Cloning of an Apobec-1-binding protein that also interacts with apolipoprotein B mRNA and evidence for its involvement in RNA editing.

Apolipoprotein (apo)B mRNA editing is mediated by a multiprotein editosome complex. Apobec-1 is the catalytic component of this complex, but other proteins involved in editing have not been identified. We used the yeast two-hybrid system to identify an apobec-1-interacting protein, ABBP-1. ABBP-1 contains 331 amino acid residues and is identical to a previously reported human type A/B hnRNP except for a 47-residue insertion at its C-terminal region. It contains typical RNP motifs at its N-terminal half and glycine-rich motifs in the C-terminal region. Northern blot analysis indicates that ABBP-1 mRNA is distributed in multiple human tissues. By deletion analysis, we mapped the apobec-1-binding region to the glycine-rich domain. ABBP-1 also binds to apoB mRNA transcripts around the editing site and can be UV-cross-linked to them in vitro. Immnodepletion of ABBP-1 from an active apoB mRNA editing tissue extract inhibits its editing activity. Down-regulation of ABBP-1 in an apobec-1-expressing HepG2 cell line by transfection with an antisense ABBP-1 cDNA construct leads to inhibition of endogenous apoB mRNA editing. We conclude that ABBP-1 is an apobec-1-interacting protein that may play an important role in apoB mRNA editing.

Apolipoprotein (apo) 1 B mRNA editing is a novel mechanism for the posttranscriptional regulation of gene expression in mammals (reviewed in Refs. 1 and 2). It consists of a C 3 U conversion of the first base of the codon CAA, encoding glutamine 2153, to UAA, an in-frame stop codon, in apoB mRNA (3,4). ApoB-100 and apoB-48 are the translation products of the unedited and edited mRNA, respectively. Although apoB-48 corresponds to the N-terminal 48% of apoB-100, the two proteins have drastically different properties and entirely different physiological functions (5).
Apobec-1 exists as a spontaneous homodimer (10). It is active against synthetic human apoB mRNA substrates in vitro only in the presence of complementation factors (7). These factors are widely distributed in mammalian tissues, including many that do not synthesize apoB mRNA (8,13). None of these factors have been characterized or cloned.
In order to understand the mechanism by which apobec-1 edits apoB mRNA and the protein composition of the editosome complex, we have used the yeast two-hybrid system to identify proteins that specifically interact with apobec-1 in vitro and in vivo. By this technique, we cloned an apobec-1-binding protein that appears to be a variant of hnRNP type A/B. This protein, named ABBP-1 (apobec-1-binding protein-1), demonstrates many properties that suggest that it plays an important role in apoB mRNA editing.

MATERIALS AND METHODS
Two-hybrid Cloning-The cDNA of human apobec-1 (10) was subcloned into PAS-2 and used as bait to screen several human two-hybrid libraries (Clontech, Inc.). About 60 positives were obtained from 6 ϫ 10 6 Trp-Leu colonies. Five passed the mating assay (14) and consist of independent clones. One of these clones, isolated from a human placenta library, contains a partial ABBP-1 cDNA. The 5Ј end of the ABBP-1 cDNA sequence was cloned by using the Clontech's 5Ј rapid amplification of cDNA ends cDNA kit.
In Vitro Binding-The glutathione S-transferase (GST)-ABBP-1 fusion protein was used for in vitro binding experiments. In 1 ml of phosphate-buffered saline, 0.2% Nonidet P-40 solution, 100 l of the GST beads (Amersham Corp) was incubated with the appropriate amount of [ 35 S]methionine-labeled apobec-1 (obtained by coupled transcription-translation using a TnT kit from Promega, Inc.) at 4°C for 1 h with gentle rocking. The beads were washed five times and were then boiled to denature in SDS loading buffer and analyzed by SDS-PAGE (15). Truncation of the GST-ABBP-1 fusion protein was done by polymerase chain reaction subcloning. The subclones were confirmed by complete sequencing and the size of the truncated proteins analyzed by SDS-PAGE.
RNA Band Mobility Shifting and UV Cross-linking-RNA gel mobility shift assay and SDS-PAGE analysis of UV-cross-linked protein-RNA complex were performed as described previously (16). 200 ng of 32 Plabeled human apoB-mRNA, a 295-mer (bases 6560 -6854), was incubated with 1 g of GST-ABBP-1 fusion protein in a total volume of 20 l of editing buffer (17) at room temperature for 30 min. 10 l was digested with T1 ribonuclease and loaded on a 5% nondenaturing polyacrylamide gel in 50 mM Tris-glycine (pH 7.9) buffer. The other 10 l was exposed to UV light for 15 min with a short-wave UV lamp and digested with T1 ribonuclease. The boiled samples were loaded on 12.5% SDS-PAGE and the gels exposed to x-ray as described (16).
In Vitro Editing Extracts and Effect of Immunodepletion-HepG2 cells transfected with human apobec-1 cDNA using the vector pFLAG-CMV-2 (International Biotechnologies, Inc., New Haven, CT) were used to generate the S-100 fractions for the in vitro editing assays as described (18). Protein A-agarose purified rabbit IgG fraction against the GST-ABBP-1 fusion protein was covalently immobilized to agarose beads (Bio-Rad) and used to incubate with the S-100 fractions at 4°C overnight in a slow rocking platform. A typical reaction contained 1 ml of phosphate-buffered saline, 0.2% Triton solution. The beads were settled on ice, and the cleared supernatant was collected for the editing assays. In vitro editing and primer extension assays were performed as described (19).
Transfection of ABBP-1 Antisense DNA and Selection of Transfected * This work was supported by a National Institutes of Health Grant HL-56668. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

