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Characterization of Apolipoprotein B mRNA Editing From Rabbit Intestine Zarah C. Garcia, Karen S. Poksay, Kristina Bostrom, David F. Johnson, Maureen E. Balestra, Ishaiahu Shechter, and Thomas L. Innerarity Apolipoprotein (apo) B-48 is generated by a unique physiological process. Cytidine 6,666 of the apo B primary transcript is posttranscriptionally converted to a uridine by an RNA editing mechanism that transforms the codon for glutamine 2,153 to a termination codon. The editing reaction can be duplicated in a cell-free extract. In this study, the apo B-48 mRNA editing activity derived from partially purified extracts of rabbit enterocytes was characterized. The optimum conditions for the editing reaction were determined to be a salt concentration of 0.125-0.150 M NaCl or KC1, a pH of 8-8.5, and a temperature of 30°C. The reaction rate was linear up to 45 minutes and was proportional to the editing extract concentration. No metal ion cofactors, DNA or RNA cofactors, or energy requirements were identified. At optimum conditions, the reaction followed Michaelis-Menten kinetics, with a Kn of 0.4 nM for the rabbit RNA substrate. In addition, the reaction rate was enhanced by the addition of 25 pig/ml heparin or 40% glycerol. The characteristics of the editing reaction suggest that it is catalyzed by a nucleotide sequence-specific cytidine deaminase that is either a single enzyme or a multimeric protein. {Arteriosclerosis and Thrombosis 1992;12:172-179)

I

n humans, apolipoprotein (apo) B occurs naturally in two forms, apo B-100 and apo B-48. Apo B-100, a 550-kd protein component of low density lipoproteins (LDLs) and very low density lipoproteins (VLDLs), serves as a ligand for the LDL receptor. Apo B-100 is synthesized in the liver and is essential for the assembly and secretion of VLDL. Apo B-48 is a 264-kd protein component of chylomicrons and is synthesized in the intestine in humans and rabbits and in the liver and the intestine in rats and mice. It is obligatory for the assembly and secretion of chylomicrons. Both apo B-100 and apo B-48 are encoded by the same apo B gene, with apo B-48 representing the amino-terminal 48% of apo B-100.1-3 Apo B-48 is produced by a unique physiological process: the apo B primary transcript is posttranscriptionally modified by a type of RNA processing known as RNA editing.4-6 Apo B nucleotide 6,666 is converted from a genomically encoded cytidine to a uridine. As a consequence, codon 2,153 of the mRNA is transformed from a glutamine codon to a translational termination codon, resulting in the for-

From the Gladstone Foundation Laboratories for Cardiovascular Disease, Cardiovascular Research Institute, University of California at San Francisco, San Francisco, Calif. Address for correspondence: Thomas L. Innerarity, PhD, Gladstone Foundation Laboratories, PO Box 40608, San Francisco, CA 94140-0608. Received August 13, 1991; accepted October 16, 1991.

mation of apo B-48.4-6 The predicted carboxy terminus of apo B-48 was confirmed by using carboxyterminal sequencing and peptide mapping combined with apo B peptide antibodies.6-7 The recent development of cell-free systems (derived from cultured rat hepatoma cells or normal rat liver) that mimic the in vivo reaction has greatly facilitated the investigation of apo B mRNA editing.8-9 Driscoll et al8 reported that in extracts from rat hepatoma cells, the cytidine-to-uridine conversion does not require ribonucleotide triphosphates, creatine phosphate, or divalent ions but does require high concentrations of EDTA. Driscoll and Casanova10 extended these studies to include the analysis of apo B mRNA editing activity in extracts of enterocytes derived from baboon small intestine. This editing activity was sensitive to heat and proteases and had an optimal salt concentration of 0.1 M. After gel filtration, the editing activity eluted with an apparent molecular mass of 125 kd.10 Our laboratory has previously shown that rabbit enterocyte S100 extracts edit synthetic apo B RNA substrate.11 The editing activity was enriched by ammonium sulfate precipitation, with most of the activity precipitating between a concentration of 10% and 40% ( N H ^ C V 1 Using this enriched extract to modify synthetic apo B RNA and then directly analyzing the resulting edited RNA, we formally snowed that the modified base is a uridine.11 Hodges et al12 obtained the same results with rat enterocyte ex-

