©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
apobec-1, the Catalytic Subunit of the Mammalian Apolipoprotein B mRNA Editing Enzyme, Is a Novel RNA-binding Protein (*)

Shrikant Anant , Andrew J. MacGinnitie , Nicholas O. Davidson (§)

From the (1)Department of Medicine, University of Chicago, Chicago, Illinois 60637

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Apolipoprotein B (apoB) mRNA editing is mediated by an enzyme complex which includes the catalytic subunit, apobec-1. Recombinant GST/APOBEC-1 binds with high specificity to a rat apoB RNA template as demonstrated by UV cross-linking and electrophoretic mobility shift assay (EMSA). ApoB RNA binding was competed by poly(U), poly(A,U), and tRNA, but not by poly(A) or other homopolymeric ribonucleotides. UV cross-linking of GST/APOBEC-1 to an apoB RNA template was uninfluenced by the binding of proteins of 60 and 44 kDa, present in S100 extracts prepared from different sources. The binding of these proteins was similarly uninfluenced by the simultaneous binding of GST/APOBEC-1. Moreover, the inclusion of heterologous S100 extracts in the RNA binding reactions completely abrogated the competitive displacement of GST/APOBEC-1 by tRNA. EMSA revealed the onset of RNA binding within 1-2 min, and its specificity was confirmed by a supershift with anti-GST/APOBEC-1 antisera. The structural specificity for apoB RNA binding, as inferred from EMSA, appears to be distinct from apoB RNA editing since wild-type chicken apoB RNA, which is not editable, and several mutant chicken apoB RNAs containing clustered mutations within the minimal apoB RNA editing cassette, bound with efficiency similar to the rat apoB RNA template. In conclusion, while the data suggest that apobec-1 binds AU-rich templates, the importance of this observation in the context of mammalian apoB mRNA editing remains unknown.


INTRODUCTION

Apolipoprotein B (apoB),()an obligate structural component of lipoproteins secreted by the small intestine and liver of mammals, circulates in two distinct forms (reviewed in Ref. 1). ApoB100 contains 4536 residues and is secreted from the liver while apoB48, which is colinear with the amino-terminal 2152 residues of apoB100, is secreted from the small intestine(1) . The molecular mechanism for this organ-specific partitioning is referred to as apoB mRNA editing(2, 3) . In this process, a post-transcriptional cytidine deamination of a glutamine (CAA) codon in the nuclear apoB mRNA produces a UAA stop codon and translational termination(2, 3) . Numerous experiments conducted over the last few years have demonstrated that this editing reaction is mediated by an enzyme complex of which the catalytic subunit, apobec-1, was recently cloned from rat small intestine(4) . More recently, homologous gene products have been cloned from human and rabbit small intestine(5, 6, 7) .

An important feature of apobec-1 activity is that preparations of the protein, produced either from cell-free translation of the cDNA or following cRNA injection into Xenopus oocytes, will not perform apoB RNA editing without a source of complementation activity (4, 8). Examples of such sources would be S100 extracts prepared from chicken small intestinal enterocytes or human liver, neither of which alone has apoB RNA editing activity(8) . By extension, an important question to emerge from these studies is whether specific elements of this complementation activity are involved in directing the catalytic subunit to the appropriate RNA sequence in the primary apoB transcript for site-specific deamination(8, 9) . In this regard, much progress has been made concerning the sequence requirements for apoB RNA editing (10). The minimal apoB RNA editing cassette consists of approximately 29 nucleotides flanking the edited base and includes a critical 11-nucleotide mooring sequence (UGAUCAGUAUA), which is sufficient to support in vitro RNA editing of both apoB and heterologous RNA templates(10, 11, 12) . Virtually all mutations within this 11-nucleotide cassette abolish in vitro apoB RNA editing (10-12). Consistent with these findings, chicken apoB mRNA, which is not edited in vitro by mammalian intestinal S100 extracts, contains 3 nucleotide alterations in this region(13) .

