Apobec-1 and apolipoprotein B mRNA editing

Biochimica et Biophysica Acta 1345 Ž1997. 11–26

Review

Apobec-1 and apolipoprotein B mRNA editing Lawrence Chan a

a,)

, Benny H.-J. Chang a , Makoto Nakamuta a , Wen-Hsiung Li b, Louis C. Smith a

Departments of Cell Biology and Medicine, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA b Human Genetics Center, SPH, UniÕersity of Texas Health Science Center at Houston, Houston, TX 77225, USA Received 15 July 1996; revised 30 September 1996; accepted 15 October 1996

Keywords: Apolipoprotein B; mRNA editing; Apobec-1; Post-transcriptional regulation; Gene expression; Action mechanism

Contents

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1. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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3. Physiology of ApoB-100 and ApoB-48 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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4. Discovery of ApoB mRNA editing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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5. Topology and physiological regulation of ApoB mRNA editing in the liver of rodents . . . .

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6. Sequence specificity of ApoB mRNA editing . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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7. Apobec-1, the catalytic subunit for a multi-protein editing enzyme complex . . . . . . . . . .

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8. Relative roles of ApoB-100 versus ApoB-48 in vivo: lessons from Apobec-1 knockout mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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9. Structure, evolution and phylogenetic analysis of apobec-1 . . . . . . . . . . . . . . . . . . . .

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10. Mechanism of action of Apobec-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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11. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Corresponding author. Fax: q1 Ž713. 7988764; E-mail: [email protected]

0005-2760r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 Ž 9 6 . 0 0 1 5 6 - 7

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L. Chan et al.r Biochimica et Biophysica Acta 1345 (1997) 11–26

1. Summary Apolipoprotein Žapo. B mRNA editing is a novel mechanism for the post-transcriptional regulation of gene expression in mammals. It consists of a C ™ U conversion of the first base of the codon CAA, encoding glutamine-2153, to UAA, an in-frame stop codon, in apoB mRNA. Since its initial description in 1987, substantial progress has been made in the last few years on the mechanism of editing. Apobec-1, the catalytic component of the apoB mRNA editing enzyme complex, has been cloned. This article begins with an overview of the general biology of apoB mRNA editing. It then provides an in-depth analysis of the structure, evolution and possible mechanism of action of apobec-1. ApoB mRNA editing is the prototype of RNA editing in mammals. What we learn from apoB mRNA editing will be useful in our understanding of other examples of RNA editing in vertebrates which are being described with increasing frequency. 2. Introduction Lipid homeostasis in mammals is regulated by intracellular lipid-related enzymes and binding proteins as well as by extracellular lipid-metabolizing enzymes and transport proteins. The extracellular lipid transport proteins are known as apolipoproteins. These proteins are an essential structural component of the plasma lipoproteins. The plasma lipoproteins are lipid microemulsion-like particles that contain a central core of nonpolar lipids and a surface monolayer of polar lipids Ž primarily phospholipids. . They vary greatly in size and are stabilized in the aqueous environment of the intra- and extravascular spaces by the amphipathic apolipoproteins. The plasma apolipoproteins can be classified into two groups, the soluble apolipoproteins Žthe major ones being apolipoprotein Ž apo. A-I, A-II, A-IV, C-I, C-II, C-III, and E. and the B apoproteins ŽapoB-100 and apoB-48.. The latter proteins differ from the former group of proteins in that they are water-insoluble and are tightly associated with the lipid components of the lipoprotein particles. Unlike the soluble apolipoproteins, they do not exchange between lipoproteins or other lipid surfaces. The two apoBs, B-100 and B-48, are involved in