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Garcia et al

tracts. This modification is most likely catalyzed by a nucleotide sequence-specific cytidine deaminase. In this study, we characterized in greater detail the apo B mRNA editing activity derived from partially purified extracts of rabbit enterocytes. The parameters we examined were the pH, ionic strength, metal ion requirements, energy requirements, substrate (synthetic RNA) size and concentration, time course, dose response, buffer composition, and nucleic acid involvement. From our results and those of others,1013 it can be concluded that either a single enzyme or a multicomponent enzyme catalyzes the editing reaction in vitro. Methods Preparation of Extracts From Rabbit Enterocytes Enterocytes were isolated from the small intestine of New Zealand White rabbits by a modification of the method of Merchant and Heller.14 The intestines were removed from anesthetized rabbits and placed into ice-cold saline. The intestinal contents were thoroughly washed with ice-cold phosphate-buffered saline (PBS) containing 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM benzamidine. After clamping off one end and cannulating the other, the intestines were filled and incubated with buffer A (1.5 mM KC1, 96 mM NaCl, 8 mM KH2PO4, 27 mM sodium citrate, 5.6 mM NazHPO,, [pH 7.3], 0.1 mM PMSF, and 1 mM benzamidine) for 15 minutes at 37°C in a shaking water bath. Buffer A was replaced with buffer B (2.7 mM KC1, 137 mM NaCl, 1.5 mM EDTA, 1.5 mM KH2PO4, 8.1 mM Na2HPO4 [pH 7.4], 0.5 mM dithiothreitol [DTT], 0.1 mM PMSF, and 1 mM benzamidine) and incubated for 30 minutes at 37°C. The enterocytes were obtained by gently squeezing the intestines and were collected on ice. Preparation of S100 Extracts The preparation of enterocyte S100 extracts was based on the method of Dignam et al.15 The isolatedcell slurry was centrifuged at 4°C for 10 minutes at 1,000 rpm in a Beckman J6B centrifuge with a JS-4.2 rotor (Beckman Instruments, Palo Alto, Calif.). The cell pellets were gentry resuspended in four cell volumes of ice-cold PBS and centrifuged at 4°C for 10 minutes at 1,700 rpm. The packed cell volume (PCV) was measured, the supernatant decanted, and the cells resuspended in 4x PCV ice-cold buffer C (10 mM hydroxyethylpiperazine-AT -2 -ethanesulfonic acid [HEPES] [pH 7.9], 1.5 mM MgCl2, 10 mM KC1, and 0.2 mM ethylene glycol-bisO-aminoethyl)N,N,N',N'-tetraacetic acid [EGTA]). The cells were incubated on ice for 10 minutes and then centrifuged at 4°C for 10 minutes at 1,700 rpm. The resulting cell pellets were resuspended in buffer C, l x PCV, containing 5 mM DTT, 0.1 mM PMSF, 1 mM benzamidine, 10 ^.M pepstatin, 10 /iM leupeptin, 20 p,g/ml soybean trypsin inhibitor, and 10 units/ml aprotinin and homogenized with a Dounce homogenizer (10 strokes with pestle B, then five strokes with

Apo B mRNA Editing

173

pestle A). The homogenate was centrifuged at 4°C for 10 minutes at 3,600 rpm and the supernatant collected and mixed with 0.11 x volume buffer E (0.3 M HEPES [pH 7.9], 30 mM MgCl^ and 1.4 M KC1). The extract was then centrifuged at 4°C for 50 minutes at 45,000 rpm in a Beckman 60 Ti rotor and the resulting S100 supernatant dialyzed overnight against 20 volumes of buffer D (20 mM HEPES [pH 7.9], 125 mM KC1, 0.2 mM EDTA, 0.2 mM EGTA, 20% glycerol, and 5 mM DTT). Ammonium Sidfate Precipitation Apo B mRNA editing activity was partially purified from the S100 enterocyte extract by first diluting it with buffer D to 10 mg protein/ml. By sequential precipitation, a 10-40% (NH4)2SO4 precipitate was obtained as previously described.11 Construction of Plasmids and In Vitro Transcription Plasmid pBS-B354 contains an EcoBlScal fragment of human apo B cDNA (nucleotides 6,507-6,860) in the vector pBSSK+.n To generate a similar construct with rabbit apo B cDNA (pRab-1), rabbit genomic DNA was amplified with two oligomers (No. 1: a 30-mer corresponding to human apo B nucleotides 6,498-6,527; No. 2: a 31-mer complementary to human apo B cDNA nucleotides 6,855-6,885). The fragment was digested with the restriction enzymes ZJcoRI and Seal and cloned into the Bluescript M13KS+ plasmid (Stratagene, La Jolla, Calif.) digested with the restriction enzymes EcoVl and Smal. To construct pBS-B63, which was transcribed to synthesize the 63-nucleotide RNA, the 63-base-pair (bp) apo B cDNA fragment from pHEB-6316 was cloned into pBSSK+ digested with the restriction enzyme £coRV. The pBS-215 plasmid contains a 215-bp EcoBl-Aiu\ fragment of human apo B cDNA (nucleotides 6,507-6,717) cloned into the vector pBSKS+ digested with the restriction enzymes £coRI and Smal. The plasmids were linearized by digestion with the restriction enzyme BamHl. The in vitro transcription was performed according to Neupert et al,17 using T7 RNA poh/merase for the pBS-B354 and pBS-B63 plasmids and T3 RNA porymerase for the pRab-1 and pBS-215 plasmids. RNA concentrations were determined by absorbance at 260 nm. In Vitro Editing and Primer Extension Assays The apo B mRNA editing assay was modified from the methods of Driscoll et al8 and Bostrom et al.11 The standard reaction contained 20 ng synthetic apo B RNA (354 nt of human or rabbit apo B sequence [see above]) and 12 units RNasin in reaction volumes of 100-600 /tl. The reactions were incubated at 30°C for either 45 minutes or 3 hours and stopped by the addition of stop buffer (100 mM tris[hydroxymethyl] aminomethane hydrochloride [pH 7.5], 10 mM EDTA, 1% sodium dodecyl sulfate [SDS], 0.2 M NaCl, and 0.2 mg/ml proteinase K) and incubation at 55°C for 15 minutes. The apo B RNA was extracted and precipitated, and primer-