In order to examine the machinery involved in apoB mRNA editing, several groups have demonstrated the presence of proteins in either nuclear or S100 extracts which will cross-link to apoB RNA(14, 15, 16) . Studies revealed a protein of approximately 40 kDa in nuclear extracts from rat liver while proteins of 60 and 44 kDa were identified in rat intestinal and hepatic S100 extracts(14, 15, 16) . Additionally, the cross-linking pattern demonstrated in rat intestinal S100 extracts was shown to be most specific for a 4-nucleotide region at the 5` end of the mooring sequence (UGAU)(15) . Examination of the association kinetics of these proteins, as inferred by UV cross linking, demonstrated an interaction prior to the onset of detectable editing of the target apoB RNA template, findings which have been argued to support the sequential assembly of a nucleoprotein editing complex referred to as an editosome(16) . Other workers, however, have demonstrated that S100 extracts, prepared from either baboon enterocytes or rat liver, yield proteins of 66 and 44 kDa following UV cross-linking to a luciferase RNA or an antisense apoB RNA template(11) . Thus, neither the identity of these proteins nor their requisite involvement in apoB mRNA editing has been conclusively demonstrated.

We now present data which demonstrate that a recombinant glutathione S-transferase/apobec-1 fusion protein (GST/APOBEC-1) has intrinsic apoB RNA binding activity as inferred from both UV cross-linking and electrophoretic mobility shift assays. However, the demonstration that GST/APOBEC-1 binds chicken apoB RNA, which is not an editable template, suggests that the structural constraints for RNA binding are distinct from those which determine editability.


MATERIALS AND METHODS

Expression of GST/APOBEC-1

apobec-1 cDNA was cloned into pGEX-4T3 (Pharmacia Biotech Inc.) and expressed as a glutathione S-transferase (GST) fusion protein in lon-protease-deficient RB791 Escherichia coli cells. The fusion protein was extracted from bacterial lysates with 1% Triton X-100, purified over glutathione-agarose beads (Sigma), and eluted in a buffer containing 50 mM Tris-HCl, pH 8.0, 10 mM reduced glutathione. Details of the production and functional analysis of the fusion protein are presented in an accompanying manuscript(26) . However, the apparent molecular mass of GST/APOBEC-1, as estimated from 8% SDS-PAGE, is 60 kDa (data not shown). Antisera to the purified fusion protein was raised in rabbits.

Plasmid Construction

A 105-base pair fragment of rat apoB cDNA (nt 6639-6743, pRB 105) and a 160-base pair fragment of wild type and mutant chick apoB cDNA (nt 6608-6768, pCB 150) were cloned into pGEM3Zf(+) and used as templates for in vitro RNA synthesis (8, 13). Single-point and clustered mutations were introduced into pCB 150 using a two-step polymerase chain reaction method and the cloned mutants sequenced on both strands(17) . A 247-base pair mouse -actin cDNA was used as previously detailed(18) . In vitro apoB RNA editing reactions were conducted as previously detailed(8) .

UV Cross-linking and Electrophoretic Mobility Shift Assays (EMSAs)

Cytoplasmic S100 extracts were prepared from whole rat liver and from isolated rat or chicken enterocytes as previously detailed(8) . P-Labeled RNA templates (50,000 cpm, 2.5-3.0 10 cpm/µg) were incubated with the indicated amounts of these extracts and/or GST/APOBEC-1 for 20 min at room temperature in a buffer containing 10 mM Hepes, pH 7.9, 100 mM KCl, 1 mM dithiothreitol and then treated sequentially with RNase T1 (1 unit/µl final concentration) and heparin (5 mg/ml final concentration) for 5 min each at room temperature unless otherwise stated(19) . Competitor RNA templates or homopolymeric RNAs were added, where indicated, at 1 µg per reaction. In supershift assays, undiluted anti-GST/APOBEC-1 antisera was added, and the incubation continued for an additional 20 min before RNase T1 and heparin addition. For cross-linking studies, the mixture was subjected to UV irradiation (254 nm) in a Stratalinker cross-linking apparatus (Stratagene, energy = 250 mJ/cm) and then separated by 10% SDS-PAGE under reducing conditions. For RNA gel shift assay, the mixture was immediately analyzed by 4% native polyacrylamide gel electrophoresis (37.5:1) using 45 mM Tris borate, 0.1 mM EDTA, pH 8.6. The gels were dried and autoradiographed at -70 °C. For the zinc-chelation experiments, 500 ng of GST/APOBEC-1 was preincubated with 1,7- or 1,10-o-phenanthroline (10 mM final concentration) or ethanol control (0.4% final concentration) for 10 min before being added to the binding assays for an additional 20-min incubation(20) .