two distinct pathways of lipoprotein biogenesis and metabolism. ApoB-100 is required for the production of very low density lipoprotein ŽVLDL. in the liver which is an essential component of the endogenous pathway. The VLDL is metabolized to intermediate density lipoprotein ŽIDL. and low density lipoprotein ŽLDL. . IDL and LDL are taken up by the LDL receptor, activating the LDL receptor pathway that has been elegantly worked out by Brown and Goldstein w1x. ApoB-48, in contrast, is required for fat absorption in the small intestine, being needed for chylomicron production. Dietary fat is an exogenous nutrient and apoB-48 is a key protein in the exogenous pathway of lipoprotein metabolism. The apoB48-containing intestinal lipoproteins are metabolized to chylomicron remnant particles which are taken up by remnant receptors, which are heterogeneous in nature w2x. There is substantial difference in lipoprotein metabolism between mammals and avians. In mammals, the intestinal chylomicrons enter lacteals and are discharged into the systemic circulation via the thoracic duct. In avians intestinal lipoproteins carrying the dietary lipids are called portamicrons and are directly discharged into the portal circulation. In these animals, both hepatic VLDL and intestinal portamicrons carry apoB-100 as their major apoprotein, and they do not have the capacity to produce apoB-48. During evolution, mammals have acquired a new species of apoB, apoB-48. Interestingly, like avians, mammals have only one apoB gene. They have the competence to produce apoB-48 because they have evolved a novel genetic mechanism, that of RNA editing, that enables them to produce apoB-48. RNA editing is a term coined by Benne et al. w3x who used it to describe the presence of nucleotides in mRNAs that are different from those encoded by the genome. The alterations in the RNA sequence occur post-transcriptionally in the protein-coding region w4x. Benne et al. w3x first described this phenomenon when they noted the occurrence of four uridines in the mitochondrial transcript for cytochrome-c oxidase Žcox. subunit 2 of trypanosomes that are not encoded in the DNA. Numerous other examples of RNA editing have now been described in trypanosomes as well as in many other organisms Ž for review see Ref. w5x.. A year after the discovery of RNA editing in

L. Chan et al.r Biochimica et Biophysica Acta 1345 (1997) 11–26

trypanosomes, Powell et al. w6x and Chen et al. w7x described apoB mRNA editing as the basis for the intestine-specific production of apoB-48 mRNA and protein in humans. ApoB mRNA editing has been the subject of several recent reviews w8–12x. A major advance in our understanding of the biochemical basis of apoB mRNA editing coincided with the cloning of apobec1, the catalytic subunit of the rat apoB mRNA editing enzyme complex w13x. In the first part of this review, we will provide a general overview of the discovery and biological consequences of apoB mRNA editing. With this background information, we will give an in-depth treatment of the structure, evolution and possible mechanism of action of apobec-1, areas that have not been covered in the previous reviews.

3. Physiology of ApoB-100 and ApoB-48 ApoB-48 is collinear with the N-terminal 48% or the first 2152 residues of apoB-100 which contains 4536 residues. However, the absence of the C-terminal portion of apoB-100 has turned apoB-48 into a protein with drastically different properties and biological functions Ž reviewed in Ref. w14x.. ApoB-100 is a highly complex protein. It is synthesized in the liver as an essential component of VLDL. As the VLDL is metabolized to IDL and LDL, the conformation of apoB-100 undergoes subtle changes. On LDL, apoB-100 consists of multiple subdomains that behave at least partially independently of one another. These subdomains differ with respect to the relative proportions of amino-acid residues that are exposed on the surface of the LDL particles and those that are buried just underneath the surface. In general, the protein appears to be extended and spans at least a hemisphere of the particle surface. A major function of apoB-100 is receptor-binding. ApoB-100 is a physiological ligand for the LDL receptor. Interestingly, only LDL apoB-100 is competent in this respect. In large IDL and in VLDL, apoE instead of apoB-100 appears to confer receptor-binding capacity to the lipoprotein particle. There is evidence that the receptor-binding domain of apoB-100 encompasses amino-acid residues 3359– 3367 Ž so-called domain B. w15,16x. However, it is