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TABLE 1. Effect of Size and Species of RNA on In Vitro Apolipoprotein B mRNA Editing

C-•U(* >) Human apo B RNA (nt) 354 215 63 Apo B RNA (nM) 0.09 0.17 0.34 0.60 0.70

82 62 0.8 Human 6.2 7.2 5.4 4.3 3.2

Rabbit 13.8 12.8 8.5 6.9 6.9

An in vitro editing assay was performed with various types of synthetic apolipoprotein (apo) B RNA as substrate. The percentage of cytidine-to-uridine (C—>U) conversion was determined as described in "Methods." Human apo B RNAs (1.4 nM) of 63, 215, and 354 nucleotides (nt) generated from plasmids pBS-B63, pBS-215, and pBS-B354, respectively, were used as substrate for the in vitro editing assay. Apo B RNAs (354 nt) from humans and rabbits were used as substrates at the concentrations shown.

extension analysis was performed as previously described.11 The percentage of cytidine-to-uridine conversion (editing activity) was determined by scanning the dried primer-extension gels on an Ambis Radioactive-Analytical Scanner (Ambis Corp., San Diego, Calif.). To determine the accuracy of the procedure, 1, 2, 4, 5, 8, and 10 yA of a 32 P-5'-end-labeled oligomer were electrophoresed and the amount of radioactivity in each lane quantified. The linear dose-response curve obtained had a correlation coefficient of 0.9987. Results A 10-40% (NHOzSCVprecipitated fraction of the S100 cytosolic extract of rabbit enterocytes was used as the source of apo B mRNA editing activity (editing extract). Synthetic apo B RNA corresponding to the 354-bp rabbit sequence (see "Methods") was used as the substrate. Apo B mRNA editing was accomplished in an in vitro assay similar to that developed by Driscoll et al.8 Edited and unedited apo B mRNAs were quantified by a primer-extension analysis in which the 33-nucleotide (unedited) and 44-nucleotide (edited) ^P-labeled primer-extension products were directly quantified on dried polyacrylamide gels by scanning. To define the optimum substrate length for the editing reaction, we compared in vitro editing activities by using synthetic RNAs of 354, 215, or 63 nt of human apo B sequence as the substrate (Table 1). The 354-nt RNA was slightly better than the 215-nt RNA and significantly better than the 63-nt RNA as a substrate for the editing reaction. Next, we examined whether the rabbit apo B sequence might be edited more efficiently than the human sequence. Although there are only 41 nucleotide differences between the rabbit and human 354-nt apo B sequences, the rabbit sequence was edited almost two-