Oligonucleotides

The following oligonucleotides were used for mutagenesis of chicken apoB cDNA and for primer extension assay. Restriction sites are underlined; underlined and bolded nucleotides represent the mutations introduced. Chicken APOB 5` primer SH1, 5`-GGGTACCGGAAAAACGCCTGGATAAT-3` (KpnI); chicken APOB 3` primer SH2, 5`-GAGATCTAGAGCTGTCATTTTTTCA-3` (BglII); chicken APOB CH150 M 5` mutagenic primer SHM1, 5`-AATTGAT-CAGTATATCAAAG-3`; chicken APOB CH150 M 3` mutagenic primer SHM2, 5`-CTTTGATATACTGATCAATT-3`; chicken APOB CH150 ML 5` mutagenic primer SHM3, 5`-GATACAATTTGATCAGTATATC-3`; chicken APOB CH150 ML 3` mutagenic primer SHM4, 5`-GATATACTGATCAAATTGTATC-3`; chicken APOB CH150 MR 5` mutagenic primer SHM5, 5`-ATTGATCAGTATATTAGAGATA-3`; chicken APOB CH150 MR 3` mutagenic primer SHM6, 5`-TATCTCTAATATACTGATCAAT-3`; rat primer extension primer [BT7]: 5`-AGTCCTGTGCATCATAATTATCTCTAATATACTGA-3`; chicken WT primer extension primer [PECH-35], 5`-TGTCAAACTGATCGTAATTCTCTTTGATGTACTGC-3`; chicken M/ML primer extension primer [PECHM/ML-35], 5`-TGTCAAACTGATCGTAATTCTCTTTGATATACTGA-3`; chicken MR/MLR primer extension primer [PECHMR/MLR-35], 5`-TGTCAAACTGATCGTAATTATCTCTAATATACTGA-3`.


RESULTS AND DISCUSSION

UV Cross-linking of GST/APOBEC-1 to an ApoB RNA Template

Incubation of GST/APOBEC-1 with a rat apoB RNA template followed by UV cross-linking produced a single band, which migrated with an apparent molecular mass of 72 kDa (Fig. 1A, lanes 3 and 4). The intensity of this band increased in a dose-dependent manner with increasing quantities of the fusion protein (Fig. 1B). ApoB RNA binding was competed for by the addition of excess rat apoB RNA (Fig. 1A, lane 5), demonstrating the specificity of binding. Additionally, poly(U) and poly(A,U) competed for apoB RNA binding, suggesting that GST/APOBEC-1 may function similarly to other AU-binding proteins(19, 21) . This suggestion is also consistent with the high (69%) AU content of the RB 105 template. Neither poly(A) (Fig. 1A, lane 7), other homopolymeric ribonucleotides (poly(G), poly(C), and poly(I):poly(C)), nor single-stranded DNA competed for binding of the apoB RNA template (data not shown). By contrast, apoB RNA binding was effectively competed for by tRNA (Fig. 1A, lane 6). This observation was unexpected since our in vitro apoB RNA editing assays contain 200-250 ng of tRNA(8, 13) . In order to confirm this observation, a series of incubations was performed in the presence of increasing quantities of tRNA. The results indicate a dose-dependent inhibition of apoB RNA binding, beginning with as little as 10 ng of tRNA (Fig. 1C). The explanation for this observation is not readily apparent although it should be emphasized that the orientation of the recombinant GST/APOBEC-1 with respect to the RNA template is likely to be different from that of the native, endogenous apobec-1, which exists within the holoenzyme and presumably functions in the context of apoB mRNA editing complementation factor(s). This issue is addressed further in experiments detailed below. The specificity of the apoB RNA binding reaction was further confirmed by the demonstration that GST/APOBEC-1 binds efficiently to the sense but not the antisense RB 105 template (Fig. 1D, compare lanes 3 and 6) and with considerably reduced efficiency to a chicken apoB RNA template or an irrelevant sense RNA (mouse -actin, Fig. 1D, lanes 7-12). Cross-linking of the chicken apoB RNA template to GST/APOBEC-1 was further explored, as detailed below.


Figure 1: Identification of GST/APOBEC-1 following UV cross-linking to a rat apoB cRNA. 500 ng of either GST- or GST/APOBEC-1 was incubated for 20 min with a P-labeled rat apoB cRNA template (RB 105), the resulting complex was cross-linked under UV for 1.5 min and analyzed by 10% SDS-PAGE. A, binding was performed either in the absence of competitor RNA or in the presence of 1 µg of unlabeled RB 105 cRNA, 125 ng of tRNA, or 1 µg of poly(A), poly(U), or poly(AU). B, binding of increasing concentrations of GST/APOBEC-1 to rat apoB cRNA (RB 105). C, competition for binding of GST/APOBEC-1 with increasing concentrations of tRNA. D, specificity of binding of GST/APOBEC-1 to a rat apoB cRNA. Incubations were conducted as above using either sense (RB 105 S) or antisense (RB 105 AS) rat apoB RNA, chicken apoB cRNA (CB 150 S), or a mouse -actin cRNA (ACT).