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clear that sequences some distance away from domain B are also important. The fact that LDL-apoB100 but not VLDL-apoB-100 is competent in receptor binding indicates that this property of apoB is very sensitive to subtle changes in protein conformation. ApoB-48, which misses domain B as well as the entire C-terminal half of apoB-100, is incompetent in LDL receptor binding. There are twenty-five cysteine residues in apoB100, sixteen of which are involved in intramolecular disulfide linkage. Eighteen of these cysteine residues are present in apoB-48. Of the seven cysteine residues in the C-terminal half of apoB-100 that is missing in apoB-48, one of them Žprobably C-4326. w17x appears to mediate the covalent linkage of apoB100 to apoŽa. in lipoprotein Ža., a unique LDL-like lipoprotein that is strongly correlated with atherosclerosis development. ApoB-48, in contrast, is incapable of contributing to lipoprotein Ža. production. It has been assumed that apoB-48 is needed for chylomicron formation and intestinal fat absorption. It now appears, however, that apoB-100 can replace apoB-48 in this function. On the other hand, there is good evidence that in rats, VLDL particles containing apoB-48 are not processed to LDL. ApoB-48 appears to lack the competence to form LDL and the apoB48-containing lipoprotein particles are metabolized via an apoE-specific receptor-mediated pathway w2x.

4. Discovery of ApoB mRNA editing ApoB-100 is a central molecule in lipoprotein and atherosclerosis research. It is a huge protein with highly unusual properties and is insoluble in water when it is delipidated. Despite the effort of a large number of laboratories all over the world, the standard protein chemistry approach failed to elucidate the primary structure of the protein. In 1986, the amino-acid sequence of apoB-100 was finally deduced from the nucleotide sequence of overlapping apoB-100 cDNAs w15,16x. Although some of the peculiar properties of apoB-100 were explained by the sequence, progress has been relatively slow in understanding this unusual protein because of its huge size and complex structure. In the meantime, investigators turned to the structure of apoB-48, which also had defied the usual biochemical approach. The

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L. Chan et al.r Biochimica et Biophysica Acta 1345 (1997) 11–26

Fig. 1. Schematic diagram of apoB mRNA editing. The genomic structure of apoB is at the top. The 29 exons are depicted by broad bars. The 28 introns are depicted by double lines between the exons both at the DNA level and at the RNA level. The nuclear compartment is enclosed by the nuclear membranes. Mature apoB mRNA is shown exported through a nuclear pore. ŽReprinted with permission from Ref. w8x..

only information available on apoB-48 was that obtained by the use of monoclonal antibodies which mapped apoB-48 to the N-terminal region of apoB100 w18x. Part of the difficulty with apoB-48 was that, unlike apoB-100 which is present in plasma at high concentrations, apoB-48 is normally barely detectable and insufficient amounts are available for detailed structural analysis. When large amounts of apoB-48 were isolated from the chyle isolated from a patient with chylous ascites, direct peptide sequencing of proteolytic fragments indicated that apoB-48 peptides span the N-terminal half of apoB-100 w7x. Finally, the structure of apoB-48 was deduced from the nucleotide sequence of apoB-48 cDNA clones w6,7x. The elucidation of apoB-48 cDNA sequence had an unexpected payoff — the discovery of apoB mRNA editing. ApoB-48 mRNA was found to differ from apoB-100 mRNA by a single base. The codon CAA for glutamine-2153 in apoB-100 mRNA was changed to UAA, an in-frame translation stop codon, in apoB-48 mRNA. The rest of the sequence appears to be identical in the two mRNAs. Subsequent research indicated that the C ™ U conversion was the consequence of RNA editing ŽFig. 1..

