Insulin increases expression of apobec-1, the catalytic subunit of the apolipoprotein B mRNA editing complex in rat hepatocytes

Two novel mRNA transcripts have been identified that result from species-and tissue-specific, alternative polyadenylation and splicing of the pre-mRNA encoding the apolipoprotein B (apoB) editing catalytic subunit 1 (APOBEC-1) complementation factor (ACF) family of related proteins. The alternatively processed mRNAs encode 43-and 45-kDa proteins that are components of the previously identified p44 cluster of apoB RNA binding, editosomal proteins. Recombinant ACF45 displaced ACF64 and ACF43 in mooring sequence RNA binding but did not demonstrate strong binding to APOBEC-1. In contrast, ACF43 bound strongly to APOBEC-1 but demonstrated weak binding to mooring sequence RNA. Consequently ACF45/43 complemented APOBEC-1 in apoB mRNA editing with less efficiency than full-length ACF64. These data, together with the finding that all ACF variants were co-expressed in rat liver nuclei (the site of apoB mRNA editing), suggested that ACF variants might compete with one another for APOBEC-1 and apoB mRNA binding and thereby contribute to the regulation of apoB mRNA editing. In support for this hypothesis, the ratio of nuclear ACF65/64 to ACF45/43 decreased when hepatic editing was inhibited by fasting and increased when editing was re-stimulated by refeeding. These findings suggested a new model for the regulation of apoB mRNA editing in which the catalytic potential of editosomes is modulated at the level of their assembly by alterations in the relative abundance of multiple related RNA-binding auxiliary proteins and the expression level of APOBEC-1.Alternative mRNA polyadenylation, splicing, and mRNA editing are mechanisms of post-transcriptional RNA processing that introduce diversity in mammalian mRNAs, thereby regulating the number of structurally and functionally different proteins encoded by a single gene (reviewed in Refs. 1 and 2). Base modification RNA editing may involve the enzymatic deamination of single nucleotides within splice site consensus motifs (3) or in protein coding sequence, often with physiologically significant effects (4). Adenosine to inosine editing of mRNAs, such as that occurring on mRNAs encoding the glutamate-gated calcium channel and serotonin 2C receptor subunits, is mediated by a family of adenosine deaminases active on RNAs that function independently of cofactors to deaminate their cognate target within double-stranded RNA (reviewed in Ref. 5). Multiple cytidine deaminases active on RNA have been predicted through their homology with the zinc-dependent cytidine deaminase domain of the mRNA editing enzyme APO-BEC-1 1 (Ref. 6 and references therein); however, only APO-BEC-1 (7, 8) and CDD1 (9) have been shown to deaminate cytidines within mRNA. Unlike adenosine deaminases active on RNA, cytidine deaminases active on RNA minimally require RNA-binding auxiliary proteins for their activity on RNA.Cytidine to uridine deamination of nucleotide 6666 of apolipoprotein B mRNA occurs in the small intestine of all, and the livers of some, mammalian species (10 -12). The glutaminespecifying CAA codon...

Section: Discussionmentioning

confidence: 99%

Section: The Expression Of Acf Splice Variants Responds To Metabolic mentioning

confidence: 90%

Identification of Novel Alternative Splice Variants of APOBEC-1 Complementation Factor with Different Capacities to Support Apolipoprotein B mRNA Editing

Sowden

Lehmann

Lin

et al. 2004

“…Differences in the abundance of endogenous SR proteins in HepG2 cells, differences in expression levels of the transfected SR protein, or context effects caused by the sequences surrounding each motif may have determined the regulatory effect of SRp40. Given the role of SRp40 in alternative RNA splicing of ACF65, it is of interest that both apoB mRNA editing and SRp40 expression are induced late during liver development (17,33) and are regulated by insulin (33)(34)(35).…”

Section: Discussionmentioning

confidence: 99%

Two Proteins Essential for Apolipoprotein B mRNA Editing Are Expressed from a Single Gene through Alternative Splicing

Dance

Sowden

Cartegni

et al. 2002

Apolipoprotein B (apoB) mRNA editing involves sitespecific 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 and ACF65 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 encoding ACF65 and ACF64, respectively. Recognition motifs for the serine/argininerich (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 express ACF65 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.

“…Insulin may regulate VLDL secretion via multiple mechanisms including effects on apoB synthesis and degradation (reviewed in Ref. 22), modulation of MTP expression (23), apoB phosphorylation (24), and apoB mRNA editing (25)(26)(27). Early studies of apoB expression suggested that apoB is under regulatory control of insulin (22).…”

mentioning

confidence: 99%

Hepatic Very Low Density Lipoprotein-ApoB Overproduction Is Associated with Attenuated Hepatic Insulin Signaling and Overexpression of Protein-tyrosine Phosphatase 1B in a Fructose-fed Hamster Model of Insulin Resistance

Taghibiglou¹,

Rashid-Kolvear²,

Iderstine³

et al. 2002

209

157

A fructose-fed hamster model of insulin resistance was previously documented to exhibit marked hepatic very low density lipoprotein (VLDL) overproduction. Here, we investigated whether VLDL overproduction was associated with down-regulation of hepatic insulin signaling and insulin resistance. Hepatocytes isolated from fructose-fed hamsters exhibited significantly reduced tyrosine phosphorylation of the insulin receptor and insulin receptor substrates 1 and 2. Phosphatidylinositol 3-kinase activity as well as insulin-stimulated Akt-Ser 473 and Akt-Thr 308 phosphorylation were also significantly reduced with fructose feeding. Interestingly, the protein mass and activity of protein-tyrosine phosphatase-1B (PTP-1B) were significantly higher in fructose-fed hamster hepatocytes. Chronic ex vivo exposure of control hamster hepatocytes to high insulin also appeared to attenuate insulin signaling and increase PTP-1B. Elevation in PTP-1B coincided with marked suppression of ER-60, a cysteine protease postulated to play a role in intracellular apoB degradation, and an increase in the synthesis and secretion of apoB. Sodium orthovanadate, a general phosphatase inhibitor, partially restored insulin receptor phosphorylation and significantly reduced apoB secretion. In summary, we hypothesize that fructose feeding induces hepatic insulin resistance at least in part via an increase in expression of PTP-1B. Induction of hepatic insulin resistance may then contribute to reduced apoB degradation and enhanced VLDL particle assembly and secretion.