In vitro apoB RNA editing reactions were conducted with GST/APOBEC-1 and chicken enterocyte S100 extracts, in the presence or absence of 250 ng of tRNA. These reactions produced similar in vitro conversion efficiency of the RB 105 template (data not shown), suggesting that the inclusion of tRNA is not detrimental to the apoB RNA editing reaction. By contrast, no apoB RNA editing activity was demonstrated when incubations of GST/APOBEC-1 were conducted without a source of complementation activity, even in the absence of tRNA (data not shown). This suggests that, despite its ability to bind the RNA template, GST/APOBEC-1 cannot perform in vitro editing alone. Since apoB RNA editing has been demonstrated to proceed at high efficiency in vitro in the presence of tRNA, the possibility that apoB RNA binding by GST/APOBEC-1 is stabilized by the presence of complementation factors was addressed.

GST/APOBEC-1 Binds to an ApoB RNA Template in the Presence of Complementation Factors and Independent of the Binding of p60 and p44

Previous studies have demonstrated UV cross-linked proteins of 60 and 44 kDa in S100 extracts from mammalian enterocytes and rat liver(11, 14, 15, 16) . UV cross-linking experiments performed in the absence of GST/APOBEC-1 yielded proteins of 44 kDa in enterocyte extracts from chicken (Fig. 2, lane 1, closed arrowhead) and rat small intestine (Fig. 2, lane 8, open arrowhead) while both 60- (open arrow, Fig. 2) and 44-kDa proteins were identified in rat liver S100 extracts (Fig. 2, lane 5). The identity of the strongly reactive cross-linked protein of 28 kDa in rat intestinal S100 extracts (Fig. 2, lanes 8-10) is unknown, but Western blotting with anti-apobec-1 antisera was unreactive (data not shown). UV cross-linking of these S100 extracts performed in the presence of GST/APOBEC-1 resulted in an indistinguishable pattern of protein binding to that found with S100 extracts alone (Fig. 2, lanes 2-4, 6, 7, and 9, 10). Conversely, the pattern of UV cross-linking noted above for GST/APOBEC-1 alone (Fig. 1) was uninfluenced by the binding of the 60- and 44-kDa proteins (Fig. 2, closed arrow) present in S100 extracts. Taken together, this suggests that apoB RNA binding by GST/APOBEC-1 is not competed for by either of the two previously identified cross-linking proteins present in S100 extracts. Furthermore, these findings imply the presence of distinct RNA binding sites on the template and also that the binding of GST/APOBEC-1 and either the 60- or 44-kDa proteins is not cooperative. In addition, apoB RNA binding by GST/APOBEC-1 is not competed for by endogenous apobec-1 in rat intestinal or hepatic S100 extracts suggesting, among other possibilities, that its concentration in these sources may be extremely low or that its presence in association with other components of the apoB mRNA editing enzyme precludes competitive interaction with the recombinant GST-fusion protein.


Figure 2: S100 extracts prepared from rat liver or intestine or from chicken intestine do not compete out cross-linking of GST/APOBEC-1. Chicken (CHICK), rat enterocyte (SI), and rat liver (LIV) S100 extracts (20 µg) were incubated with the RB 105 probe, either alone (-) or in the presence of 500 ng of GST/APOBEC-1 (+). The position of GST/APOBEC-1 is shown by the solid arrow. The position of p60 is shown by the open arrow. The position of bands corresponding to p44 is shown by arrowheads (closed = chicken, open = rat).



Further studies conducted in the presence of high concentrations (250 ng) of tRNA and GST/APOBEC-1, together with 20 µg of rat liver or chicken intestinal S100 extract, produced an indistinguishable UV cross-linking pattern to that shown in Fig. 2(data not shown). This suggests either that the complementation factors stabilize or position GST/APOBEC-1 in a more favorable orientation with respect to the apoB RNA template, despite the presence of tRNA, or in some way sequester the tRNA and abrogate its inhibitory effects. It is equally possible that the complementation factors function as adaptors to bring the RNA template and apobec-1 into proximity. Further studies will be necessary to resolve these possibilities. It also bears emphasis that, under the conditions of in vitro editing (high concentrations of tRNA and the presence of complementation factors), apobec-1 would be anticipated to bind the RNA template.

