deamination editing of apolipoprotein B (apoB) mRNA catalyzed by APOBEC-1 (apoB mRNA editing catalytic subunit 1) is a nuclear process. The signals in APOBEC-1 responsible for its dual cytoplasmic/nuclear distribution have been evaluated. Residues 97-172 in the middle of APOBEC-1 together with its N-terminal 56 residues affect nuclear localization. Mutagenesis studies however revealed no discrete nuclear localization signal in APOBEC-1. Fifteen amino acids (Leu 173-Leu 187) within the previously identified C-terminal domain of APOBEC-1 were sufficient as a determinant for cytoplasmic distribution in that context. These residues failed to demonstrate nuclear export function in a reporter assay. Further, the distribution of APOBEC-1 in the cytoplasm did not respond to leptomycin B, suggesting that APOBEC-1 did not have nuclear export activity. The data suggested that there are at least three regions in APOBEC-1 that participate in its distribution in both the nucleus and the cytoplasm of editing competent cells; however, none of these meet the functional criteria of nuclear localization or nuclear export signals. The findings are discussed in terms of their implications in the regulation of nuclear editing activity and the possibility that interactions with chaperones may play a role in the cellular distribution of APOBEC-1.

Induction of cytidine to uridine editing on cytoplasmic apolipoprotein B mRNA by overexpressing APOBEC-1.

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, APOBEC-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.

APOBEC-1 dependent cytidine to uridine editing of apolipoprotein B RNA in yeast.

Cytidine to uridine editing of apolipoprotein B (apoB) mRNA requires the cytidine deaminase APOBEC-1 as well as a tripartite sequence motif flanking a target cytidine in apoB mRNA and an undefined number of auxiliary proteins that mediate RNA recognition and determine site-specific editing. Yeast engineered to express APOBEC-1 and apoB mRNA supported editing under conditions of late log phase growth and stationary phase. The cis -acting sequence requirements and the intracellular distribution of APOBEC-1 in yeast were similar to those described in mammalian cells. These findings suggest that auxiliary protein functions necessary for the assembly of editing complexes, or 'editosomes', are expressed in yeast and that the distribution of editing activity is to the cell nucleus.

Disproportionate relationship between APOBEC-1 expression and apolipoprotein B mRNA editing activity.

Apolipoprotein B (apoB) mRNA editing is a site-specific (nucleotide 6666) cytidine to uridine transition catalyzed by a cytidine deaminase, APOBEC-1, in the context of a multiprotein complex referred to as the C/U editosome. This report quantifies for the first time the effect of altering APOBEC-1 protein abundance on the proportion of edited apoB mRNAs using transfected McArdle rat hepatoma cells which had been sorted by flow cytometry into populations expressing different levels of green fluorescent protein-APOBEC-1 chimera, GFP-APOBEC. A correlation was observed in which increased expression of GFP-APOBEC protein resulted in a higher proportion of edited apoB mRNA. The number of enzyme molecules required to increase the proportion of edited apoB RNAs was disproportionately high relative to that which might have been predicted from a typical catalytic relationship. Moreover, editing of apoB mRNA at inappropriate sites (promiscuous editing) occurred in response to overexpressing GFP-APOBEC. The data suggest that experimental manipulation of APOBEC-1 abundance in the absence of other regulatory considerations will always result in some level of promiscuous editing. Coordinate expression of APOBEC-1 and the auxiliary proteins and/or regulation of their interactions may be required to increase editing activity without losing editing-site fidelity.

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

The cDNA encoding rat betaine-homocysteine S-methyltransferase (BHMT) was isolated through production of monoclonal antibodies against protein fractions enriched with apolipoprotein B (apo B)-mRNA-editing complexes. BHMT mRNA was expressed predominantly in liver, and also in kidney, but not in small intestine. In stable McArdle RH-7777 (McA) cell lines expressing differing levels of BHMT, the editing efficiency of apo B mRNA was unchanged. Evaluation of apo B-mRNA expression revealed that steady-state levels were increased significantly and in parallel with BHMT protein expression. The highest levels of BHMT mRNA and BHMT enzyme activity expressed in stably transfected McA cells were comparable with those found in rat hepatocytes. In contrast to the changes in apo B-mRNA abundance, levels of other apolipoprotein-encoding mRNAs and several liver-specific and ubiquitously expressed mRNAs were unchanged by BHMT expression. In the cell line expressing the highest level of BHMT, apo B-containing lipoprotein secretion was increased, indicating utilization of increased endogenous message. Results suggest that apo B-mRNA abundance in McA cells is related to the expression of BHMT, an enzyme important in homocysteine metabolism.

