Ô|Identification of GRY-RBP as an Apolipoprotein B RNA-bindingProtein That Interacts with Both Apobec-1 and Apobec-1Complementation Factor to Modulate C to U Editing*

Received for publication, July 19, 2000, and in revised form, December 12, 2000Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M006435200

Valerie Blanc‡, Naveenan Navaratnam§¶, Jeffrey O. Henderson‡, Shrikant Anant‡,Susan Kennedy‡, Adam Jarmuz§¶, James Scott§i, and Nicholas O. Davidson‡i**‡‡

From the ‡Department of Internal Medicine and **Department of Pharmacology and Molecular Biology,Washington University School of Medicine, St. Louis, Missouri 63110 and the §MRC Molecular Medicine Group,Clinical Science Center and Division of National Heart and Lung Institute, Imperial College School of Medicine,Hammersmith Hospital, London W12 0NN, United Kingdom

C to U editing of apolipoprotein B (apoB) mRNA in-volves the interaction of a multicomponent editing en-zyme complex with a requisite RNA sequence embeddedwithin an AU-rich context. This enzyme complex in-cludes apobec-1, an RNA-specific cytidine deaminase,and apobec-1 complementation factor (ACF), a novel 65-kDa RNA-binding protein, that together represent theminimal core of the editing enzyme complex. The pre-cise composition of the holo-enzyme, however, remainsunknown. We have previously isolated an enriched frac-tion of S100 extracts, prepared from chicken intestinalcells, that displays apoB RNA binding and which, follow-ing supplementation with apobec-1, permits efficient Cto U editing. Peptide sequencing of this most active frac-tion reveals the presence of ACF as well as GRY-RBP, anRNA-binding protein with ;50% homology to ACF. GRY-RBP was independently isolated from a two-hybridscreen of chicken intestinal cDNA. GRY-RBP binds toACF, to apobec-1, and also binds apoB RNA. Experi-ments using recombinant proteins demonstrate thatGRY-RBP binds to ACF and inhibits both the binding ofACF to apoB RNA and C to U RNA editing. This compet-itive inhibition is rescued by addition of ACF, suggest-ing that GRY-RBP binds to and sequesters ACF. As fur-ther evidence of the role of GRY-RBP, rat hepatoma cellstreated with an antisense oligonucleotide to GRY-RBPdemonstrated an increase in C to U editing of endoge-nous apoB RNA. ACF and GRY-RBP colocalize in thenucleus of transfected cells and, in cotransfection exper-iments with apobec-1, each appears to colocalize in apredominantly nuclear distribution. Taken together,the results indicate that GRY-RBP is a member of theACF gene family that may function to modulate C to URNA editing through binding either to ACF or to apo-bec-1 or, alternatively, to the target RNA itself.

Apolipoprotein B (apoB)1 is an abundant gene product ex-pressed in the mammalian small intestine and liver and playsa central role in the transport of cholesterol and triglyceride inplasma (1). Two forms of apoB exist, designated on a centilescale as apoB100 and apoB48 (2). ApoB48 is generated as aresult of a site-specific, posttranscriptional C to U deaminationof the nuclear transcript that in turn results in translationaltermination of the (edited) apoB RNA (3, 4). ApoB RNA editingis particularly active in the mammalian small intestine butalso in the liver of certain species, including the mouse and rat(5). Since apoB RNA editing eliminates an important func-tional domain from the C terminus of the protein (reviewed inRef. 2), the molecular mechanisms underlying this organ-spe-cific partitioning have been extensively investigated to under-stand the presumed advantage of this specialized geneticadaptation.

Enzymatic deamination of apoB RNA exhibits importantrequirements in both the cis-acting RNA elements and in thetrans-acting protein factors that restrict this reaction largely toa single, canonical site (6–15). The cis-acting elements havebeen well characterized and include an 11-nucleotide (nt) re-gion referred to as a “mooring sequence,” located 4 nt down-stream of the edited C, an AU-rich bulk RNA context, and other“efficiency elements” flanking a ;30-nt region representing theminimal editing cassette (6–15). This cassette is located withina region that exhibits important secondary structure, includinga stem-loop conformation that is predicted to position the ed-ited C in a favorable configuration relative to the active site ofthe deaminase (10, 12, 16, 17). C to U editing of apoB RNA ismediated by an enzyme complex that includes a single catalyticsubunit, apobec-1, as well as additional proteins that togetherrepresent the holoenzyme (18). Apobec-1 is a zinc-dependentcytidine deaminase that exhibits RNA-binding affinity for AU-rich substrates (19, 20), such as apoB, as well as other tem-plates including c-Myc, interleukin-2, granulocyte-macro-phage-colony stimulating factor, and tumor necrosis factor-a(16). Targeted deletion of apobec-1 eliminated C to U editing ofapoB mRNA, demonstrating that no functional redundancyexists in the catalytic deamination of this RNA (21–23). Nev-ertheless, although absolutely required for C to U editing of

* This work was supported in part by National Institutes of HealthGrants HL-38180 and DK-56260 (to N. O. D.) and the Morphology Coreof the Digestive Disease Research Core Center Grant DK-52574. Thecosts of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked “adver-tisement” in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

¶ Supported by the Medical Research Council (UK).i Both laboratories contributed equally to this work.‡‡ To whom correspondence should be addressed: Gastroenterology

Division, Washington University Medical School, Box 8124, 660 S.Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-2027; Fax: 314-362-2023; E-mail: NOD@IM.WUSTL.EDU.

1 The abbreviations used are: apoB, apolipoprotein B; nt, nucleotide;PAGE, polyacrylamide gel electrophoresis; ACF, apobec-1 complemen-tation factor; MS, mass spectrometry; PMSF, phenylmethylsulfonylfluoride; PCR, polymerase chain reaction; DTT, dithiothreitol; FITC,fluorescein isothiocyanate; DAPI, 4,6-diamidino-2-phenylindole; PTB,pyrimidine tract-binding protein; hnRNP, heterogeneous nuclearribonucleoprotein.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 13, Issue of March 30, pp. 10272–10283, 2001© 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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apoB RNA, apobec-1 alone is not sufficient (11, 18). Specifi-cally, recombinant apobec-1 will deaminate a monomeric cyti-dine substrate but alone exhibits no deaminase activity on anRNA template (24, 25). This observation coupled with earlierstudies in which C to U editing activity was found to exist in ahigher order complex provide support for the proposal thatapoB RNA editing is mediated by a heteromeric enzyme com-plex whose composition and functional organization facilitatescatalytic deamination of the targeted base (11, 26, 27).

