Abstract
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.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF442133–AF442135
These data, together with the finding that all ACF variants were co-expressed in rat liver nuclei, suggested that ACF variants might compete with one another for APOBEC-1 and apolipoprotein B (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
ApoB mRNA editing is catalyzed by a multiprotein complex, or editosome [16, 17], that assembles upon an 11-nucleotide mooring sequence positioned 3Ј of the edited cytidine at nucleotide position 6666 [18, 19]
Summary
CDNA Isolation, RACE, and DNA Manipulations—Plaque hybridization screening of a gt11-based rat liver 5Ј stretch cDNA library (Stratagene) with radioactively labeled probes (RTS Rad-Prime system; Invitrogen) and 3Ј-RACE on Marathon ready rat liver cDNAs (Clontech) were performed according to the recommendations from the manufacturer. Rat genomic DNA (Clontech) was amplified using Expand High Fidelity PCR System (Roche Molecular Biochemicals). Rat liver nuclear extracts were UV cross-linked to apoB RNA [38] and the complexes immunoprecipitated with NH2- or COOH-terminal specific antibody at 4 °C overnight. Transfections were performed using FuGENE 6 (Roche Molecular Biochemicals), and total cellular RNA or protein was isolated as previously described [40]. PCR-amplified acf cDNA variants, apobec-1, and apoB DNA (nucleotides 6413– 6802) were subcloned into pAc 5.1-V5-His (Invitrogen). RNA Binding Assay—Ultraviolet cross-linking assays were performed as previously described using 498-nucleotide in vitro transcripts of apoB RNA [38], NF-1 RNA [42], or WT-1 (a control RNA lacking mooring sequence motifs) [17]. For protein excess competition, purified recombinant ACF64, ACF45, and/or ACF43 were combined in editing buffer prior to the addition of radiolabeled apoB RNA and UV cross-linking. Oligonucleotides Used in RT-PCR Analyses—Oligonucleotides used were (5Ј-CAGTTACGTTAGGTACACCC), (5Ј-TATATGCCAAATACCCAC), 43 (5Ј-AAGCCAGTGGACAAGGAC), and 45 (5Ј-ACATGGATGTGGTGCACACC)
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