Lipoprotein alterations in 10- and 20-week-old Zucker diabetic fatty rats: hyperinsulinemic versus insulinopenic hyperglycemia.

Lipoprotein and apolipoprotein parameters were studied in the male Zucker diabetic fatty (ZDF) rat at 10 and 20 weeks of age, corresponding to hyperinsulinemic and insulinopenic type 2 diabetes mellitus, respectively. At both ages, ZDF rats had elevated serum triglycerides, free fatty acids, and corticosterone, whereas 20-week ZDF rats had reduced thyroid hormones. At 10 weeks, the hyperlipidemia was confined to elevations in pre-beta triglyceride-rich (d < 1.006 g/mL) lipoproteins. By 20 weeks, all lipoprotein density fractions were increased compared with lean rats, with substantial increases in both low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol. In ZDF rats, there was a progressive increase in apolipoprotein B (apo B) from 1.9 times control at 10 weeks to three times control at 20 weeks. The increase in apo B was accompanied by a shift of apo B, particularly B100, from very-low-density lipoprotein (VLDL) into denser lipoproteins corresponding to intermediate-density lipoproteins plus LDLs (1.006 < d < 1.063 g/mL). In Zucker and 10-week ZDF rats, in the presence of hyperinsulinemia, the increase in serum apo B was predominantly apo B48 present in VLDL. By 20 weeks, when ZDF rats are insulinopenic, the mass ratio of B48:B100 shifted from 2.7 to 0.7. The shift was associated with a decrease in hepatic-edited apo B mRNA. Apo E increased in lean rats between 10 and 20 weeks of age. Although apo E also increased in ZDF rats, the increase by 20 weeks was less than that of lean rats. The molar ratio of apo E to B in VLDL was decreased in ZDF rats. In lean rats, greater than 50% of apo E was present in HDL, in contrast to ZDF rats, where less than 20% of apo E was present in HDL. VLDL apo E shifted to denser fractions by 20 weeks of age, similar to apo B. The apo C level was more than double compared with the level in lean rats and was redistributed from the HDL fraction to lipoprotein fractions containing apo B. Both apo A-I and apo A-IV levels more than doubled between 10 and 20 weeks in ZDF rats. The ZDF rat model may be useful in comparative studies of lipoproteins during diabetic progression from hyperinsulinemia to insulinopenia.

Ethanol increases apolipoprotein B mRNA editing in rat primary hepatocytes and McArdle cells.

Apolipoprotein B (apoB) mRNA editing involves a site-specific cytidine to uridine transition catalyzed by the cytidine deaminase, APOBEC-1, in the context of and regulated by a multi-protein-containing editosome. ApoB mRNA editing in vivo is subject to tissue specific, developmental and metabolic regulation. We demonstrate for the first time that the amount of edited apoB mRNA in rat primary hepatocytes is markedly increased subsequent to transient treatment with ethanol in vitro. The apparent change in editing efficiency was dose-dependent (from 0.1%-2.4% initial ethanol dose) and occurred with rapid onset. The proportion of edited apoB mRNA was also markedly enhanced in a rat hepatoma cell line, McArdle RH7777 cells and in a stable McArdle cell line over-expressing APOBEC-1 by transient treatment with 2.5 % ethanol. In contrast, the apoB mRNA editing in a human hepatoma cell line, HepG2 cells and a stable HepG2 cell line over-expressing APOBEC-1 did not respond to ethanol treatment. The data support the possibility that editing activity is ethanol-responsive but suggest that this change is cell type-specific.

Apolipoprotein B RNA sequence 3' of the mooring sequence and cellular sources of auxiliary factors determine the location and extent of promiscuous editing.

Apolipoprotein B (apoB) RNA editing involves a cytidine to uridine transition at nucleotide 6666 (C6666) 5' of an essential cis -acting 11 nucleotide motif known as the mooring sequence. APOBEC-1 (apoB editing catalytic sub-unit 1) serves as the site-specific cytidine deaminase in the context of a multiprotein assembly, the editosome. Experimental over-expression of APOBEC-1 resulted in an increased proportion of apoB mRNAs edited at C6666, as well as editing of sites that would otherwise not be recognized (promiscuous editing). In the rat hepatoma McArdle cell line, these sites occurred predominantly 5' of the mooring sequence on either rat or human apoB mRNA expressed from transfected cDNA. In comparison, over-expression of APOBEC-1 in HepG2 (HepG2-APOBEC) human hepatoma cells, induced promiscuous editing primarily 5' of the mooring sequence, but sites 3' of the C6666 were also used more efficiently. The capacity for promiscuous editing was common to rat, rabbit and human sources of APOBEC-1. The data suggested that differences in the distribution of promiscuous editing sites and in the efficiency of their utilization may reflect cell-type-specific differences in auxiliary proteins. Deletion of the mooring sequence abolished editing at the wild type site and markedly reduced, but did not eliminate, promiscuous editing. In contrast, deletion of a pair of tandem UGAU motifs 3' of the mooring sequence in human apoB mRNA selectively reduced promiscuous editing, leaving the efficiency of editing at the wild type site essentially unaffected. ApoB RNA constructs and naturally occurring mRNAs such as NAT-1 (novel APOBEC-1 target-1) that lack this downstream element were not promiscuously edited in McArdle or HepG2 cells. These findings underscore the importance of RNA sequences and the cellular context of auxiliary factors in regulating editing site utilization.