Over the last several years, many different proteins havebeen proposed to function as auxiliary or complementationfactors, including candidates ranging in molecular mass from40 to 240 kDa (11, 13, 26–30). Despite intensive effort, untilvery recently none of these has met the criteria of an authenticenzymatic cofactor in C to U editing of apoB RNA. The work ofDriscoll and colleagues (31) has very recently resulted in theidentification of a candidate protein, referred to as apobec-1complementation factor (ACF), that most plausibly represents,with apobec-1, the minimal editing enzyme complex. ACF is an;65-kDa RNA-binding protein, with three distinct RNA bind-ing domains, that binds both to apobec-1 and to apoB RNA.Similar findings were also reported by Greeve et al. (32) whoisolated a cDNA identical to ACF,2 with the exception of an8-residue insertion, that exhibits complementation activitythat was enhanced in the presence of additional proteins pres-ent in nuclear extracts. These most recent findings, taken inconjunction with earlier reports that demonstrated the pres-ence of apoB editing activity in a higher order complex, suggestthat the native editing enzyme complex may consist of apobec-1and ACF as well as additional proteins (32).

We previously reported the presence of apobec-1 complemen-tation activity in chicken intestinal S100 extracts that fraction-ated in the range of 49–65 kDa (17, 33). During the purificationof this complementing activity, using both biochemical andgenetic isolation techniques, we have isolated another RNA-binding protein, glycine-arginine-tyrosine-rich RNA-bindingprotein (GRY-RBP), that exhibits ;50% amino acid sequencesimilarity to ACF. GRY-RBP binds ACF both in vitro and invivo and in addition binds apoB RNA. GRY-RBP binding toACF competitively inhibits both its binding to apoB RNA and Cto U RNA editing. ACF and GRY-RBP are both localized in thenucleus of transfected cells, including cells that are competentto edit apoB RNA as well as cells that lack this activity. Fur-thermore, in cotransfection experiments, ACF and GRY-RBPeach colocalize with apobec-1 in both a cytoplasmic as well as anuclear distribution. Taken together, the data suggest thatGRY-RBP, a member of the ACF gene family, may function tomodulate C to U RNA editing of mammalian apoB RNAthrough binding to ACF as well as to apobec-1 and/or apoBRNA.

MATERIALS AND METHODS

Identification of GRY-RBP by Protein Sequence Analysis—Chickenenterocyte S100 extracts were prepared as described previously andfractionated through a 25-ml Blue-Sepharose column (Amersham Phar-macia Biotech) as detailed in Ref. 17. The material eluting in fractions16–19 was pooled and used for UV cross-linking to a 55-nucleotide32P-labeled synthetic apoB RNA (17). Cross-linked proteins were sepa-rated by 10% SDS-PAGE, and the cross-linked bands were identified byautoradiography. The cross-linked band migrating at ;65 kDa (;p65)was excised and electroeluted from the gel. This material was subjectedto sequence analysis at the Harvard Microchemistry Facility, usingmicrocapillary reverse-phase high pressure liquid chromatography and

nano-electrospray tandem mass spectrometry (MS/MS) on a FinniganLCQ quadrupole ion trap mass spectrometer. The MS/MS peptide se-quences were then analyzed for consensus to known proteins, and theresults were manually confirmed for fidelity.

Cloning and Expression of Recombinant GRY-RBP—Full-lengthGRY-RBP cDNA was generated by polymerase chain reaction (PCR)from human liver cDNA and introduced into pPCR-Script (Stratagene).The primers used were GRYsense, 59-GCGGTCGACATGGCTACAGA-ACATGTTAATGGAAAT-39, and GRY antisense, 59-GCGGGGCCCGC-GGCCGCCTACTTCCACTGTTGCCCAAAAGTATCCTGATAA-39.GRY-RBP cDNA was then subcloned into a mammalian expressionvector pCMV-Tag3B (Stratagene) using the SalI-ApaI sites to generatean N-terminal c-Myc-tagged fusion protein. By using GRY-RBP-specificprimers flanked with an NheI restriction site at the end of the 59 primer,the DNA was PCR-amplified from the recombinant pPCR-Script vectorand introduced into the NheI-SmaI sites of the expression vector pTYB2(Biolabs) to generate the recombinant protein. The primers used wereIMP5 sense, 59-GGTGCGGCTAGCATGGCTACAGAACATGTTAATG-39, and IMP3 antisense, 59-CTTCCACTGTTGCCCAAAAGTATC-39.GRY-RBP was fused to a self-cleavable intein tag containing a chitin-binding domain for affinity purification as a fusion protein. Expressionof the fusion protein was conducted in ER2566 cells, and cultures weregrown as recommended by the manufacturer (New England Biolabs).The cell pellet obtained from a 1-liter culture was resuspended in 50 mlof lysis buffer (20 mM HEPES (pH 8.0), 500 mM NaCl, 1 mM EDTA, 0.5%Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride), and the cells werelysed by sonication, followed by 30 min of incubation at 4 °C. The lysatewas then centrifuged, and the clarified cell extract was loaded onto a10-ml chitin column. The column was washed extensively and GRY-RBP released in the presence of 50 mM dithiothreitol. Fractions wereanalyzed by denaturing 10% SDS-PAGE and Western-blotted usingaffinity-purified antipeptide antiserum, generated using residues 105–122 of human GRY-RBP (Research Genetics). Fractions containingGRY-RBP were pooled and dialyzed against 20 mM HEPES (pH 8.0),100 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 20% glycerol, 0.5 mM PMSF.

Cloning and Expression of Recombinant ACF—ACF cDNA was am-plified by reverse transcriptase-PCR from human (small intestinal andhepatic) RNA and RACE-ready RNA (CLONTECH) and cloned intopGem-T easy (Promega), using the following primers: ACF1 sense, 59-GGATCCCATATGGAATCAAATCACAAATCCG-39; ACF2 antisense,59-CTCGAGTCAGAAGGTGCCATATCCATC-39. ACF cDNA was thensequenced and subcloned into pCMV-Tag 2B using the BamHI-XhoIsites, generating an N-terminal FLAG-tagged fusion protein. ACFcDNA was also subcloned into pTYB1 and expressed as an intein fusionprotein as described above. Preparations of the recombinant proteinwere stored at 220 °C in small aliquots and used at the indicatedconcentrations. ACF cDNA was also cloned into the expression vectorpET28a using the BamHI-XhoI sites to generate a His-tagged protein.The protein was overexpressed in Novablue (DE3) cells according to themanufacturer’s instructions (Novagen). The bacterial supernatant wasincubated with 2 ml of Ni21-nitrilotriacetic acid metal affinity resin(Qiagen) and His6-tagged ACF eluted with sequential imidazole stepsup to 400 mM. Fractions containing His6-ACF were pooled and dialyzed.A full-length chicken intestinal ACF cDNA was also amplified by re-verse transcriptase-PCR using the human primers, above. Antiseraagainst human ACF (N- and C-terminal antipeptide antisera) weregenerously provided by D. Driscoll as detailed previously (31).