EMSA of GST/APOBEC-1 following Incubation with an ApoB RNA Template

The finding that GST/APOBEC-1 binds an apoB RNA template was further pursued by means of EMSA. A single-shifted band was found with increasing amounts of GST/APOBEC-1 (Fig. 3A). A supershifted band was demonstrated following the addition of anti-GST/APOBEC-1 (Fig. 3B, open arrow). The supershifted complex contains virtually 100% of the GST/APOBEC-1, confirming the identity of the EMSA gel shift. Timed incubations demonstrated an RNA gel shift within 3 min of the addition of GST/APOBEC-1 (Fig. 3C). Further analysis of the time course of apoB RNA binding demonstrated a detectable gel shift as early as 1-2 min of incubation (Fig. 3D). Although the time frame for apoB RNA editing was not specifically addressed in these studies, previous work from our own and other laboratories suggest that at least a 5-min incubation is required for the detection of C to U conversion(13, 16, 22) . The current findings are thus consistent with previous suggestions that the binding of proteins to the apoB RNA template precedes editing(16) . The addition of 10 mM 1,10-o-phenanthroline, but not the inactive isomer or ethanol vehicle, decreased, but did not abolish, apoB RNA binding, as inferred by EMSA (Fig. 3E). These findings are broadly consistent with the recent demonstration that apobec-1 is a zinc-dependent cytidine deaminase(20) . However, the observation that 10 mM 1,10-o-phenanthroline abolishes in vitro apoB RNA editing (20) but does not completely inhibit apoB RNA binding suggests that these properties are distinct.


Figure 3: Formation of an apoB RNAGST/APOBEC-1 complex demonstrated by EMSA. Either GST- or GST/APOBEC-1 was incubated with the P-labeled apoB cRNA template, and the complex was analyzed by nondenaturing polyacrylamide gel electrophoresis. A, GST- (500 ng, lane 2) and GST/APOBEC-1 (250 ng, lane 3; 500 ng, lane 4). B, supershift of the GST/APOBEC-1apoB RNA complex with increasing concentrations of anti-apobec-1 antisera (APOBEC-1) (open arrow, lanes 3-6). The shifted GST/APOBEC-1apoB RNA complex is shown by the closed arrow. A nonspecific band (arrowhead) is also seen due to interaction with a protein in serum (lane 2, NRS, normal rabbit serum). C, time course of GST/APOBEC-1 binding from 0-30 min. RNase T1 and heparin incubations were performed simultaneously for 5 min following incubation. D, time course of binding from 0-3 min. RNase T1 and heparin incubations were performed simultaneously for 1 min following incubation. Bold arrows indicate shifted complex; two smaller arrows indicate nonspecific artifact. E, effect of zinc chelation with o-phenanthroline on apoB RNA binding by GST/APOBEC-1. The protein was incubated with 10 mM 1,7- (lane 2) or 1,10-o-phenanthroline (lane 3) or with ethanol control (lane 1) prior to addition of the RNA template. Migration of the free transcript (F) and position of the complex formed with GST/APOBEC-1 (closed arrow) are indicated in all panels.



Sequence Requirements for ApoB RNA Binding Are Distinct from the Requirements for ApoB RNA Editing

Previous studies have demonstrated that a conserved 29-nucleotide cassette flanking the edited base contains sufficient structural information to confer editing capacity upon heterologous RNA templates(10, 11) . Chicken apoB RNA, which is not edited, contains several differences from the conserved mammalian sequence within the minimal apoB RNA editing cassette, including three nucleotide alterations within the 11-nucleotide mooring sequence previously shown to be essential for apoB RNA editing(10, 11, 12) . The nucleotide sequence of rat and chicken apoB RNA are illustrated in Fig. 4A, in order to demonstrate these differences and also to illustrate the mutations that were introduced into the chicken apoB cDNA fragment in order to revert groups of nucleotides back to the rat sequence. Four groups of mutations were tested for RNA editing and RNA binding, each centering on the mooring sequence, for reasons detailed above. These were (i) mooring sequence alone (M); (ii) mooring sequence and 3 nucleotides upstream (i.e. to the left (ML)); (iii) mooring sequence and 3 nucleotides downstream (i.e. to the right (MR)); (iv) the entire 29-nucleotide cassette, containing both upstream and downstream alterations (MLR). Each of these mutants, along with the wild-type chicken apoB RNA and the control rat apoB RNA, was incubated in an in vitro RNA editing assay. As shown in Fig. 4B, the rat apoB RNA template is effectively edited in vitro (35% UAA), but of the chicken apoB RNA templates, only the MLR mutant supports editing activity (4% UAA). These findings demonstrate that the entire 29-nucleotide mammalian apoB RNA cassette is necessary for editing of a chicken apoB RNA template, a conclusion broadly consistent with the results of other studies(10, 11, 12) . Further studies of the chimeric chicken/rat apoB RNA templates will be necessary to determine the structural requirements in the regions flanking the minimal editing cassette which restore wild type levels of RNA editing.