Effects of fatty acids on apolipoprotein B secretion by McArdle RH-7777 rat hepatoma cells.

The effect of oleic acid (OA), stearic acid (SA) and elaidic acid (EA) on cellular and secreted apolipoprotein (apo) B was examined in McArdle RH-7777 (McArdle) hepatoma cells and in primary rat hepatocytes. ApoB secretion by McArdle cells was significantly inhibited by 20% in 8 h incubations in medium containing EA and SA and by 50% in medium containing OA. In contrast, apo B secretion and cellular apo B of primary rat hepatocytes was relatively unaffected by incubations in medium containing fatty acids. Both B100 and B48 secretion in McArdle wild type and B48 in apo B mRNA editing enzyme catalytic polypeptide transfectants expressing B48 were inhibited to a similar extent indicating an effect of OA on both apo B species. The effect of OA occurred without changes in cellular apo B or in apo B mRNA abundance suggesting a post-transcriptional mechanism. Time course studies indicate that the suppressive effect of OA requires 4 h of incubation suggesting the depletion of a limiting factor important in apoB secretion. By increasing the proportion of palmitic acid to OA in the medium, apoB secretion by McArdle cells was progressively restored to control levels implicating an unique role for newly synthesized saturated fatty acid.

Base-modification mRNA editing through deamination--the good, the bad and the unregulated.

RNA editing is a co- or post-transcriptional process in which select nucleotide sequences in RNA are altered from that originally encoded in the genome. The mRNAs encoding apolipoprotein B and some glutamate receptor subunits of ionotropic membrane channels are edited by site-specific base-deamination systems. Although these editing systems differ markedly in their mechanism for RNA-substrate binding and in their catalytic subunits, recent results suggest potentially common solutions to the problem of editing-site selectivity. The data suggest that there are multiple editing complexes or 'editosomes', which manifest editing-site preferences due to their macromolecular composition.

Regulation of hepatic apolipoprotein B RNA editing in the genetically obese Zucker rat.

Hepatic apolipoprotein (apo) B RNA editing was examined in the genetically obese hyperinsulinemic and hypertriglyceridemic Zucker rat. In obese Zucker rats, apo B RNA editing was increased 42% relative to that in lean controls. Correspondingly, the proportion of serum triglyceride-rich lipoprotein containing apo B48 increased 4.7-fold in the obese Zucker rat. Quantification of hepatic total apo B mRNA showed no difference between obese Zucker and lean control rats. In contrast, the hepatic mRNA encoding APOBEC-1, the catalytic subunit of the RNA editing activity, demonstrated an increased abundance of 1.8-fold in obese Zucker rats versus lean controls.

Efficient activation of transcription in yeast by the BPV1 E2 protein.

The full-length gene product encoded by the E2 open reading frame (ORF) of bovine papillomavirus type 1 (BPV1) is a transcriptional transactivator. It is believed to mediate its effect on the BPV1 long control region (LCR) by binding to motifs with the consensus sequence ACCN6GGT. The minimal functional cis active site, called the E2 response element (E2RE), in mammalian cells comprises two copies of this motif. Here we have shown that E2 can function in Saccharomyces cerevisiae by placing an E2RE upstream of a synthetic yeast assay promoter which consists of a TATA motif and an mRNA initiation site, spaced correctly. This E2RE-minimal promoter is only transcriptionally active in the presence of E2 protein and the resulting mRNA is initiated at the authentic start site. This is the first report of a mammalian viral transactivator functioning in yeast. The level of activation by E2 via the E2RE was the same as observed with the highly efficient authentic PGK promoter where the upstream activation sequence is composed of three distinct elements. Furthermore a single E2 motif which is insufficient in mammalian cells as an activation site was as efficiently utilized in yeast as the E2RE (2 motifs). Previous studies have shown that mammalian cellular activators can function in yeast and our data now extend this to viral-specific activators. Our data indicate however that while the mechanism of transactivation is broadly conserved there may be significant differences at the detailed level.