UV Cross-linking Analysis of Protein-RNA Interaction—A 32P-la-beled rat apoB RNA template (50,000–70,000 cpm at 4 3 108 cpm/mg)was incubated with the indicated amounts of purified recombinantprotein for 15 min at room temperature in a binding buffer containing10 mM HEPES (pH 8.0), 100 mM KCl, 1 mM EDTA, 0.25 mM DTT, and2.5% glycerol. The RNA was then sequentially treated with RNase T1 (1unit/ml final concentration) and heparin (2 mg/ml final concentration)prior to UV irradiation on ice in a Stratalinker (Stratagene) at 250mJ/cm2. The cross-linked material was analyzed by 10% SDS-PAGE.Competition experiments were performed using wild-type or mutants ofapoB mRNA (55-mer) or actin RNA as described previously (13).

Far Western Analysis of Protein-Protein Interaction—1 mg of recom-binant GRY-RBP was separated by SDS-PAGE and Western-blottedonto Immobilon P (Millipore). The immobilized protein was denaturedwith 6 M guanidine hydrochloride in buffer A (20 mM HEPES (pH 7.9),100 mM KCl, 0.2 mM EDTA, 20% glycerol, 0.5 mM DTT, 0.5 mM PMSF,0.5 mM benzamidine) at room temperature for 1 h. Proteins were rena-tured by diluting the denaturation buffer 1:1 in buffer A for 12 cycles of10 min each (29, 34). Filters were blocked overnight at 4 °C with 5%bovine serum albumin and 5% skim milk in buffer A and then incubatedwith 35S-labeled apobec-1 (5 3 105 cpm/ml) in buffer B (buffer A plus 2.5

2 We use the term ACF to denote the complementation factor identi-fied by Driscoll and colleagues (31). ACF is of almost identical sequenceto the cDNA recently cloned by Greeve and colleagues (32), referred toas ASP. For the purposes of this report, we refer to ACF to describe thisprotein and its functions.

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mM MgCl2, 0.5% skim milk; 2% bovine serum albumin; 0.1% Tween 20)for 16 h at 30 °C. Filters were washed three times in buffer C (buffer Aplus 2.5 mM MgCl2, 0.1% Tween 20), dried at room temperature, andsubjected to autoradiography.

Antisense Oligonucleotide Experiments—An antisense morpholino-oligonucleotide to GRY-RBP (59-GCTCCGTTCATCTTGTTGGCTGTG-C-39, 59 located at nt 1479) and a scrambled control morpholino-oligo-nucleotide were prepared by Gene Tools, LLC, Corvallis, OR. McArdlerat hepatoma cells were plated on a 35-mm culture dish and grown to70% confluence. Oligonucleotides (final concentration, 5 mM) weremixed with delivery reagent (EPEI, Gene Tools) in serum-free Dulbec-co’s modified Eagle’s medium and layered over the cells. After incuba-tion for 3 h, the delivery solution was removed, replaced with completeDulbecco’s modified Eagle’s medium containing 10% fetal bovine serum,and the cells incubated for 48 h. RNA was extracted, and apoB RNAediting was determined by primer extension assay.

Immunofluorescence Microscopy—Rat-apobec-1 cDNA was clonedinto pCMV-Tag 2B or 3B and expressed either as an N-terminal FLAG-tagged or an N-terminal c-Myc-tagged fusion protein, respectively. Hu-man GRY-RBP cDNA was cloned into pCMV-Tag 3B and expressed asan N-terminal c-Myc-tagged fusion protein. Human ACF was expressedfrom pCMV-Tag2B vector as an N-terminal FLAG-tagged fusion pro-tein. COS-7, HepG2, and McArdle cells (ATCC) were grown on cover-slips and transiently transfected with 1–2 mg of plasmid DNA usingFUGENE 6 (Roche Molecular Biochemicals). The cells were fixed 48 hafter transfection with 3.7% formaldehyde, permeabilized with 0.5%Triton X-100, and probed with mouse monoclonal anti-Myc IgG (SC-40,Santa Cruz Biotechnology) and rabbit anti-FLAG IgG (71-5400, ZymedLaboratories Inc.), followed by Cy3- or FITC-conjugated secondary IgG,respectively (Jackson ImmunoResearch). For confocal microscopy, nu-clei were visualized using TO-PRO-3 iodide (Molecular Probes). Prepa-rations were imaged with a 633 Zeiss plan apochromatic objective anda Bio-Rad MRC 1024 confocal adaptor. A krypton-argon laser was usedwith epifluorescence filter sets designed for Texas Red (Cy3), fluores-cein (FITC), and cyanine (Cy5). The confocal aperture was set at 1.8.Usually, 15–40 images at planes separated by 0.5 mm were obtained.This increment allows sectioning of the entire image giving a range ofsignals covering every plane of the cells in that image. Pictures wereprocessed using Adobe Photoshop 4.0 software. For standard immuno-fluorescence microscopy, transfection and antibody staining was carriedout as described above using cells grown on coverslips. Stained cellswere mounted with Vectashield and nuclei imaged with DAPI (Vector).Images were obtained with a Zeiss Axioskop 2 MOT microscopeequipped with a 403 plan neofluar objective and a 3CCR camera(DAGE-MTI, Inc.). A Zeiss Attoarc variable intensity lamp was usedwith filter sets designed for Cy3, FITC, and DAPI. Images were pro-cessed using Adobe Photoshop 4.0 software.

Miscellaneous Methods—Primer extension analysis of apoB RNAediting was conducted as detailed previously (24). Coimmunoprecipita-tion assays were conducted using Myc-GRY-RBP, FLAG-ACF, apo-bec-1, and GST-apobec-1, expressed either as recombinant proteins or,where indicated, from in vitro translation using a TnT Coupled Reticu-

locyte Lysate (Promega). Interaction studies were performed at 30 °Cfor 30 min in 200 ml of binding buffer (20 mM HEPES (pH 7.9), 100 mM

KCl, 1 mM EDTA, 10% glycerol, 1 mM PMSF, 0.4% Nonidet P-40). ACFand GRY-RBP were coimmunoprecipitated from cell lysates followingtransient transfection as described above. Following nondenaturing celllysis, the target proteins were immunoprecipitated with 5 ml of theappropriate antisera, including mouse monoclonal anti-FLAG (Sigma)or rabbit polyclonal anti-Myc (Sigma) antibody at 4 °C for 60 min withagitation. The complexes were collected on protein A-agarose beads thatwere washed extensively and analyzed by 12% SDS-PAGE and autora-diography. Where indicated, the material coprecipitating on the proteinA-agarose beads was eluted in denaturing buffer, subjected to SDS-PAGE, and visualized by Western blotting with the appropriate anti-sera. Yeast two-hybrid interaction assays were conducted with ACF andGRY-RBP cDNAs, which were cloned into either pSB202 or pJG vectors(35) or both, and their interaction with wild-type apobec-1 and variousC- or N-terminal deletion mutants was determined as described (35).Construction of a yeast two-hybrid library used 5 mg of poly(A)1 RNA,isolated from chicken enterocytes, packaged in pAD-GAL4 using theHybriZAP two-hybrid gigapack cloning kit (Stratagene). Rat apobec-1cDNA was cloned into pBD-Gal4 as the bait using standard methods,and 10 mg of each plasmid was used to cotransform yeast (YRG-2) asdetailed by the manufacturer (Stratagene).