Figure 4: Binding of GST/APOBEC-1 to an apoB RNA template is distinct from editing. A, nucleotide sequence of rat and chicken apoB RNA in the homologous region flanking the edited base. The 11-nucleotide mooring sequence is bracketed. Mutations were performed as detailed under ``Materials and Methods'' to introduce clustered revertants in the chicken template back to the rat sequence. B, in vitro RNA editing reactions were performed on the various templates using GST/APOBEC-1 and chicken intestinal S100 extracts (see ``Materials and Methods''). The reaction products were resolved on an 8% polyacrylamide gel, and the mobility of unedited (CAA) and edited templates (UAA and UAA) are shown by the arrows. C, EMSA was performed using the various RNA templates and GST/APOBEC-1. The arrow shows the mobility of the shifted band. In the right panel, various competitor RNAs were examined for their ability to displace the rat apoB cRNA template (RB 105). These included excess sense (S) or antisense (AS) rat apoB cRNA and excess wild-type chicken apoB cRNA (CB 150). Antisense rat apoB cRNA forms a duplex with the sense template (open arrowhead). F = free probe. D, supershift of the GST/APOBEC-1 complexes demonstrated following the addition of antisera as detailed in Fig. 3. The open arrow indicates the supershifted band while the closed arrow shows the remaining unshifted probe. The open arrowhead is a nonspecific band. F = free probe.



In order to examine the relationship of the structural constraints on apoB RNA editing to apoB RNA binding, these templates were used in gel-shift and super-shift assays. GST/APOBEC-1 produced an indistinguishable gel shift with all the apoB RNA templates examined (Fig. 4C), suggesting that there is a loose stringency for apoB RNA binding. Additionally, the gel shift with the rat apoB RNA template was competed with either excess unlabeled rat or chicken apoB RNA (Fig. 4C, right panel). The absence of a shift in the presence of excess antisense rat apoB RNA suggests that the double-stranded RNA is not a target for apobec-1 binding. Finally, a comparable supershift was obtained using antisera to GST/APOBEC-1 in the presence of the different RNA templates (Fig. 4D), demonstrating that the recombinant protein binds to all the templates examined, regardless of their intrinsic ability to undergo editing. The features of these chicken apoB RNA templates which allow binding of GST/APOBEC-1 have not been elucidated, but the implication arising from the data in this study is that the AU content of the template is of fundamental importance. In this regard, the AU content of the chicken apoB RNA templates used in this study range from 63-67%. A further conclusion is that the stringency required for apoB RNA binding by apobec-1 is looser than that for RNA editing.

There are no known RNA binding motifs in the primary amino acid sequence of apobec-1, suggesting that this is a novel RNA binding protein(23, 24, 25) . The implications and biological relevance of this observation in the context of the intact mammalian apoB mRNA editing enzyme await the further identification of its component subunits. In the meantime, the functional domains of apobec-1 involved in this interaction will be the focus of future investigation.


FOOTNOTES

*
These studies were supported by National Institutes of Health Grants HL-38180, HL-18577, and DK-42086 (to N. O. D.), Training Grants HD-07136 (to S. A.), GM-07281 and HL-07237 (to A. J. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: MC 4076, Dept. of Medicine, University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637. Tel.: 312-702-6480; Fax: 312-702-2182.

The abbreviations used are: apoB, apolipoprotein B; apobec-1, apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1; GST, glutathione S-transferase; EMSA, electrophoretic mobility shift assay; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Susan Skarosi, Trish Glascoff, and Annalise Hausman for outstanding technical assistance and Federico Giannoni for discussions and the provision of cell extracts.


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