Mark P. Sowden

MESSENGER RNA EDITING IN MAMMALS: NEW MEMBERS OF THE APOBEC FAMILY SEEKING ROLES IN THE FAMILY BUSINESS

Alteration of mRNA sequence through base modification mRNA editing frequently generates protein diversity. Several proteins have been identified as being similar to C-to-U mRNA editing enzymes based on their structural domains and the occurrence of a catalytic domain characteristic of cytidine deaminases. In light of the hypothesis that these proteins might represent novel mRNA editing systems that could affect proteome diversity, we consider their structure, expression and relevance to biomedically significant processes or pathologies.

Overexpression of APOBEC-1 results in mooring sequence-dependent promiscuous RNA editing.

Apolipoprotein B (apoB) RNA editing involves site-specific deamination of a cytidine to a uridine. A mooring sequence, a spacer region, and a regulator region are components of the apoB RNA editing motif of which only the mooring sequence is both necessary and sufficient for editosome assembly and editing. The catalytic component of the editosome is APOBEC-1. In rat hepatoma, stable cell lines, overexpression of APOBEC-1 resulted in 3-6-fold stimulation of the editing efficiency on either rat endogenous apoB RNA or transiently expressed human apoB RNA. In these cell lines, cytidines in addition to the one at the wild type site were edited. The occurrence and efficiency of this “promiscuous” editing increased with increasing expression of APOBEC-1. Promiscuous editing was restricted to cytidines 5′ of the mooring sequence and only occurred on RNAs that had been edited at the wild type site. Moreover, RNAs with mutant editing motifs supported high efficiency but low fidelity editing in the presence of high levels of APOBEC-1. This study demonstrates that overexpression of APOBEC-1 can increase the efficiency of site-specific editing but can also result in promiscuous editing.

Hepatic very-low-density lipoprotein and apolipoprotein B production are increased following in vivo induction of betaine–homocysteine S-methyltransferase

We have previously reported a positive correlation between the expression of BHMT (betaine-homocysteine S-methyltransferase) and ApoB (apolipoprotein B) in rat hepatoma McA (McArdle RH-7777) cells [Sowden, Collins, Smith, Garrow, Sparks and Sparks (1999) Biochem. J. 341, 639-645]. To examine whether a similar relationship occurs in vivo, hepatic BHMT expression was induced by feeding rats a Met (L-methionine)-restricted betaine-containing diet, and parameters of ApoB metabolism were evaluated. There were no generalized metabolic abnormalities associated with Met restriction for 7 days, as evidenced by control levels of serum glucose, ketones, alanine aminotransferase and L-homocysteine levels. Betaine plus the Met restriction resulted in lower serum insulin and non-esterified fatty acid levels. Betaine plus Met restriction induced hepatic BHMT 4-fold and ApoB mRNA 3-fold compared with Met restriction alone. No changes in percentage of edited ApoB mRNA were observed on the test diets. An increase in liver ApoB mRNA correlated with an 82% and 46% increase in ApoB and triacylglycerol production respectively using in vivo Triton WR 1339. Increased secretion of VLDL (very-low-density lipoprotein) with Met restriction plus betaine was associated with a 45% reduction in liver triacylglycerol compared with control. Nuclear run-off assays established that transcription of both bhmt and apob genes was also increased in Met-restricted plus betaine diets. No change in ApoB mRNA stability was detected in BHMT-transfected McA cells. Hepatic ApoB and BHMT mRNA levels were also increased by 1.8- and 3-fold respectively by betaine supplementation of Met-replete diets. Since dietary betaine increased ApoB mRNA, VLDL ApoB and triacylglycerol production and decreased hepatic triacylglycerol, results suggest that induction of apob transcription may provide a potential mechanism for mobilizing hepatic triacylglycerol by increasing ApoB available for VLDL assembly and secretion.

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.

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 editingcatalytic 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-1complementation 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.