5. Topology and physiological regulation of ApoB mRNA editing in the liver of rodents In the small intestine of most mammals, apoB-48

mRNA is the predominant apoB mRNA species and generally accounts for some 70–95% of the total apoB mRNA. In rodents, the proportion of apoB-48 mRNA in the intestine stays fairly constant at 85–95% and is generally not regulated by physiological or pharmacological manipulations. In contrast, the amount of apoB-48 mRNA in the liver varies greatly among different mammals, from undetectable or barely detectable in most species, to about 20% in dog, 40% in horse and 60–70% in rat and mouse w19x. Studies in rat liver indicate that apoB mRNA editing occurs in the nucleus w20x ŽFig. 1. . The nascent apoB mRNA transcript is essentially totally unedited. As it is processed by splicing and polyadenylation, a higher and higher proportion of the mRNA is in the edited form. The fully mature spliced and polyadenylated apoB mRNA in the nucleus is edited to about the same extent as the cytoplasmic apoB mRNA, which indicates that apoB mRNA editing is predominantly an intranuclear event. Very little, if any, additional editing occurs following export of the mature mRNA into the cytoplasm w20x. The regulation of hepatic apoB mRNA editing has not been examined in dog or horse, but has been studied extensively in the rat. In this animal, nutritional factors affect hepatic apoB mRNA editing. When a rat is fasted and then refed a high carbohydrate diet, a manoeuvre that produces a 30-fold increase in hepatic triglyceride content, the propor-

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tion of apoB-48 mRNA decreases from 60–70% to about 30–40% during fasting and increases to 80– 90% in 2–3 days of refeeding w21,22x. The changes in apoB-48 mRNA are reflected by changes in the capacity of liver extracts for editing synthetic apoB mRNA substrates in vitro w23x. Ethanol is another dietary factor that modulates apoB mRNA editing. Experimentally, ethanol ingestion in rats has been shown to stimulate VLDL synthesis and secretion w24x. Lau et al. w25x fed male Wistar rats one of three different diets: Ži. a regular chow, Ž ii. an isocaloric liquid diet, or Žiii. an isocaloric ethanol-liquid diet where ethanol accounts for 35.5% of the total calories. They found that the ethanol diet resulted in a time-dependent increase in the proportion of hepatic apoB-48 mRNA which reached 100% by day 40 of the ethanol diet. There was no change in apoB-48 mRNA in animals on the regular chow or the isocaloric liquid diet. The change in the amount of edited apoB mRNA was accompanied by an increase in the relative amount of newly synthesized apoB-48 protein, from 30–50% to ) 99% of the total plasma apoB. The ethanol diet concomitantly induced hypertriglyceridemia as a result of a marked elevation of the plasma VLDL. There is a positive correlation between plasma triglyceride concentration and the proportion of apoB-48 mRNA in the liver, but no correlation of the latter with the intrahepatic triglyceride content. A number of hormones regulate apoB mRNA editing. Supraphysiological doses of estrogen decreased the proportion of apoB-48 mRNA in the rat liver w26x. Thyroid hormone treatment of hypothyroid rats increased the amount of apoB-48 mRNA in the liver w27x at the same time that it increased hepatic lipogenesis. In cultured primary rat hepatocytes, highdose insulin treatment caused an increase in the apoB-48rB-100 mRNA ratio from f 1 to f 7 w28x. Yamane et al. w29x examined a diabetic animal model, the GK ŽGoto–Kakizaki. rat, that has hyperglycemia, insulin resistance, hyperinsulinemia, diabetic nephropathy and neuropathy. They found that these animals had increased steady state plasma apoB-48 associated with an increased proportion of apoB-48 mRNA in the liver. The increased editing, however, could not be attributed to hyperinsulinemia because GK animals that developed hypoinsulinemia as a result of streptozotocin treatment, or animals with

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pancreatic islet b-cell exhaustion secondary to induced ventromedial hypothalamus lesions, also exhibited increased ratio of hepatic apoB-48rB-100 mRNA. Other factors, genetic or otherwise, probably induce the increased apoB mRNA editing in the GK rats, a genetic model of noninsulin-dependent diabetes mellitus.