fold more efficiently than the human sequence and was therefore used for the majority of the experiments. The kinetic and biochemical properties of the apo B mRNA editing reaction were also investigated. The editing reaction could be detected after 5 minutes and was linear for 45 minutes, and the amount of edited apo B mRNA increased up to 6 hours (Figure 1A). In other experiments (data not shown), the editing reaction was shown to continue at reduced levels for at least 48 hours. The best preparations of editing extract (150 /ig) edited more than 65% of the synthetic RNA in 16 hours. At the incubation time of 45 minutes, which measures the initial rate of the reaction, the reaction rate was directly proportional to the amount of editing extract added (Figure IB). The editing activity had a broad pH range, with an optimum at 8-8.5 (Figure 1C). Other properties of the apo B mRNA editing reaction were also examined. We explored the effects of reaction volume on reaction rate. The reaction volume was varied from 0.025 to 1.6 ml. As would be expected from diluting the substrate and editing extract, the total amount of edited apo B mRNA decreased slightly, but it was apparent that the reaction volume could be varied without greatly influencing the reaction rate (Figure 2A). In contrast, the editing reaction was very sensitive to ionic strength. A KC1 or NaCl concentration of 0.1250.150 M was optimal for editing activity (Figures 2B and 2C). Using the optimum reaction conditions determined from the previous experiments, we determined the effect of increasing RNA substrate concentrations on the rate of apo B mRNA editing (Figure 3). The reaction rate was maximal at a substrate concentration of about 2 nM. The reaction exhibited Michaelis-Menten kinetics, as a doublereciprocal (Lineweaver-Burke) plot was linear, with a correlation coefficient of 0.9908. The Michaelis constant (Km) and the maximum initial velocity (Vm) as determined from the Lineweaver-Burke plot were 0.4 nM and 0.13 nmol/1-hr, respectively. Previously, Smith et al18 proposed that apo B mRNA editing occurs in a large complex similar to a spliceosome, which they designated an "editosome." Because spliceosomes contain a number of small RNAs necessary for splicing and because small RNAs are necessary for RNA editing in kinetoplastic mitochondria, we considered the possibility that RNA or DNA cofactors could be involved in apo B mRNA editing. To determine whether this was the case, we examined the effect of ribonuclease (RNase) or deoxyribonuclease (DNase) treatment on apo B mRNA editing. When preincubated with the editing extract, neither DNase nor RNase had any effect on apo B mRNA editing (Figure 4). Incubation of the editing extract with trypsin, however, abolished editing (data not shown). Thus, these results, which are similar to those of Driscoll and Casanova10 and Greeve et al,13 indicate that protein factors and/or enzymes are sufficient for apo B mRNA editing to occur and that RNA or DNA cofactors are

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Garcia et al

so

pH 6

10

100

Apo B mRNA Editing

150 200 (ig Protein

175

250 300

FIGURE 1. Panel A: Tune-course curve of apolipoprotein (apo) B mRNA editing activity. A 50-fil aliquot containing 150 fjg editing extract and 20 ng synthetic rabbit apo B RNA was removed from a 30°C reaction mixture at each time point and the reaction terminated as described The apo B RNA was extracted and analyzed by primer-extension analysis, and the percentage of cytidine-touridine (C—*U) conversion (editing activity) was determined as described in "Methods." Panel B: Plot of effect of increasing concentrations of editing extract on apo B mRNA editing. Increasing amounts of editing extract were added to the standard reaction mix and incubated at 30°C for 45 minutes and analyzed as described Panel C: Curve showing effect of pH on apo B mRNA editing. Forty-millimolar (2x) buffer solutions were prepared and pH-adjusted at 0.5 pH-unit intervals across the following ranges: Bis-Trispropane, pH 6.0-9.5; Tris, pH 7.0-9.5; and HEPES, pH 6.5-8.5. Equal volumes of 2 x buffer solutions and 2x reaction mix were combined The final reaction mix was identical to the standard reaction mix except for buffer and pH. The reaction mix was incubated for 45 minutes at 30°C and analyzed as described

not required. We also determined that no other cofactors are required. Extensively diatyzed extracts retained full editing activity, and the addition of ribonucleotide triphosphates or mono- or divalent metal ions to the extract did not affect activity (data not shown). To determine which conditions were optimal for the purification of the apo B mRNA editing factor(s), we examined the effect of a number of agents on activity. For example, editing activity was abolished by preincubation of editing extract with either 0.1% SDS, 4 M guanidine, or 6 M urea followed by dialysis in buffer D (data not shown). Activity was retained in the presence of low concentrations of DTT, whereas higher concentrations inhibited the editing reaction (data not shown). Heparin has been used to reduce the nonspecific binding of nuclear binding proteins to either RNA or DNA.19 We found that 25 ng/fd heparin in the in vitro assay increased the apo B mRNA editing activity (Figure 5). We also examined the effect of various enzymestabilizing agents on apo B mRNA editing. Many enzymes, especially multienzyme complexes, are stabilized by the addition of protein stabilizers such as glycerol or volume-excluding polymers such as polyethylene glycol (PEG) and ethylene glycol (EG). We examined various concentrations of glycerol, PEG, and EG in buffer D. As shown in Figure 6, all three reagents increased the activity of the editing reaction. Glycerol was the most effective, with an optimum concentration of 40%, whereas

15% PEG and 20% EG were the most effective concentrations of these stabilizers. Discussion We have characterized the apo B mRNA editing activity from cytosolic extracts of rabbit enterocytes. After defining the optimal conditions for the editing reaction, we determined that the Km for the reaction is 0.4 nM under these conditions, using synthetic rabbit apo B RNA as the substrate. In comparison, Greeve et al13 found the Km of the editing reaction for the rat enterocyte extract to be 2 nM, using human RNA as the substrate. Because we found higher editing activity (1.9-fold) using a homologous RNA substrate (compared with human RNA), it is likely that Greeve et al would have observed a lower Ka if they had used a homologous synthetic rat apo B RNA substrate. We also have found that the reaction does not require an energy source such as ATP or guanosine triphosphate, nor does it require divalent metal ions. Like Driscoll and Casanova 10 and Greeve et al,13 we found that no cofactors were needed for the editing reaction. We have shown, however, that the editing reaction is sensitive to ionic strength, pH, heat, denaturants, and proteases. In addition, treatment of the editing extract with RNase or DNase does not affect editing activity. Thus, the editing factor(s) appear to be composed of protein, and unlike many other types of RNA processing such as splicing, polyadenylation, and RNA editing in kinetoplastic mitochondria, no additional small RNAs ap-