RESULTS
Cloning, Structure, and Expression of ABBP-1-Using the yeast two-hybrid system and apobec-1 as bait, we have identified five independent cDNA clones. The in vivo interactions in the yeast system were confirmed by binding in vitro to human apobec-1 (see below). Partial sequence analysis revealed that one of these clones, ABBP-1, contains RNA-binding motifs (RNP motifs, also known as RNA recognition motifs, or RNAbinding domain (20)) that suggest that it may be involved in apoB mRNA editing. The complete deduced amino acid sequence of ABBP-1 is shown in Fig. 1. It contains 331 amino acid residues with a calculated M r 36,615 and is identical in structure to a human type A/B hnRNP (21), except for a 47-amino acid insertion close to the C-terminal region of the molecule. This insertion is the result of alternate RNA splicing, the inserted sequence corresponding to an exon that is excluded in the mature cytoplasmic ABBP-1 mRNA in about half of the mRNA population on HepG2 cells (data not shown). The structural domains of ABBP-1 are depicted in Fig. 2B. ABBP-1 contains two 80-amino acid-long RNA-binding domains, each containing two short sequences, RNP1 (RNP octamer) and RNP2 (RNP hexamer), that are typical of many RNA-binding proteins (20,22). The C-terminal part of the protein is very glycine-rich and contains a stretch of 8 glycine residues immediately preceding the extra exon sequence. Such glycine-rich domains have been found in other RNP proteins and appear to be auxiliary domains that mediate protein-protein interactions (23). The 47-amino acid insert is also rich in glycine, containing 13 glycine residues, of which 9 are followed immediately by a tyrosine residue. The function of such GY repeats is unknown. A potential ATP/GTP binding fold is present in the C-terminal region immediately following the 47-amino acid insertion. Northern blot analysis of RNAs isolated from various human tissues indicates that ABBP-1 mRNA expression is widely distributed; a ϳ2-kilobase band was identified in all the tissues examined, including spleen, thymus, prostate, testis, ovary, small intestine, pancreas, colon, leukocyte, heart, brain, placenta, lung, liver, skeletal muscle, and kidney (data not shown).
Mapping of ABBP-1 Binding to Human Apobec-1-The ability of ABBP-1 to bind to apobec-1 synthesized in vitro was examined by using full-length and various deletion mutants of ABBP-1 conjugated at the N terminus to GST. As shown in Fig.  2, full-length GST-ABBP-1 binds to human apobec-1 in vitro, confirming the yeast two-hybrid interactions in vivo. This binding is mediated by the C-terminal region of the molecule, since removal of this part of the molecule completely abolishes binding (mutant 4). The 47-amino acid insertion (mutants 8 and 9) by itself appears competent in binding, but is not required for binding because its deletion from ABBP-1 (in mutants 2 and 3) does not impair binding. A stretch of 4 glutamine residues upstream of the glycine-rich domain does not by itself confer apobec-1 binding (mutant 4, which contains this stretch at its C terminus, does not bind). The ATP/GTP fold immediately downstream to the 47-amino acid insertion is not required for binding (mutant 3, which misses this fold, binds well). Taken together, it is evident that sequences rich in glycine in ABBP-1 mediate apobec-1 binding.
ABBP-1 Binds to Human ApoB mRNA Fragment Around the Edited Site and Can Be UV Cross-linked to It-Since ABBP-1 contains typical RNA-binding motifs, we tested whether it would bind to apoB mRNA in vitro. Binding of 32 P-labeled human apoB mRNA (containing nucleotides 6560 -6854) to GST-ABBP-1 was examined by band mobility-shift analysis. As   FIG. 1. DNA-deduced amino acid sequence of ABBP-1. The original yeast two-hybrid clone contains the codons for residues 56 -331. The N-terminal 55 residues (underlined) are deduced from a cDNA clone obtained by 5Ј rapid amplification of cDNA ends. The sequence is identical to a previously published human type A/B hnRNP (21) except for a 47-amino acid insert (double-underlined). The two pairs of RNP2 (hexamer) and RNP1 (octamer) sequences are boxed. The ATP/GTP fold is overlined.