6. Sequence specificity of ApoB mRNA editing ApoB mRNA editing involves C-6666, a nucleotide right in the middle of a 14-kilobase long mRNA. A simple assay for in vitro editing was developed by Driscoll et al. w30x that permitted the mapping of apoB mRNA sequence elements that appear to be important for editing in vitro. The most important mRNA sequence signals that direct the editing machinery to specifically deaminate C-6666 reside very close to C-6666. The signal motif can be divided into three parts, from the 3X end going in a 5X direction, they are a mooring sequence, a spacer sequence immediately 3X of C-6666 and a regulator region immediately 5X of the canonical C Ž reviewed in Refs. w31,32x and references therein.. The mooring sequence is both necessary and sufficient for conferring editing susceptibility to a C located 3–5 bases 5X upstream to the mooring sequence. Ligation of the mooring sequence downstream of any unrelated RNA sequence will also result in the editing of C’s in that sequence if they happen to be located 3–5 bases 5X to the mooring sequence w33x. In addition, sequences further removed from the editing site also contribute a poorly understood bulk RNA ‘context’ that enhances editing in vitro; the enhancing activity of the bulk RNA is related to the length and AU content of the sequence. Information on the sequence specificity of apoB mRNA editing is based on studies on tissue extracts or transfected cells in vitro. The protein componentŽs. that interact with the tripartite sequence motif, i.e., the mooring, spacer or regulator region, have not been identified. Under in vitro conditions, synthetic apoB mRNA fragments that encompass the tripartite sequence motif can be UV cross-linked to a f 40 kDa and a f 60 kDa rat liver protein w34–36x. The role of these proteins in apoB mRNA editing, if any, is unknown.

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7. Apobec-1, the catalytic subunit for a multi-protein editing enzyme complex Much of the early information on apoB mRNA editing was based on studies using tissue extracts from rat small intestine or liver, or small intestine extracts from other mammals. Some of the early experiments, primarily from Smith and coworkers w35,37x, indicate that there are macromolecular complexes, or ‘editosomes’, that are involved in apoB mRNA editing. Because none of the components of the editosome complexes were purified, the multicomponent editosome hypothesis remained controversial until the catalytic component of the editing enzyme complex, called apobec-1, was cloned w13x. Apobec-1, produced by translation in Xenopus oocytes, was found to be competent in editing apoB mRNA in vitro only in the presence of complementation factors in the form of either chicken or rat intestinal cytosolic extracts w13x. Subsequently, these complementation factorŽs. were found to be widely distributed in various mammalian tissues including those that do not produce apoB mRNA w38,39x. These observations provide support for the multi-component nature of the apoB mRNA editing apparatus. The primary structure of apobec-1 has been deduced from cDNA sequences from human, rabbit, rat and mouse. It is a 229-amino acid Žrat w13x and mouse w40x. to 236-amino acid Ž human w41,42x and rabbit w39x. protein that shows substantial sequence similarity to cytidinercytidylate deaminases from both mammals and bacteriophages w40,43x Ž vide infra. . Apobec-1 protein expression is extremely low and is undetectable in rat or mouse liver or intestine extracts by immunoblotting w44x. Apobec-1 mRNA is widely distributed in numerous tissues in rats and mice, but is detected only in the small intestine in humans. Thus, the tissue-specific pattern of apobec-1 mRNA expression suggests that apobec-1 may be limiting in the expression of apoB mRNA editing activity in humans. In mice, the mRNA for apobec-1 in the small intestine is smaller than that in the other tissues including the liver. The size difference was the result of differential promoter utilization and alternative splicing of the apobec-1 gene in the tissue w40x. The mouse apobec-1 gene contains eight exons Ž Fig. 2.. The major apobec-1 mRNA transcript in the liver is transcribed from all eight exons, whereas the major

Fig. 2. Structure of the mouse apobec-1 gene and its major mRNA transcripts. This schematic drawing is based on the study of Nakamuta et al. w40x.