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30 min @ 30° 45min@30°

20

c KCI [mM]

I 16

I

^

512

^ ^ £ N

st -UAA

CAA%UAA .025ml

.06ml

.18ml

.54ml

2

7

5

4

2

1.6ml

Volume of Reaction

FIGURE 2. Panel A: Bar graph of the effect of volume on apolipoprotein (apo) B mRNA editing. A reaction mix containing 200 ug editing extract was incubated with synthetic rabbit apo B RNA (20 ng) for 45 minutes in various assay volumes and analyzed as described. Also shown are autoradiograms demonstrating the effect of potassium chloride or sodium chloride concentration on apo B mRNA editing. Reaction mixtures containing 150 ug editing extract were incubated with synthetic human apo B RNA (20 ng) in the presence of 0-300 mM KCI (panel B) or 0-300 mM NaCI (panel C) for 3 hours at 30°C. Reactions were stopped and analyzed as described. The percentage of cytidine-to-uridine (C^*U) conversion or editing activity (%UAA) is listed under each lane.

NaCI [mM]

-UAA

CAA

-

%UAA

13 6

1,000-fold lower than that for a non-site-specific cytidine deaminase or for cytosine deaminase. For example, human liver cytidine deaminase has a Km of 9.2-49 /AM,20-21 whereas cytosine deaminase has a Km of 0.89 fiM.22 Thus, the sequence-specific cytidine deaminase would bind with high affinity to the apo B pre-mRNA, indicating that it could reach its maximum catalytic rate at the low concentrations of apo B pre-mRNA that occur in the nucleus. Based on an analogy to gene regulatory proteins,23 we speculate that in addition to binding with high affinity to the

pear to be involved. As shown in Table 2, which compares properties of the editing factors from enterocyte extracts of rats and rabbits as well as cultured rat hepatoma cells, the characteristics of the editing factor(s) from different sources are remarkably similar. These results are consistent with the concept that the editing reaction is catalyzed by a nucleotide sequence-specific cytidine deaminase. It is interesting to note that the Km for the apo B mRNA editing reaction is in a nanomolar range that is more than

0.14 0.12 •

0.10 0.08

o

•5 f

0.06 : 0.04 0.02

/

: T5

180 160 140 120

1 100

X "•

1 •/ /

y-

f

0.00 0.0

FIGURE 3. The effect of increasing RNA substrate concentration on the rate of apolipoprotein (apo) B mRNA editing. Increasing amounts of synthetic rabbit apo B RNA were incubated at 30*C for 45 minutes with 150 ug editing extract The reaction was stopped and analyzed as described. A Michaelis-Menten plot of the reaction is shown. Inset Lineweaver-Burke plot Regression analysis of 1/s vs. 1/v was performed according to least-squares analysis.

40 20

.10'0

10

ao

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40

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so

1/S (1/nM)

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1.0

1.5

2.0

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3.5

RNA Concentration (nM)

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Garcia et al

Apo B mRNA Editing

177

300,-

200 1

1

4

ou 10u

9

MU

a>u

FIGURE 4. Bar graphs of effects of ribonuclease (RNase; panel A) and deoxyribonuclease (DNase; panel B) treatment on apolipoprotein (apo) B mRNA editing. Forpanel A, editing extract (150 \ig) was preincubated at 23°C as follows: 1) (control) 30 minutes without RNase or RNasin; 2) 30 minutes with 5 ng RNase, followed by 15 minutes with 200 units RNasin; 3) 30 minutes with 200 units RNasin; 4) 15 minutes with 200 units RNasin, followed by 30 minutes with 5 ng RNase; 5) (positive control) 5 ng RNase added to a reaction mixture containing editing extract and synthetic human apo B RNA with no RNasin and incubated for 30 minutes at 23°C. Synthetic human apo B RNA (20 ng) was added to treatments 1-4, incubated at 30°Cfor 3 hours, extracted, and analyzed as described. For panel B, editing extract was incubated at 30°C for 15 minutes with 0,10, 20, or 30 units DNase. Samples were incubated with 20 ng synthetic human apo B mRNA at 30°C for 3 hours and analyzed as described Percentage of editing is shown on y axis (% TAA).