RESULTS AND DISCUSSION

Biochemical and Genetic Isolation of Proteins That Bind toApoB RNA and to Apobec-1—We reasoned that proteins thatfunction as integral components in the apoB RNA editing com-plex should exhibit binding activity toward both the substrate(apoB RNA) and also the catalytic subunit of the holoenzyme(apobec-1). Partially purified chick intestinal S100 extractswere UV cross-linked to radiolabeled apoB mRNA (13), and thecross-linked protein(s) of molecular weight ;p65 were identi-fied by autoradiography (Fig. 1). The material contained inthese pooled fractions was previously demonstrated to be en-riched in apoB RNA editing complementation activity, al-though its composition was unknown (17). 70 peptides wereobtained in this screen of which the sequence of 6, shown in Fig.1, matched unambiguously to GRY-RBP, whereas 5 othersmatched to a sequence recently identified as ACF by Driscolland co-workers (31). These results suggest that fractionatedchicken intestinal extracts, a source of enriched complementa-tion activity, contain at least two proteins that can be identifiedthrough interaction with apoB RNA. The demonstration thatACF copurifies with other protein(s) is reminiscent of the re-cent report of Greeve and colleagues (32) in which fractionatedrat liver nuclear extracts revealed the presence of other pro-

FIG. 1. Peptide sequencing from fractionated chicken S100 extracts. Chicken enterocyte S100 extracts were subjected to 10–70%ammonium sulfate precipitation and fractionated through Blue-Sepharose (Amersham Pharmacia Biotech) to enrich for RNA binding and C to Uediting complementation activity (13). The most enriched fractions (fractions 16–19) (13) were used for UV cross-linking to a 55-mer apoB RNAand then autoradiographed. The cross-linking material was silver-stained and eluted for peptide sequence. The peptide sequences obtained fromthe mass spectroscopy analysis are indicated. The assignments of leucine and isoleucine were not distinguished and are indicated as alternatives(L/I).

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teins in addition to ACF/ASP in the most highly enrichedediting fraction.

Independent examination of interacting proteins was under-taken using a chicken intestinal library cloned into a yeasttwo-hybrid expression system, using rat apobec-1 as bait. Thisapproach also revealed a strongly interacting clone, which wasidentified as GRY-RBP (data not shown). Searches with vari-ous regions of GRY-RBP identified several homologs includingthe EST image clone (N77737) recently identified as ACF byDriscoll et al. (31) and ASP by Greeve and colleagues (32).Clone N77737 was directly sequenced, and five separate nucle-otide differences were identified from the reported sequence ofDriscoll and coworkers (31), (C deletions at 84,191, C insertionsat 364, 437, and a G at 189). After correcting these differences,clone N77737 was found to align completely with the cDNA ofthe recently identified complementation factor, ACF (31). Anal-ysis of the full-length chicken ACF cDNA demonstrated thepredicted amino acid sequence (Fig. 2A) is identical to thehuman clone. This apparent conservation suggests an interest-ing paradox, since chicken apoB RNA is not edited (33). Thisfunctional limitation reflects at least two components. First,chicken intestine lacks apobec-1 (33, 36) and thus cannot me-diate catalytic deamination of a target apoB RNA and, second,chicken apoB RNA is itself not an editable template (33). Thus,the evolutionary and functional significance of ACF expressionin this setting must await further study.

Sequence Alignment and Phylogeny of GRY-RBP and ACFIsoforms—Full-length cDNAs encoding GRY-RBP and ACFwere isolated from human liver and intestinal RNA as de-scribed, and their sequence alignments are shown in Fig. 2A.GRY-RBP contains a distinctive N-terminal extension (Fig. 2A)as well as three nonidentical RNA recognition motifs (RRMs)that appear conserved with those found in ACF (Fig. 2A). Inaddition, GRY-RBP contains a C-terminal, putative bipartite,nuclear localization sequence (Lys564-Arg-Lys-X9-Lys-Arg-Arg578) that differs in sequence and location from that proposedin ACF (31). The C-terminal region of GRY-RBP, betweenresidues 446 and 623, contains 20 RG clusters and 8 RGGrepeats in an overall context of 36% arginine or glycine resi-dues. This region, in particular the RGG repeats and RG clus-ters, may signify an RNA binding domain (37) that spans over170 residues. By contrast, the C-terminal region of ACF con-tains 6 RG clusters within a domain spanning residues 311–402 that is composed of 28% arginine/glycine residues. Furtheranalysis revealed three isoforms of ACF cDNA in human liver(data not shown). These include the cDNA clone reported byDriscoll et al. (31), a second isoform containing an 8-amino acidinsertion (ASP, described by Greeve et al. (32), and a third formwith a 55-amino acid deletion (data not shown). Sequence anal-ysis of 10 independent clones isolated from adult human liverRNA revealed 6 encode the clone identified by Driscoll andco-workers (31) and 2 contain the 8-amino acid insertion and 2contain the 55-amino acid deletion. Analysis of ACF cDNAsfrom human small intestine revealed that 3 of 9 clones containthe 8-amino acid insertion (data not shown), recently demon-strated by Greeve and colleagues (32) in their liver-derivedclone. This insertion was present in the single clone analyzedfrom chicken small intestine. Phylogenetic analysis suggeststhat ACF and GRY-RBP represent two distinct members of acommon ancestral gene family that is conserved from Dictyos-telium to mammals (Fig. 2B). Examination of the UNIGENEdata base reveals that the EST corresponding to ACF is locatedon human chromosome 10, whereas GRY-RBP is located onchromosome 20.

Protein-Protein Interaction of GRY-RBP with ACF and withApobec-1—Protein-protein interaction of GRY-RBP with ACF

was examined by several complementary strategies. We firstundertook immunoprecipitation of the products of radiolabeledin vitro translation mixed with epitope-tagged recombinant,unlabeled protein. In vitro translated Myc-GRY-RBP yielded apredominant species of ;80 kDa with smaller products thatrepresent either internal methionine residues or partial degra-dation products, whereas in vitro translation of FLAG-ACFyielded a protein of ;67 kDa (Fig. 3A, lanes 1 and 2, respec-tively). Mixing radiolabeled ACF with cold, unlabeled Myc-GRY-RBP followed by immunoprecipitation with anti-Myc IgGrevealed a physical interaction of these two proteins as dem-onstrated in lane 2 of Fig. 3B, showing a radiolabeled bandcorresponding to the dominant translation product of ACFalone. The converse experiment, mixing radiolabeled GRY-RBP with cold, unlabeled FLAG-ACF followed by immunopre-cipitation with FLAG IgG, similarly revealed a single radiola-beled band corresponding to the dominant translation productof GRY-RBP alone (Fig. 3B, lane 4). Control immunoprecipita-tions, performed with the radiolabeled ligand and anti-epitopeIgG, but without the target protein, revealed no coprecipitation(Fig. 3B, lanes 1 and 3).