small intestinal transcript contains exons 4–8. Two alternatively spliced hepatic apobec-1 mRNAs have also been observed Ž Fig. 2.. The human apobec-1 genomic structure has not been reported. The human apobec-1 gene is located in chromosome band 12p13.1–p13.2 w42x. The mouse gene is located on chromosome 6 in a region syntenic to human chromosome 12p12–p13, indicating that it is a true homolog of the human apobec-1 gene w40x. Following the cloning of apobec-1 cDNA, the regulation of apoB mRNA editing by various dietary and hormonal manipulations has been re-examined. The stimulation of editing in the rat liver by fasting followed by high-carbohydrate diet feeding was found to be associated with a marked increase in apobec-1 mRNA w45x. In contrast, when rat hepatic apoB mRNA editing was stimulated by thyroid hormone administration, hepatic apobec-1 mRNA concentration remained unchanged, which suggests that the increased editing activity was mediated by alterations in the complementation factors w46x. Similarly, the marked accumulation of apoB-48 mRNA relative to apoB-100 mRNA in the liver of rats fed a liquid ethanol diet was not accompanied by any change in the hepatic apobec-1 mRNA content w24x. Finally, the rat small intestine and liver became competent in apoB mRNA editing during embryonic and early postnatal development with distinct developmental patterns which were not directly correlated with apobec-1 mRNA abundance w46x. Thus, although apobec-1 is essential for apoB mRNA editing, it is

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only one of the many factors that determine apoB mRNA editing. The extent of editing of apoB mRNA in a cell is determined by the activity of the enzyme complex as well as other mechanisms. Chen and Chan w47x found that kinetic parameters, such as the apoB mRNA transcription and degradation rates, are also important in controlling the degree of apoB mRNA editing. For example, the rate of transcription of apoB mRNA has a transient effect on the extent of editing, whereas changes in mRNA degradation rate during a particular stage of mRNA maturation tend to alter the degree of editing only at the specific maturation stage. In transfection experiments, Sowden et al. w48x found that endogenous apoB gene transcripts and transcripts from transfected apoB minigenes were edited with different efficiencies. They proposed a complicated ‘gating’ hypothesis postulating a ‘nuclear restriction point’ limiting the degree of editing. The data of Sowden et al. w48x can also be readily explained by simple kinetic arguments formulated by Chen and Chan w47x.

8. Relative roles of ApoB-100 versus ApoB-48 in vivo: lessons from Apobec-1 knockout mice That apobec-1 is essential for apoB mRNA editing is supported by the complete absence of editing in apobec-1 knockout mice created by gene targeting w49–51x and its restoration by adenovirus-mediated transfer of apobec-1 cDNA w51x. These interesting animals provide valuable information on the relative roles of apoB-100 and apoB-48 in lipoprotein metabolism that were not evident previously. It is generally accepted that apoB-48 is essential for fat absorption from the small intestine because patients with abetalipoproteinemia suffer from steatorrhea w52x. Because chylomicrons are much larger than VLDL, it has been widely assumed that apoB100 and apoB-48 produce different types of lipoprotein particles and that animals that produce only apoB-100 might have difficulty in forming chylomicrons and manifest fat malabsorption. It turned out that apobec-1 knockout mice which produce only apoB-100 in both the liver and small intestine have normal growth and development whether they are fed a regular chow or a high-fat

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diet. They also form normal sized chylomicrons w50x. Therefore, like apoB-48, apoB-100 appears to be fully competent in supporting chylomicron production and fat absorption in the small intestine. This conclusion is also supported by observations in transgenic mice that express only apoB-100 w53x. Another unexpected finding in these apoB-100only animals is the presence of relatively normal plasma lipids and lipoproteins, although minor reductions in high density lipoproteins w51x and in LDLsized particles w50x were noted in two studies. The absence of the expected lipoprotein phenotype in the presence of markedly increased plasma apoB-100 prompted Nakamuta et al. w51x to cross-breed the knockout mice with human apoB transgenic mice. They found that the absence of apoB mRNA editing in apoB transgenic mice is associated with elevated total plasma cholesterol and triglyceride, as well as increases in VLDL and LDL. Since apoB-100 is secreted in VLDL and is an essential component of LDL, an increased concentration of these lipoproteins is to be expected in the presence of the hyperapoB100, which in the apobec-1 knockout animals is exaggerated in animals that overexpress the human apoB transgene w51x.