editing site, the apo B mRNA editing enzyme has a weak affinity for any RNA sequence. This would give the enzyme the advantage of being able to "bind and slide," thereby scanning long lengths of RNA for the specific recognition and binding site for apo B mRNA editing. Once the enzyme finds the very long apo B transcript, the probability of finding the specific editing site would be facilitated by one-dimensional diffusion. It has been proposed by Smith et al18 that apo B mRNA editing occurs in a very large complex (an editosome) that is analogous to a spliceosome. When synthetic apo B RNA was added to a hepatic editing extract, a huge complex with a sedimentation coefficient of 27S was formed, suggesting that the assembly of this complex is a prerequisite for editing activity.18 Heparin [ng

8

st

St

-UAA CAA%UAA

*lvtt?v« 9

8

11 15 8

6

0

FIGURE 5. Autoradiogram showing the effect of heparin on apolipoprotein (apo) B mRNA editing. A reaction mix containing 20 ng synthetic human apo B RNA, 150 ug editing extract, and increasing amounts of heparin (0-2,500 ngjul) was incubated for 3.5 hours at 30°C. The apo B RNA was isolated and analyzed as described. The percentage of cytidine-to-uridine (C-*U) conversion (%UAA) is listed under each lane.

o * o | 100

o 0

5

10

1

1

0

6

1 0

1 20

40

JO

Ethyfanoglycol i 1 i 10 Potytlhyton* glycd i

1



40

Qtycsrol

1 15

il

I 20

I 80

SO

% Reagent in Assay FIGURE 6. The effect of stabilizing agents and volumeexcluding agents on apolipoprotein (apo) B mRNA editing. An editing assay was performed under standard conditions for 3 hours in the presence of increasing amounts of gtycerol, ethylene gfycol, or polyethylene gfycoL The rabbit apo B RNA was isolated and analyzed as described The percentage of cytidine-to-uridine (C-*U) conversion (editing activity) in the presence of these agents is plotted

The results from our work on extracts of intestinal origin, while not specifically designed to examine this concept, do not support the notion of an editosome requirement for in vitro apo B mRNA editing. First, we found evidence of apo B mRNA editing after 5 minutes, and the amount of edited product is linear with time for the first 45 minutes of the reaction. Unless editosome assembly is a very rapid process or the binding affinities among the individual components are too high to permit dissociation, a lag period corresponding to the time necessary for editosome assembly would be expected. Second, the reaction kinetics represents a very simple one-substrate reaction that is linear when plotted according to the method of lineweaver and Burke. Moreover, when the editing reaction mixture was diluted 64-fold, there was only about a 40% decrease in editing activity. If the editing reaction required the assembly of a number of components, then dilution would have severely retarded the reaction. Third, we found no evidence for the involvement of nucleic acid cofactors in the reaction. Furthermore, Driscoll and Casanova10 also concluded that editing in vitro does not require a large macromolecular complex such as a spliceosome. They showed that editing activity in extracts from baboon enterocytes was unaffected by

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Arteriosclerosis and Thrombosis TABLE 2.

Vol 12, No 2 February 1992

Properties of ApoUpoprotein B mRNA Editing Activity From Different Species

Property Sensitivity to RNase DNase Protease pH optimum ATP, GTP requirement Ka Optimal concentration of NaCl or KC1 Metal cofactors EDTA (50 mM) Temperature conditions at which activity is abolished

Rabbit intestine*

Baboon intestinef

Rat intestine^

No No Yes 8-8.5 No 0.4 nM 0.125-0.15 M None Inhibits activity

No No Yes ND ND ND 0.1 M None No effect on activity

No No Yes 8.5 No 2.2 nM ND None Enhanced activity

40°C, 2 hours

40°C, 10 minutes

60°C, 5 minutes

RNase, ribonuclease; DNase, deoxyribonuclease; GTP, guanosine triphosphate; Km, Michaelis constant; ND, not determined. 'Data from rabbit intestine are presented in this article. tData from baboon intestine are from Driscoll and Casanova.10 tData from rat intestine are from Greeve et al. u

micrococcal nuclease treatment and that the addition of ribonucleotide triphosphates did not increase editing activity. On gel filtration chromatography, baboon enterocyte apo B mRNA editing activity eluted with an apparent molecular weight of 125,000. Very recently, Greeve et al13 characterized the apo B mRNA editing activity from rat enterocyte cytosolic extracts. Their results were very similar to our own and to those of Driscoll and Casanova10 in that they found no evidence of a nucleotide cofactor requirement or lag period for the reaction, and the reaction kinetics fits the Michaelis-Menten equation. Furthermore, Greeve et al found that the editing activity has a density corresponding to pure protein that has no nucleic acid content. They concluded that the apo B mRNA editing reaction bears little analogy to more complex types of RNA processing such as splicing or polyadenylation or to more complex types of RNA editing such as those that occur in kinetoplastic mitochondria. They also concluded that a single enzyme mediates apo B mRNA editing in vitro. Although we agree that apo B mRNA editing is a much simpler process than splicing and have observed no evidence of a protein complex analogous to a spliceosome, it is premature to conclude that the editing reaction is catalyzed by a single enzyme or protein. Probably the simplest model for apo B mRNA editing would be one in which a single pofypeptide identifies and binds the apo B mRNA as well as catalyzes the conversion of cytidine 6,666 to uridine. However, none of the data rule out the possibility that a preformed multicomponent protein complex deaminates cytidine 6,666. For example, in polyadenylation, pory(A) polymerase adds multiple adenylates to the 3' cleavage site. Alone, it lacks specificity and will polyadenylate any RNA.24-26 A second factor, the specificity factor, is necessary for the reaction to recognize the AAUAAA-containing RNAs specifically and must be present for the in vitro reaction to