To demonstrate the interaction of ACF and GRY-RBP in aphysiological context, cotransfection experiments were con-ducted in which epitope-tagged ACF, GRY-RBP, and apobec-1were introduced either alone or in combinations into COS cells.Cell lysates were prepared and examined by Western blottingto demonstrate expression of the relevant protein. Cells trans-fected with ACF, apobec-1, or GRY-RBP cDNA alone demon-strated comparable expression of the cognate protein (Fig. 3C,lanes 2–4). Cotransfection of ACF and apobec-1 (Fig. 3C, lane5) or of ACF and GRY-RBP (Fig. 3C, lane 6) also revealedexpression of the relevant proteins in cell lysates. Immunopre-cipitation of cell lysates was then conducted to demonstrateprotein-protein interaction in vivo. Apobec-1 and ACF wascoimmunoprecipitated from COS cells transfected with bothcDNAs (Fig. 3D, lane 5), confirming the recent findings ofDriscoll and colleagues (31). Extending these findings, COScells transfected with ACF, and GRY-RBP demonstrated copre-cipitation of GRY-RBP following immunoprecipitation of ACF(Fig. 3D, lane 6). These cumulative findings provide furtherevidence for a physical interaction of ACF and GRY-RBP invivo.

The interaction of GRY-RBP, and of ACF, with apobec-1 wasfurther examined using a yeast two-hybrid binding assay. Asindicated in Fig. 3E, the data reveal strong (11) interactionbetween ACF and apobec-1 and comparable interaction be-tween apobec-1 and GRY-RBP. Apobec-1 has been demon-strated previously to exist as a homodimer (35, 38), and itsself-interaction (111 in this assay) is shown by way of apositive control. Both C- and N-terminal deletions of apobec-1failed to support an interaction with GRY-RBP, suggestingthat these domains may be of importance in stabilizing andmaintaining the interaction with additional proteins in theholoenzyme. This speculation is supported by the earlier dem-onstration that these very N- and C-terminal deletions in apo-bec-1 eliminate homodimerization, RNA binding, and apoBRNA editing activity (35). Nevertheless, it bears emphasis thatother interpretations, such as an effect on apobec-1 folding,cannot be excluded.

Finally, the interaction of GRY-RBP and apobec-1 was ex-amined by far Western blotting using immobilized GRY-RBPand 35S-labeled apobec-1, generated from in vitro translation(Fig. 3F). The ability of apobec-1 to bind to GRY-RBP in thisrenaturation assay further suggests that these two proteinshave the capacity to interact biochemically.

Taken together, the results from several independent lines of

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FIG. 2. Sequence alignment of GRY-RBP and ACF. A, the deduced amino acid sequence of GRY-RBP and human/chicken (h/Ch) ACF (seetext) are aligned. The three RNA recognition motifs (RRMs) are indicated in shaded text. Peptides identified through mass spectroscopy areindicated by the bold line above the sequence. The putative nuclear localization motif in GRY-RBP is indicated by a broken line. B, phylogeneticanalysis of GRY-RBP and ACF. The RRM domains were aligned, and a distance matrix was calculated using the PROTDIST and Phylip programs.

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FIG. 3. GRY-RBP interacts with ACF and with apobec-1. A, Myc-GRY-RBP and FLAG-ACF were synthesized in vitro using a coupled TnTlysate (Promega) in the presence of [35S]methionine. Lanes 1 and 2 are representative examples of the products revealed by autoradiography of 10%denaturing SDS-PAGE. The mobility of the respective species is indicated by the arrowhead or the bold arrow with molecular weight markersindicated on the left. B, co-Immunoprecipitation of ACF and GRY-RBP. The products of 35S-radiolabeled in vitro translation of ACF (lanes 1 and2) or GRY-RBP (lanes 3 and 4) were mixed with unlabeled Myc-tagged GRY-RBP (lane 2) or FLAG-tagged ACF (lane 4), respectively. After mixingfor 30 min at 30 °C, the indicated IgG (a-Myc or a-FLAG, respectively) was added to all incubations and then was gently rotated at 4 °C for 90 minand collected on protein A-Sepharose beads. Lanes 2 and 4 indicate the coimmunoprecipitation of each species. C, COS cells were transfected withvector DNA (lane 1) or with DNA encoding ACF, apobec-1, or GRY-RBP (lanes 2–4). Other transfections were conducted in which apobec-1 and ACFor GRY-RBP and ACF were simultaneously introduced (lanes 5 and 6). After 48 h, cell lysates were prepared and extracts analyzed by Westernblotting with anti-FLAG or anti-Myc IgG. D, ACF coimmunoprecipitates with GRY-RBP and with apobec-1 in transfected cells. The extracts fromCOS cells prepared as in C were immunoprecipitated with anti-FLAG IgG, and the immunoprecipitates were resolved by SDS-PAGE andWestern-blotted with anti-Myc IgG. The data indicate coimmunoprecipitation of apobec-1 with ACF (lane 5) and of GRY-RBP with ACF (lane 6),with specificity demonstrated by the absence of corresponding bands in the other lanes. E, protein-protein interaction of GRY-RBP with apobec-1using yeast two-hybrid assay. Apobec-1 or mutants thereof, GRY-RBP and ACF cDNAs, were cloned into the indicated yeast vectors (see under“Materials and Methods”) and interactions were determined using O-nitrophenyl-b-D-galactopyranoside staining as previously validated (33).Descriptions of the different apobec-1 mutations are detailed (33). F, far Western blotting reveals interaction between GRY-RBP and apobec-1. Leftpanel, GRY-RBP (1 mg) was resolved on a 10% SDS-PAGE and stained with Coomassie Blue. Middle panel, GRY-RBP was resolved by SDS-PAGEand transferred to a polyvinylidene difluoride membrane. Immobilized GRY-RBP was submitted to 12 cycles of denaturation-renaturation andprobed with 35S-labeled apobec-1. Right panel, the identity of GRY-RBP was confirmed by Western blot analysis using affinity-purified GRY-RBPIgG. In each case, a representative result is shown from replicate experiments.

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evidence indicate that GRY-RBP interacts with ACF and withapobec-1.