9. Structure, evolution and phylogenetic analysis of apobec-1 The similarity between apobec-1 and the cytidinercytidylate deaminases suggests that apobec1 has evolved from these housekeeping enzymes. Before the availability of the crystal structure of apobec-1, careful sequence comparisons and phylogenetic analysis have allowed the mapping of possible functional domains in apobec-1. To date, four mammalian Žhuman, mouse, rat and rabbit. apobec-1 cDNA sequences have been determined. The divergences among these four homologous sequences are relatively high w40x ŽTable 1.. Using the mouse and rat apobec-1 as an example, the synonymous divergences between these two sequences is 15.8%, which is only slightly higher than the average divergence among various genes between mouse and rat Ž14.4%, w54x.. In contrast, the substitution rate at the nonsynonymous sites in apobec-1 Ž4.0%. is almost 3-fold higher than the average of

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Table 1 Rates of nucleotide substitution in apobec-1 as defined by number of substitutions per synonymous site Žbelow diagonal. and per nonsynonymous site Žabove diagonal. using W.-H. Li’s method w54x GenBank a L07114 U22262 U10695 L26234

Rat rat mouse rabbit human

Mouse 4.0 " 0.9

15.8 " 3.5 60.7 " 9.4 54.2 " 8.4

67.1 " 10.0 53.5 " 8.0

various rodent genes Ž1.5%, w54x; also see Table 1.. These data indicate that apobec-1 is not a conservative protein. Apobec-1 is an essential component of the apoB mRNA-editing enzyme complex w55x. The high substitution rate in apobec-1 may reflect a coadaptation of this protein with its substrate, apoB mRNA, which seems to have an even higher substitution rate w54x. However, this hypothesis remains to be tested when the specific interactions between apobec1 and apoB mRNA are better understood. Using a sliding window approach on the aligned apobec-1 protein sequences ŽFig. 3., several conservative regions within apobec-1 can be identified ŽFig. 4. w40,43x. These include positions 15–20, 31– 35, 37–42, 61–66, 84–93, 127–131, 155–159 and 189–193. Positions 15–20 and 31–35 represent a potential bipartite nuclear localization signal. Posi-

Rabbit

Human

21.8 " 2.4 19.5 " 2.2

18.9 " 2.2 17.2 " 2.0 15.3 " 1.9

47.8 " 7.7

tions 61–66 and 84–93 are part of the apobec-1 catalytic domain in which His-61 and Cys-93 are potential zinc-chelating sites and Glu-63 is the hypothetical residue for chemical reaction of deamination Žvide infra.. Another hypothetical zinc-coordinating residue, Cys-96, is also conserved among these four mammalian species. Positions 189–193 contain two completely conserved proline residues flanked on either side by leucine-rich sequences. Deletion of the leucine-rich region in rat apobec-1 leads to the elimination of editing activity w13x. The leucine-rich sequence in apobec-1 is not a leucine-zipper motif because it is interrupted by proline residues w40,43x. Similar leucine-rich repeats found in other proteins are thought to be involved in protein–protein interaction w55x. The leucine-rich region in apobec-1 may be important for the dimerization of apobec-1 w42x as

Fig. 3. Average proportion of substitutions per amino-acid position per sequence pair computed by using a sliding window size of 5 amino-acid residues. The smaller divergence values correspond to the better conserved positions. ŽReprinted with permission from Ref. w40x..

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Fig. 4. General structure of apobec-1. The protein which contains 236 amino acids in the human and rabbit enzyme is depicted linearly from the N- to the C-terminus. The interrupted line close to the C-terminus marks the position of the C-terminus of mouse and rat apobec-1 which is shorter by 7 residues than the human and rabbit apobec-1. Amino-acid residue numbers are displayed below the protein. The zinc-coordinating residues, H61, C93 and C96, are shown above the protein. The putative bipartite nuclear localization signal ŽNLS. is as shown; the second half of the signal is PRXXRK in human, rat and mouse, but PQXXRK in rabbit. Of the three ‘deaminase homology’ boxes, only the Nterminal two have a counterpart in all the other cytidinercytidylate deaminases Žsee Table 2.; the third ŽC-terminal. box shows similarity to a similar region found in E. coli cytidine deaminase only, because this region is missing in the other deaminases which tend to be much shorter proteins. The vertical bars for the leucine-rich domain mark the position of individual leucine residues in this domain.