mimic in vivo mRNA polyadenylation. Although the specific molecular reaction has not yet been elucidated, poly(A) polymerase, when combined with the specificity factor, adds poly(A) to AAUAAA-containing RNAs at substrate concentrations 1,000-fold lower than those of non-AAUAAA-containing RNA substrates.24 A model analogous to that of polyadenylation would be a multicomponent protein complex for apo B mRNA editing, in which one protein or subunit deaminates the cytidine after another recognizes and binds the RNA, thus providing the substrate specificity. Purification of the proteins responsible for apo B mRNA editing will clarify the protein/ complex requirements and the editing mechanism. Assuming that apo B mRNA editing occurs in the nucleus (for which there is circumstantial evidence16), then it probably is not an isolated event, and in vivo, the editing protein(s) probably are associated with other proteins involved in RNA processing as part of a complex or with extrinsic proteins as part of a transient complex. For example, there appears to be a connection between apo B mRNA editing and the activation of cryptic polyadenylation sites. The selection of alternate pory(A) sites was noted in edited apo B RNA isolated from human and rabbit intestine.46 This observation was mimicked by transfecting a vector containing 354 bp of apo B into intestinally derived human CaCo-2 cells.16 Two different-sized apo B messages were produced. The smaller message, in which an alternate polyadenylation site had been activated, was edited, while the larger apo B message contained predominantly the unmodified glutamine codon. These results suggest that the editing process leads to the recognition of a cryptic polyadenylation signal immediately downstream from the edited nucleotide, resulting in cleavage of the apo B mRNA and the addition of a pory(A) tail at the alternate site. In addition, it is known that pre-mRNAs in the nucleus occur as complexes with specific proteins. These macromolec-

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Garcia et al

ular complexes of proteins and RNA are designated heterogeneous nuclear ribonucleoprotein (hnRNP) particles, or hnRNP complexes. Proteins that are part of these complexes are, except for histones, the most abundant proteins in the nucleus. The hnRNP particles, which appear under electron microscopy like "beads on an RNA string," appear to play a role in RNA processing, both in splicing and polyadenylation.27 Because it appears that apo B mRNA editing is, in some cases, coupled with polyadenylation in vivo and that most forms of RNA processing involve macromolecular complexes, the possibility remains that the protein(s) responsible for apo B mRNA editing are part of a larger protein complex in vivo. Acknowledgments We thank Makram Michail for isolating the rabbit intestines used for these studies. We also thank Tom Rolain for graphics, Al Averbach for editorial assistance, and William Doolittle for manuscript preparation. Special thanks to B J. McCarthy for suggesting the volume dilution experiment and for critically reviewing the manuscript. References 1. Innerarity TL: Familial hypobetalipoproteinemia and familial defective apolipoprotein B10o: Genetic disorders associated with apolipoprotein B. Curr Opin Lipidol 1990;l:104-109 2. Scott J: Regulation of the biosynthesis of apolipoprotein B1Oo and apolipoprotein B^. Curr Opin Lipidol 1990;1:96-103 3. Young SG: Recent progress in understanding apolipoprotein B. Circulation 199O;82:1574-1594 4. Powell LM, Wallis SC, Pease RJ, Edwards YH, Knott TJ, Scott J: A novel form of tissue-specific RNA processing produces apolipoprotein-B48 in intestine. Cell 1987;50: 831-840 5. Hospattankar AV, Higuchi K, Law SW, Meglin N, Brewer HB Jn Identification of a novel in-frame translational stop codon in human intestine apoB mRNA. Biochem Biophys Res Commun 1987;148:279-285 6. Chen SH, Habib G, Yang CY, Gu ZW, Lee BR, Weng SA, Silbennan SR, Cai SJ, Desfypere JP, Rosseneu M, Gotto AM Jr, Li WH, Chan L: Apolipoprotein B-48 is the product of a messenger RNA with an organ-specific in-frame stop codon. Science 1987^38:363-366 7. Innerarity TL, Young SG, Poksay KS, Mahley RW, Smith RS, Milne RW, Marcel YL, Weisgraber KH: Structural relationship of human apolipoprotein B48 to apolipoprotein B100. / Clin Invest 1987;80:1794-1798 8. Driscoll DM, Wynne JK, Wallis SC, Scott J: An in vitro system for the editing of apolipoprotein B mRNA. Cell 1989-48: 519-525 9. Chen SH, Li X, Iiao WSL, Wu JH, Chan L: RNA editing of apolipoprotein B mRNA: Sequence specificity determined by in vitro coupled transcription editing. / Biol Chem 1990,265: 6811-6816