Two proteins essential for apolipoprotein B mRNA editing are expressed from a single gene through alternative splicing

Apolipoprotein B (apoB) mRNA editing involves site-specific deamination of cytidine to form uridine, resulting in the production of an in-frame stop codon. Protein translated from edited mRNA is associated with a reduced risk of atherosclerosis, and hence the protein factors that regulate hepatic apoB mRNA editing are of interest. A human protein essential for apoB mRNA editing and an eight-amino acid-longer variant of no known function have been recently cloned. We report that both proteins, henceforth referred to as ACF64 and ACF65, supported APOBEC-1 (the catalytic subunit of the editosome) equivalently in editing of apoB mRNA. They are encoded by a single 82-kb gene on chromosome 10. The transcripts are encoded by 15 exons that are expressed from a tissue-specific promoter minimally contained within the −0.33-kb DNA sequence. ACF64 andACF65 mRNAs are expressed in both liver and intestinal cells in an approximate 1:4 ratio. Exon 11 is alternatively spliced to include or exclude 24 nucleotides of exon 12, thereby encodingACF65 and ACF64, respectively. Recognition motifs for the serine/arginine-rich (SR) proteins SC35, SRp40, SRp55, and SF2/ASF involved in alternative RNA splicing were predicted in exon 12. Overexpression of these SR proteins in liver cells demonstrated that alternative splicing of a minigene-derived transcript to expressACF65 was enhanced 6-fold by SRp40. The data account for the expression of two editing factors and provide a possible explanation for their different levels of expression.

Structural Phylogenetic Analysis of Activation-Induced Deaminase Function

In mammals, activation-induced deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) of Ig genes. SHM and CSR activities require separate regions within AID. A chromosome region maintenance 1 (CRM1)-dependent nuclear export signal (NES) at the AID C terminus is necessary for CSR, and has been suggested to associate with CSR-specific cofactors. CSR appeared late in AID evolution, during the emergence of land vertebrates from bony fish, which only display SHM. Here, we show that AID from African clawed frog (Xenopus laevis), but not pufferfish (Takifugu rubripes), can induce CSR in AID-deficient mouse B cells, although both are catalytically active in bacteria and mammalian cell systems, albeit at decreased level. Like mammalian AID, Takifugu AID is actively exported from the cell nucleus by CRM1, and the Takifugu NES can substitute for the equivalent region in human AID, indicating that all the CSR-essential NES motif functions evolutionarily predated CSR activity. We also show that fusion of the Takifugu AID catalytic domain to the entire human noncatalytic domain restores activity in mammalian cells, suggesting that AID features mapping within the noncatalytic domain, but outside the NES, influence its function.

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.

Cyclophilin A Is Required for Angiotensin II–Induced p47phox Translocation to Caveolae in Vascular Smooth Muscle Cells

Objective—Angiotensin II (AngII) signal transduction in vascular smooth muscle cells (VSMC) is mediated by reactive oxygen species (ROS). Cyclophilin A (CyPA) is a ubiquitously expressed cytosolic protein that possesses peptidyl-prolyl cis-trans isomerase activity, scaffold function, and significantly enhances AngII-induced ROS production in VSMC. We hypothesized that CyPA regulates AngII-induced ROS generation by promoting translocation of NADPH oxidase cytosolic subunit p47phox to caveolae of the plasma membrane. Approach and Results—Overexpression of CyPA in CyPA-deficient VSMC (CyPA−/−VSMC) significantly increased AngII-stimulated ROS production. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors (VAS2870 or diphenylene iodonium) significantly attenuated AngII-induced ROS production in CyPA and p47phox-overexpressing CyPA−/−VSMC. Cell fractionation and sucrose gradient analyses showed that AngII-induced p47phox plasma membrane translocation, specifically to the caveolae, was reduced in CyPA−/−VSMC compared with wild-type–VSMC. Immunofluorescence studies demonstrated that AngII increased p47phox and CyPA colocalization and translocation to the plasma membrane. In addition, immunoprecipitation of CyPA followed by immunoblotting of p47phox and actin showed that AngII increased CyPA and p47phox interaction. AngII-induced p47phox and actin cell cytoskeleton association was attenuated in CyPA−/−VSMC. Mechanistically, inhibition of p47phox phosphorylation and phox homology domain deletion attenuated CyPA and p47phox interaction. Finally, cyclosporine A and CyPA-peptidyl-prolyl cis-trans isomerase mutant, R55A, inhibited AngII-stimulated CyPA and p47phox association in VSMC, suggesting that peptidyl-prolyl cis-trans isomerase activity was required for their interaction. Conclusions—These findings provide the mechanism by which CyPA is an important regulator for AngII-induced ROS generation in VSMC through interaction with p47phox and cell cytoskeleton, which enhances the translocation of p47phox to caveolae.

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.