FIG. 2. Structure of wild-type and deletion mutant ABBP-1s
and their binding to apobec-1. Binding was performed using GST-ABBP-1 fusion proteins. The full-length wild-type (wt) protein binds well, so does the initial clone obtained by the yeast two-hybrid system (mu1). Mutants (mu) 2-7 were obtained from mutant 1 by C-terminal truncation. GST fusion proteins of mutants 8 and 9 were obtained independently. A, SDS-PAGE and autoradiograph of GST-fusion ABBP-1 bound 35 S-apobec-1. B, schematic drawing of wild-type and mutant ABBP-1s. The RNP2 and RNP1 sequences are indicated by light stippling. The 47-amino acid insert containing the 9 GY repeats is marked by heavy stippling.
shown in Fig. 3A, ABBP-1 binds to 32 P-apoB mRNA. Binding was effectively competed in the presence of excess unlabeled apoB mRNA, but not yeast RNA. Furthermore, ABBP-1 could be specifically cross-linked to 32 P-apoB mRNA by UV light (Fig.  3B). UV cross-linking showed the same specificity as the gelshift analysis.
Down-regulation of ABBP-1 Inhibits ApoB mRNA Editing-We transfected a human apobec-1 cDNA vector into the human hepatoma cell line HepG2. S-100 extracts from these cells are competent in editing human apoB mRNA in vitro (Fig.  4A). To examine the potential role of ABBP-1 in apoB mRNA editing, we examined the effect of immunodepletion with anti ABBP-1 IgG on in vitro editing activity (Fig. 4A). We found that immunodepletion of ABBP-1 in the editing extracts results in a significant inhibition of editing; the extent of editing is reduced from 14.75 Ϯ 4.97% in control extracts to 4.43 Ϯ 1.95% (p Ͻ 0.05) in immunodepleted extracts. The use of preimmune serum and sham incubation of the extract under the same conditions resulted in minimal changes in editing. This experiment suggests that ABBP-1 is a component of the apoB mRNA editing complex in vitro.
To further investigate the role of ABBP-1 in editing-competent HepG2 cells (produced by stable transfection with apobec-1 cDNA (10)) under in vivo conditions, we transfected them with either an ABBP-1 antisense cDNA expression vector clone or with the expression vector (pHook-3) only (Fig. 4B). Total RNA was isolated from successfully transfected cells (selected by magnetic bead-coupled hapten capture of pHook-3-directed antigen expression) and assayed for the extent of apoB mRNA editing. We found that transfection with antisense ABBP-1 led to a marked inhibition in endogenous apoB mRNA editing in these cells from 10.83 Ϯ 2.84% to 3.87 Ϯ 1.79% (p Ͻ 0.05), a ϳ75% reduction. Transfection with the vector alone had no effect. Thus, down-regulation of ABBP-1 expression by antisense expression inhibits endogenous apoB mRNA editing in vivo.

DISCUSSION
The search for complementation proteins for apoB mRNA editing started with the demonstration of a 40-kDa protein (16) and later both a ϳ44-kDa and a ϳ66-kDa protein (24,25)  demonstrated to interact with either apobec-1 or apoB mRNA and its role in apoB mRNA editing in vivo remains to be determined.
In this study we have used the yeast two-hybrid technique to identify a protein, ABBP-1, that specifically binds to apobec-1. The deduced amino acid sequence of ABBP-1 indicates that it is an RNA-binding protein that shows sequence identity with a human hnRNP type A/B protein (21), except for a 47-amino acid insertion produced by alternative RNA splicing. The previously described hnRNP protein was identified as an RNAbinding protein of unknown function in HeLa cells. Here we show that this protein interacts specifically with apobec-1. Apart from the classical RNP-2 and RNP-1 motifs in the Nterminal half of ABBP-1, there is a glycine-rich region in the C-terminal region of the molecule. Such glycine-rich sequences in RNP proteins are typically involved in protein-protein interactions (23), and in ABBP-1 they appear to mediate its binding to apobec-1. We have identified two isoforms of ABBP-1 produced by alternatively spliced mRNAs. Both forms seem to be competent in binding to apobec-1 in vitro.
ABBP-1 also binds to apoB mRNA and can be UV-crosslinked to it in vitro. Immunodepletion of ABBP-1 in editing extracts appears to inhibit their editing activity. Most important, expression of antisense ABBP-1 transcript by transfection reduces endogenous apoB mRNA editing in apobec-1-expressing HepG2 cells by 75%. These experiments provide evidence for the possible involvement of ABBP-1 in apoB mRNA editing in vivo.
We speculate that ABBP-1 may be involved in recruiting and possibly disrupting the secondary structure of apoB mRNA and bringing it to the vicinity of apobec-1 (and possibly other complementation proteins) for editing. We note that ABBP-1 is the first apobec-1-interacting protein identified and cloned. Furthermore, to date, other than apobec-1 itself, it is the only protein whose down-regulation seems to affect both the editing activity of a cellular editing extract assayed in vitro and the level of endogenous apoB mRNA editing in a cell. Future research in this area will be aimed at the identification of other factor(s) involved in apoB mRNA editing and their possible interaction with apobec-1 and ABBP-1.