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10. Driscoll DM, Casanova E: Characterization of the apolipoprotein B mRNA editing activity in enterocyte extracts. / BM Chem 1990;265:21401-21403 11. Bostrdm K, Garcia Z, Poksay KS, Johnson DF, Lusis AJ, Innerarity TL: Apolipoprotein B mRNA editing: Direct determination of the edited base and occurrence in nonapolipoprotein B-producing cell lines. / Biol Chem 1990;265: 22446-22452 12. Hodges PE, Navaratnam N, Greeve JC, Scott J: Site-specific creation of undine from cytidine in apolipoprotein B mRNA editing. Nucleic Acids Res 1991;19:1197-1201 13. Greeve J, Navaratnam N, Scott J: Characterization of the apolipoprotein B mRNA editing enzyme: No similarity to the proposed mechanism of RNA editing in kinetoplastid protozoa. Nucleic Acids Res 1991;19J569-3576 14. Merchant JL, Heller RA: 3-Hydroxy-3-methylglutaryl coenzyme A reductase in isolated villous and crypt cells of the rat fleum. / Lipid Res 1977;18:722-733 15. Dignam JD, Lebovitz RM, Roeder RG: Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 1983;11: 1475-1489 16. Bostrom K, Lauer SJ, Poksay KS, Garcia Z, Taylor JM, Innerarity TL: Apolipoprotein B48 RNA editing in chimeric apolipoprotein EB mRNA. IBM Chem 1989-^64:15701-15708 17. Neupert B, Thompson NA, Meyer C, Kflhn LC: A high yield affinity purification method for specific RNA-binding proteins: Isolation of the iron regulatory factor from human placenta. Nucleic Acids Res 199OA&S1-55 18. Smith HC, Kuo SR, Backus JW, Harris SG, Sparks CE, Sparks JD: In vitro apolipoprotein B mRNA editing: Identification of a 27S editing complex. Proc Nad Acad Sci U S A 1991;88: 1489-1493 19. Briggs MR, Kadonaga JT, Bell SP, Tjian R: Purification and biochemical characterization of the promoter-specific transcription factor, Spl. Science 1986;234:47-52 20. Wentworth DF, Wolfenden R: Cytidine deaminases (from Escherichia coli and human liver). Methods Enzymol 1978^51: 401-407 21. Fanucchi MP, Watanabe KA, Fox JJ, Chou TC: Kinetics and substrate specificity of human and canine cytidine deaminase. Biochem Pharmacol 1986^5:1199-1201 22. West TP, Shanley MS, O'Donovan GA: Purification and some properties of cytosine deaminase from Salmonella typhimunum. Biochim Biophys Acta 1982;719:251-258 23. Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD: Molecular Biology of the Cell, ed 2. New York, Garland Publishing, 1989, p 127 24. Wickens M: How the messenger got its tail: Addition of pofy(A) in the nucleus. Trends Biol Sci 1990;15:277-281 25. Christofori G, Keller W: Pofy(A) polymerase purified from HeLa cell nuclear extract is required for both cleavage and polyadenylation of pre-mRNA in vitro. Mol Cell Biol 1989;9: 193-203 26. Wilusz J, Shenk T, Takagaki Y, Manley JL: A multicomponent complex is required for the AAUAAA-dependent crossUnking of a 64-kilodalton protein to poh/adenylation substrates. Mol Cell Biol 1990;10:1244-1248 27. Dreyfuss G, Swanson MS, Pinol-Roma S: Heterogeneous nuclear ribonucleoprotein particles and the pathway of mRNA formation. Trends Biochem Sci 1988;13:86-91 KEY WORDS • RNA processing • apolipoprotein B • RNA editing • apolipoprotein B-48

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Characterization of apolipoprotein B mRNA editing from rabbit intestine. Z C Garcia, K S Poksay, K Boström, D F Johnson, M E Balestra, I Shechter and T L Innerarity Arterioscler Thromb Vasc Biol. 1992;12:172-179 doi: 10.1161/01.ATV.12.2.172 Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1992 American Heart Association, Inc. All rights reserved. Print ISSN: 1079-5642. Online ISSN: 1524-4636

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Characterization of apolipoprotein B mRNA editing from rabbit intestine.

Apolipoprotein (apo) B-48 is generated by a unique physiological process. Cytidine 6,666 of the apo B primary transcript is posttranscriptionally conv...
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