RNA Binding Activity of ACF and GRY-RBP—RecombinantACF and GRY-RBP (Fig. 4A) were used to establish the RNAbinding activity of the respective proteins with an apoB tem-plate. GRY-RBP binds to apoB RNA as evidenced by the dom-inant UV cross-linked band (Fig. 4B). The binding of ACF toapoB RNA, previously demonstrated by Driscoll and colleagues(31), was confirmed in our hands (Fig. 4C). Additionally, incre-mental supplementation with ACF inhibited GRY-RBP bindingto apoB (Fig. 4C). GRY-RBP additions in turn appeared tointerfere with ACF binding to apoB RNA, the data suggestingthat binding of GRY-RBP to apoB RNA was of lower affinitythan that of ACF (compare 1000 ng of GRY-RBP in D to 500 ngof ACF in C of Fig. 4). These findings are of interest in light ofthe recent demonstration by Greeve and colleagues (32) thatbinding of ACF to apoB RNA was competitively displaced byKSRP, suggesting that other RNA-binding proteins, in additionto ACF and apobec-1, may regulate the assembly and compo-sition of the holoediting enzyme. The findings do not allow us todistinguish whether this apparent displacement is mediateddirectly, through competitive binding for a shared site on theRNA, or alternatively through the binding of ACF and GRY-RBP to one another indirectly modulating RNA binding affin-ity. However, we will return to the functional consequences ofACF and GRY-RBP binding below.

RNA Binding Specificity of GRY-RBP and ACF—The RNAbinding activity of ACF and GRY-RBP was further examinedusing homopolymeric RNAs to compete their binding to apoBRNA. Poly(U), poly(AU), poly(I), and poly(G) competed bothproteins but poly(A) competed only ACF (Fig. 5A). Poly(C) andpoly(IC) failed to compete RNA binding with either ACF orGRY-RBP (Fig. 5A). These data suggest that there are differ-ences in the specificity of RNA binding between GRY-RBP and

ACF, particularly with respect to A-rich templates. Despitethese differences, however, the results suggest that both pro-teins bind to U- and AU-rich targets. This feature would beanticipated in light of their binding to the apoB RNA template,which is ;70% AU-rich in the region flanking the edited base(14).

To refine the binding specificity, the recombinant proteinswere used in a binding assay with apoB RNAs, into whichvarious scrambling mutations have been introduced. TheseapoB mutants, each of identical length and spanning the min-imal editing cassette, were created by changing 6-nucleotidesections to the complementary sequence, as previously de-scribed (Fig. 5B) (13, 17). The results of these experimentssuggest that GRY-RBP binds with low specificity throughoutthe apoB template, as evidenced by the comparable reductionin binding with all the scrambled mutations, compared withthat observed with a nonspecific, actin RNA (Fig. 5B). On theother hand, ACF appears to bind preferentially to two regionswithin the minimal apoB RNA, as evidenced by the abrogationof competition with mutants C and D (Fig. 5B). One site isupstream of the edited base (nt 6660–6665) and another isdownstream (nt 6667–6673), the latter spanning the proximalend of the mooring sequence and partially overlapping thebinding site recently identified for apobec-1 (16, 17, 20). Theseresults are similar to those obtained using crude rat enterocyteS100 extracts where binding activity of p60 was localized to aregion spanning nts 6671–6674 (25). The current data alsoextend the results of Driscoll and colleagues (29, 31) who dem-onstrated that ACF failed to bind to a 280-nt baboon apoB RNAcontaining three mutations within the mooring sequence (6671,6675, and 6678). The cumulative interpretation of these earlierfindings, along with the current data, suggests that regionsimmediately flanking the edited base may represent bindingsites for ACF. Nevertheless, fine mapping of the binding site of

FIG. 4. RNA binding activity of GRY-RBP and ACF. A, 1 mg of protein was analyzed by denaturing SDS-PAGE and stained with CoomassieBlue. B, UV cross-linking was performed by incubating increasing amounts of GRY-RBP (50 ng-2 mg) with a 32P-labeled 105-nt rat apoB cRNA(RB105) flanking the edited base. C, 100 ng of GRY-RBP was cross-linked to radiolabeled RB105 RNA in the presence of increasing amounts ofHis-ACF (50–500 ng), representing up to a 10-fold molar excess of recombinant ACF. D, 100 ng of recombinant His-ACF was incubated withincreasing amounts of GRY-RBP (50–1000 ng), representing up to 20-fold molar excess and cross-linked to RB 105 RNA. This is a representativeof three such experiments.

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ACF and its role in the cooperative assembly of the apoB RNAediting enzyme will require further study.

Regulation of C to U RNA Editing Activity through ACF-GRY-RBP Interaction—ACF and apobec-1 were used in an invitro editing assay to confirm the demonstration that these twoproteins represent the minimal editing enzyme complex for Cto U deamination of apoB RNA. The data reveal a linear in-crease in C to U editing activity in an assay using 250 ng ofGST-apobec-1 and 1–40 ng of ACF (Fig. 6A). Optimal editingactivity (.80% C to U conversion) was noted in assays contain-ing ;40 ng of ACF and ;250 ng of GST-apobec-1 (Fig. 6A). Thiscorresponds to a predicted molar ratio of apobec-1:ACF of 3:1,assuming that all the available protein exists in a functionallyactive complex. As noted previously in assays utilizing chickenintestinal S100 extracts, further addition of complementationactivity (in the present studies, authentic recombinant ACF)led to a progressive decline in editing activity (Fig. 6A). Thesefindings emphasize the crucial stoichiometry of apobec-1 andACF in this assay and lend indirect support to the hypothesisthat other proteins may participate in a regulatory capacity invivo. Although antisera against GRY-RBP, ACF, and apobec-1are available, the abundance of ACF and GRY-RBP is belowdetection levels by Western blotting of fractionated cell or tis-sue extracts (data not shown, but in agreement with Driscoll etal. (29)), precluding estimates of their relative proportionswithin a functional editing enzyme. Accordingly, the precisemolar concentrations of apobec-1, ACF, and potentially GRY-RBP within the holoenzyme remain to be ascertained directly.

The addition of increasing amounts of recombinant GRY-RBP to assays containing ACF and apobec-1 demonstratedprogressive inhibition of RNA editing, with complete abroga-tion of C to U deamination of apoB RNA noted at the highestamounts tested (Fig. 6B). By contrast, assays containing apo-bec-1 and up to 100 ng of GRY-RBP failed to demonstrate

evidence of C to U editing activity, indicating that GRY-RBPitself lacks the ability to complement apobec-1 (Fig. 6B). Assayswere then conducted using a range of input RNA in the pres-ence of apobec-1 and ACF and with increasing amounts ofGRY-RBP. C to U conversion demonstrated saturable kineticswith an apparent Km of 7 6 1.1 nM (Fig. 6C). The presence ofincreasing amounts of GRY-RBP (10 and 100 nM) altered theKm for this reaction to 24 and 40 nM, respectively. Lineweaver-Burk plots of the data suggest that this is the result of com-petitive inhibition (Fig. 6C, inset). To examine further themechanism of this inhibition, experiments were conducted inwhich assays, containing amounts of ACF and apobec-1 suffi-cient to yield ;25–50% editing, were modified through additionof GRY-RBP and then rescued with the addition of ACF, apo-bec-1, or both. The results of a representative series of suchexperiments indicate that the addition of ACF (Fig. 6D) but notapobec-1 (data not shown) rescues the inhibition produced byGRY-RBP. Furthermore, supplemental apobec-1 failed to pro-duce incremental effects in assays rescued with ACF (data notshown), suggesting that GRY-RBP exerts its inhibitory effectsthrough binding to and sequestering ACF. This suggestion mayalso account for the effects noted on apoB RNA binding, alludedto above (Fig. 4).

