Phylogenetic Analysis of the Apolipoprotein B mRNA-editing Region

C to U editing of apolipoprotein B (apoB) RNA requires a multicomponent holoenzyme complex in which minimal constituents include apobec-1 and apobec-1 complementation factor (ACF). We have examined the predicted functional domains in ACF in binding apoB RNA, interaction with apobec-1, and complementation of RNA editing. We demonstrate that apoB RNA binding and apobec-1-interacting domains are defined by two partially overlapping regions containing the NH 2 -terminal RNA recognition motifs of ACF. Both apoB RNA binding and apobec-1 interaction are required for editing complementation activity. ACF is a nuclear protein that upon cotransfection with apobec-1 results in nuclear colocalization and redistribution of apobec-1 from the cytoplasm. ACF constructs with deletions or mutations in the putative nuclear localization signal (NLS) still localize in the nucleus of transfected cells but do not colocalize with apobec-1, the latter remaining predominantly cytoplasmic. These observations suggest that the putative NLS motif in ACF is not responsible for its nucleo-cytoplasmic trafficking. By contrast, proteinprotein interaction is important for the nuclear import of apobec-1. Taken together, these data suggest that functional complementation of C to U RNA editing by apobec-1 involves the NH 2 -terminal 380 residues of ACF.

Section: Deletional Analysis Of Acf: Functional Implications Frommentioning

confidence: 94%

Mutagenesis of Apobec-1 Complementation Factor Reveals Distinct Domains That Modulate RNA Binding, Protein-Protein Interaction with Apobec-1, and Complementation of C to U RNA-editing Activity

Blanc¹,

Henderson²,

Kennedy³

et al. 2001

“…The weak cross-linking of ASP may be explained by the presence of KSRP in our fractions. Two recent studies re-examined the secondary RNA structure requirements for efficient editing at C 6666 (60,61). Both studies concluded that C 6666 is located at a junction between a double-stranded and a single-stranded RNA region.…”

Section: Discussionmentioning

confidence: 99%

“…Both studies concluded that C 6666 is located at a junction between a double-stranded and a single-stranded RNA region. This junction was localized either at the loop of a hairpin (60) or at the beginning of a doublestranded stem formed by the mooring sequence (nucleotides 6671-6681) and a 3Ј-efficiency element (nucleotides 6718 -6746) (61). A protein that specifically recognizes this junction between a double-stranded and a single-stranded region would fit these models.…”

Section: Discussionmentioning

confidence: 99%

Purification and Molecular Cloning of a Novel Essential Component of the Apolipoprotein B mRNA Editing Enzyme-Complex

Lellek¹,

Kirsten²,

Diehl³

et al. 2000

159

179

Editing of apolipoprotein B (apoB) mRNA requires the catalytic component APOBEC-1 together with "auxiliary" proteins that have not been conclusively characterized so far. Here we report the purification of these additional components of the apoB mRNA editing enzyme-complex from rat liver and the cDNA cloning of the novel APOBEC-1-stimulating protein (ASP). Two proteins copurified into the final active fraction and were characterized by peptide sequencing and mass spectrometry: KSRP, a 75-kDa protein originally described as a splicing regulating factor, and ASP, a hitherto unknown 65-kDa protein. Separation of these two proteins resulted in a reduction of APOBEC-1-stimulating activity. ASP represents a novel type of RNA-binding protein and contains three single-stranded RNA-binding domains in the amino-terminal half and a putative double-stranded RNA-binding domain at the carboxyl terminus. Purified recombinant glutathione S-transferase (GST)-ASP, but not recombinant GST-KSRP, stimulated recombinant GST-APOBEC-1 to edit apoB RNA in vitro. These data demonstrate that ASP is the second essential component of the apoB mRNA editing enzymecomplex. In rat liver, ASP is apparently associated with KSRP, which may confer stability to the editing enzymecomplex with its substrate apoB RNA serving as an additional auxiliary component.

“…A tripartite RNA sequence motif, consisting of an 11-nucleotide mooring sequence, a spacer, and a regulatory element, is required for site-specific RNA editing (4 -8). Recently, a stem-loop model of secondary structure involving the essential sequence required for editing has been proposed (9,10). These cis-acting elements are required for the assembly of an editing complex, the C/U editosome (5,11), and site-specific editing activity.…”

mentioning

confidence: 99%

Induction of Cytidine to Uridine Editing on Cytoplasmic Apolipoprotein B mRNA by Overexpressing APOBEC-1

Yang¹,

Sowden²,

Smith³

2000

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, APO-BEC-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. Apolipoprotein B (apoB)1 mRNA editing involves a site-specific deamination reaction wherein a cytidine at nucleotide 6666 is changed to a uridine thereby placing an in frame UAA stop codon within the 14-kilobase apoB mRNA (1, 2). Translation of the unedited and edited variants of apoB mRNA generates two isoforms of apoB proteins, apoB100 and apoB48, which behave differently in terms of lipoprotein secretion and uptake by peripheral cells (3). A tripartite RNA sequence motif, consisting of an 11-nucleotide mooring sequence, a spacer, and a regulatory element, is required for site-specific RNA editing (4 -8). Recently, a stem-loop model of secondary structure involving the essential sequence required for editing has been proposed (9, 10). These cis-acting elements are required for the assembly of an editing complex, the C/U editosome (5, 11), and site-specific editing activity. APOBEC-1, the cytidine deaminase responsible for editing (12), is the catalytic subunit of the editosome (9) and may function in this capacity as a dimer (13,14).APOBEC-1 alone cannot edit apoB mRNA and requires multiple yet-to-be characterized proteins referred to collectively as the auxiliary proteins (15-20). Auxiliary proteins are broadly expressed in mammalian cell lines and tissues, independent of the expression of APOBEC-1 and apoB mRNA (12, 16 -18, 21). Recently, APOBEC-1 complementation factor (ACF) has been cloned (40). ACF is 64.3-kDa RNA binding protein, it binds to apoB mRNA in vitro and in vivo. ACF and APOBEC-1 comprise the minimal protein requirements for specific and efficient editing of apoB mRNA in vitro. Other candida...