Effects of Other ApoB RNA-binding Proteins on C to U Edit-ing Activity—The demonstration that GRY-RBP binds to ACFand to apoB RNA and also inhibits C to U RNA editing raisedthe possibility that other apoB RNA-binding proteins may alsomodulate this process, as previously demonstrated withhnRNP C and D (26, 39). However, as demonstrated in Fig. 7,pyrimidine tract-binding protein (PTB), hnRNP-A, andhnRNP-F, all bind to apoB RNA (Fig. 7, upper panel), yet noneproduce significant inhibition of apoB RNA editing (Fig. 7,lower panel). These results imply that the interaction of GRY-RBP with apoB RNA and the consequent inhibition of C to U

FIG. 5. ApoB RNA binding activityof GRY-RBP and ACF, competitionwith homopolymeric RNA and apoBRNA mutants. A, 250 ng of unlabeledhomopolymeric RNA was added to bind-ing reactions containing radiolabeledRB105 and 200 ng of either GRY-RBP(left panel) or ACF (right panel). Aftertreatment with RNase T1 and UV irradi-ation, the cross-linked products were an-alyzed on a 10% SDS-PAGE. Molecularweight markers are indicated at the left ofeach gel. B, competition with mutantapoB RNAs. Upper panel, cross-linkingwas carried out with a 55-nt apoB RNAflanking the edited base, in the absence(2) or presence of competitor RNA; 55-ntwild-type RNA (WT), scanning mutantsB–I, representing 6 nucleotide sectionsimmediately upstream (B and C) or down-stream (D-I), of the edited base (shown inlower panel). As nonspecific control, anactin cRNA was used at equivalent con-centrations. Lower panel, the cross-linkedmaterial was stained with CoomassieBlue to demonstrate equivalent amountsof protein in each lane. These results arerepresentative of triplicate experiments.

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editing is not a general phenomenon associated with AU-richRNA-binding proteins.

Antisense Inhibition of GRY-RBP Expression, Effects onApoB mRNA Editing—To demonstrate a potential physiologi-cal role for GRY-RBP in regulating apoB RNA editing, weundertook antisense oligonucleotide treatment of rat hepatomacells to decrease expression of GRY-RBP. McA cells were se-lected since they express the requisite trans-acting proteins toedit endogenous apoB mRNA. Our prediction, based upon thefindings reported above, was that decreased expression of GRY-RBP might result in increased editing activity. Control, un-

transfected cells demonstrated ;15% C to U editing of endog-enous apoB mRNA (Fig. 8, lane 1), a range frequentlyencountered with this cell line (24). Cells transfected with anantisense oligonucleotide to GRY-RBP, by contrast, demon-strated a 2-fold increase in endogenous apoB RNA editing(.30%, Fig. 8A, lanes 2–4), whereas a scrambled oligonucleo-tide was without effect. The results are summarized in Fig. 8Band support our prediction that GRY-RBP may play a physio-logical role in the regulation of apoB RNA editing in vivo.Further study will be required to address the regulatory mech-anisms involved.

Colocalization of ACF, Apobec-1, and GRY-RBP in Trans-

FIG. 6. In vitro RNA editing with ACF and apobec-1, competitive inhibition by GRY-RBP. A, apobec-1 and ACF alone edit an apoB RNAtemplate. Increasing amounts of recombinant ACF were added to 250 ng of GST-apobec-1 together with 20 fmol of a 470-nt rat apoB cRNA andin vitro C to U editing assayed by primer extension (see under “Materials and Methods”). B, increasing amounts of purified GRY-RBP were addedto assays containing 20 fmol of apoB RNA and 250 ng of recombinant GST-apobec-1 together with 2 ng of purified ACF (lanes 1–5). The RNA wasextracted and C to U editing quantitated by primer extension assay. The relative mobility of the edited (UAA) and unedited (CAA) products areindicated. Editing is expressed as % U. This is a representative of 2–4 experiments at each concentration of GRY-RBP. As control, 10 or 100 ngGRY-RBP was added to an assay containing 250 ng of GST-apobec-1 and apoB RNA but without ACF (lanes 6 and 7). C, substrate dependence ofC to U editing activity. Editing assays were conducted with increasing concentrations of RNA substrate under optimal conditions, using a 3-h timepoint. Edited RNA was quantitated by phosphorimaging. Regression analysis using Lineweaver-Burk kinetics was performed at increasingconcentrations of GRY-RBP (inset) to demonstrate competitive inhibition. Each point is the average of three independent determinations. D, rescueof GRY-RBP inhibition with ACF. C to U editing assays were conducted using 250 ng of GST-apobec-1 and 2 ng of ACF together with 20 fmol ofapoB RNA. 4 ng of GRY-RBP produced ;50% inhibition, which was overcome by rescue with 8 or 16 ng of ACF. A representative experiment fromthree independent determinations is shown.

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fected Cells—To characterize further the interaction of ACF,GRY-RBP, and apobec-1 within the cell, epitope-tagged pro-teins were introduced into HepG2 and McA cells and localizedusing immunofluorescence microscopy. Transfection of epitope-tagged proteins permitted the detection of exogenous protein incells that express endogenous ACF, with (McA) or without(HepG2) coexpression of endogenous apobec-1. Cotransfectionof ACF and GRY-RBP suggests a nuclear localization of bothproteins in HepG2 and McA cells (Fig. 9, upper left panel).Cotransfection of GRY-RBP and apobec-1 suggests that GRY-RBP is again found in a nuclear distribution, whereas apobec-1is distributed in a predominantly nuclear localization patternalso but with evidence of cytoplasmic staining (Fig. 9, upperright panel). Cotransfection of ACF and apobec-1 indicates apredominantly nuclear staining pattern of ACF, again withevidence of cytoplasmic staining of apobec-1 along with theintense nuclear staining pattern (Fig. 9, lower panel). It mustbe emphasized, however, that the interpretation of these re-sults is based upon the distribution of epitope-tagged proteinswithin cells that express a small but presumably functionalpool of endogenous ACF, GRY-RBP, and for McA cells, apo-bec-1. In all cases, the size of this pool is unknown, since theseproteins defy quantitation using conventional immunochemi-cal approaches (data not shown). Accordingly, resolution of thecrucial question of whether these findings reflect the pattern ofendogenous proteins must await the development of more sen-sitive methodology and reagents.

To refine the colocalization data hinted at above, confocalmicroscopy was undertaken in COS-7 cells, which express verylow levels of ACF, GRY-RBP, and undetectable levels of apo-bec-1 (data not shown). Apobec-1 was found predominantly in

the nucleus, although some staining was noted within in thecytoplasm (Fig. 10A). In both locations, apobec-1 staining colo-calized with ACF (Fig. 10, A–C). Similarly, cotransfection ofapobec-1 and GRY-RBP demonstrated a predominantly nu-clear colocalization of both proteins, with diffuse cytoplasmicstaining also evident (Fig. 10, E–G). By contrast, cotransfectionof ACF and GRY-RBP demonstrated almost exclusively nuclearstaining, with both proteins again colocalizing in the confocal,merged image (Fig. 10, I–K). Taken together, the imaging re-sults support the proposal that ACF and GRY-RBP are colocal-ized nuclear proteins and demonstrate that apobec-1 associatesin vivo with both GRY-RBP and with ACF.

The pattern of apobec-1 distribution (predominantly nuclearwith some cytoplasmic staining) is consistent with the workingmodel of C to U editing of apoB RNA as a nuclear event andraises the additional possibility that apobec-1 may shuttle froma cytoplasmic to nuclear pool. Such speculation requires formalproof but is consistent with recent data demonstrating a rolefor apobec-1 in regulating the stability of AU-rich transcriptsthrough its binding activity, a function presumed to imply acytoplasmic location (15). In addition, recent results fromSmith and colleagues (40) imply the possibility that apobec-1may edit apoB RNA in the cytoplasm of rat hepatoma cells,again consistent with its localization in this compartmentrather than the nucleus, as had been earlier concluded (37).However, the current findings are somewhat at variance withthose of Smith and colleagues (40) in regard to the localizationof ACF. Their findings suggest both a cytoplasmic and nucleardistribution as evidenced by standard immunofluorescence mi-croscopy, whereas our data, using confocal microscopy, suggest

FIG. 7. Other apoB RNA-binding proteins fail to modulate C toU editing. Upper panel, UV cross-linking assays were conducted withrecombinant hnRNP A1, F, and PTB together with a radiolabeled apoBRNA (see under “Materials and Methods”). The bound transcript wasresolved by denaturing SDS-PAGE. Lower panel, C to U editing assayswere conducted with recombinant apobec-1 and ACF (lane 3) to whichwas added 2 mg recombinant hnRNP A1 (lanes 4 and 5), F (lanes 6 and7), or PTB (lanes 8 and 9). There was no detectable difference in theextent of C to U editing in any of the incubations containing ACF andapobec-1. A representative assay is shown.

FIG. 8. Antisense oligonucleotide inhibition of GRY-RBP ex-pression increases apoB RNA editing in McA rat hepatoma cells.A, McA hepatoma cells were incubated with 5 mM antisense GRY-RBP(a-GRY-RBP, lanes 2–4) or a scrambled oligonucleotide (lanes 5–7) andRNA analyzed by primer extension. The products were resolved byPAGE and fluorography; p, primer; C, unedited apoB RNA; U, editedapoB RNA. B, apoB RNA editing was quantitated by phosphorimagingof the gel and expressed as % U. Antisense GRY-RBP treated cellsshowed a significant increase in apoB RNA editing.

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that ACF exhibits a predominantly nuclear localization (Figs. 8and 9). Further examination of this apparent compartmental-ization is clearly warranted.

In summary, the findings of this report indicate that theS100 fraction of chicken enterocyte extracts, a source estab-lished to be enriched for apoB editing complementation activ-ity, contains ACF as well as a related protein, GRY-RBP. Bothproteins bind to apoB RNA and also bind to one another.GRY-RBP and ACF both exhibit binding activity for apoB RNA,the binding site for ACF localizing to a region flanking theedited base. In addition, these proteins colocalize with one

another in the nucleus of transfected cells, and each appears tocolocalize with apobec-1. These data, taken together with thefinding that addition of GRY-RBP to the minimal editing reac-tion components (apobec-1 plus ACF) produces a competitiveinhibition of C to U editing and the demonstration that anti-sense inhibition of GRY-RBP expression increases apoB RNAediting in rat hepatoma cells, suggest that GRY-RBP may playa role in the regulation of apoB RNA editing. One possibleinterpretation is that this regulation may be exerted through acomplex interaction that reflects the role of additional compo-nents that compose the holoediting enzyme. The mechanism of

FIG. 9. Immunofluorescence microscopy of transfected GRY-RBP, ACF, and apobec-1 in HepG2 and McA cells. The indicated cellswere grown on coverslips and transfected with epitope-tagged protein as indicated under “Materials and Methods.” Left upper panel cotransfectionof GRY-RBP (A) and ACF (B). Right upper panel, cotransfection of GRY-RBP (A) and apobec-1 (B). Lower panel, cotransfection of apobec-1 (A) andACF (B). In all cases, these are representative images derived from three independent experiments. Nuclear counterstaining was performed withDAPI (C), the arrows indicating cells expressing the relevant protein products for each panel.

FIG. 10. Immunofluorescence con-focal microscopy of transfected apo-bec-1, ACF, and GRY-RBP. COS-7 cellswere transfected with Myc-apobec-1 (A)and FLAG-ACF (B) or with FLAG-apo-bec-1 (E) and Myc-GRY-RBP (F). In bothcases, the merged images (C and G) indi-cate colocalization of the signals. In addi-tion, FLAG-ACF (I) was coexpressed withMyc-GRY-RBP (J) and the confocal im-ages merged (K) revealing colocalizationin a nuclear distribution (compare To-Pro3 iodide nuclear staining in D, H and L).These images are representative of threeindependent transfections.

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interaction of these and other candidate genes will be the focusof future reports.

Acknowledgments—We acknowledge the initial contributions of JingMin in the two-hybrid screen. We thank Karen Hutton (Digestive Dis-ease Research Core Center) for technical assistance in the confocalmicroscopy studies, W. Lane (Harvard Microchemistry Facility, Har-vard University) for protein sequencing, and D. Driscoll for providingantiserum to ACF. We also thank Clare Gooding and Chris Smith forsupplying purified PTB and Shern Chew and Ian Eperon for hnRNP A1.

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Identification of GRY-RBP as an ApoB RNA-binding Protein 10283

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Kennedy, Adam Jarmuz, James Scott and Nicholas O. DavidsonValerie Blanc, Naveenan Navaratnam, Jeffrey O. Henderson, Shrikant Anant, Susan

to U EditingCInteracts with Both Apobec-1 and Apobec-1 Complementation Factor to Modulate

Ô?Identification of GRY-RBP as an Apolipoprotein B RNA-binding Protein That

doi: 10.1074/jbc.M006435200 originally published online December 27, 20002001, 276:10272-10283.J. Biol. Chem. 

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