well as the interaction of apobec-1 with complementation factors. These putative functional regions are imbedded in the otherwise rapidly evolving sequences and are likely maintained by negative selection pressure. There are several cytidine and cytidylate deaminases from different organisms that show different degrees of homology with apobec-1. These deaminases seem to be very similar functionally because they all require zinc at the catalytic center for coordinating deamination activities and, in fact, the short putative zinc-chelating motifs CHAE and PCG are conserved across most of these proteins. Phylogenetic analysis ŽFig. 5. suggests that mammalian apobec-1 and other deaminases diverged very early in evolution. The position of the root, as determined by UPGMA analysis, also suggests that the cytidylate deaminases ŽCMPrdCMP deaminases. belong to a more ancient group than the cytidinerdeoxycytidine deaminases. Included in the analysis are the two known double-stranded RNA-specific adenosine deaminases, human dsRAD and RED1, which are candidate enzymes for glutamate receptor subunit pre-mRNA editing in mammalian brain, and a related

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mitochondrial sequence of unknown function from C. elegans ŽT20H4.4. . These three sequences are quite diverged except for the catalytic domains, but they are closer to one another than to the other deaminases, forming a separate group offshooting from the cytidylate deaminases. It is intriguing that the dsRNA adenosine deaminases essentially share little homology with other known adenosine deaminases which utilize 3 histidine residues for zinc coordination w56x. As discussed in the section on the Section 10 below, a comparison of the crystal structures of E. coli cytidine deaminase and adenosine deaminase suggests that any similarity in reaction mechanism between these two enzymes is the result of convergent evolution Ž vide infra. . Kim et al. w57x suggested that the catalytic mechanism of human dsRAD might be more similar to that of cytidinercytidylate deaminase gene family, especially to apobec-1, since they share similarities both in signature peptide residues, i.e., HAE and PCG, and in enzyme kinetics. Two other deaminases also show homology to cytidinercytidylate deaminases. The Bacillus cereus blasticidin-S deaminase converts blasticidin-S, a nucleoside antibiotic, to a non-toxic deaminohydroxy derivative w58x. This protein is closer evolutionarily to the cytidine deaminases Ž Fig. 5. , which is not unexpected, because they share substrates with similar structures, i.e., nucleosides. The E. coli ribG protein catalyzes the conversion of 2,5-diamino-4-oxy-6ribosylamino-pyrimidine 5X-phosphate to 2,4-dioxy5-amino-6-ribosylaminopyrimidine 5X-phosphate ŽTable 2.. Both chemicals are intermediates of riboflavin biosynthesis w59x. Since the chemical structure of the ribG substrate is very similar to that of cytosine monophosphate, it is not surprising to find ribG sequence showing more similarity to those of cytidylate deaminases ŽTable 2, legend.. T2 and T4 phages as well as human cytidylate deaminases seem to be composed of homohexamers ŽTable 2.. Cytidine deaminases appear to function either as homodimers, as in the case of the E. coli enzyme, or as homotetramers, as in the case of the human and Bacillus subtilis enzymes. Apobec-1 is a nonglycosylated protein that exists as a spontaneous homodimer w42x. Homodimerization is not mediated by disulfide bridge formation between the cysteinerich apobec-1 monomers. Since many other cytidinercytidylate deaminases also exist as homodi-

apobec-1 apobec-1 apobec-1 apobec-1 cytidylate deaminase cytidylate deaminase cytidylate deaminase cytidylate deaminase cytidylate deaminase unknown unknown unknown ribG

unknown dsRAD

RED1

cytidine deaminase

cytidine deaminase

cytidine deaminase

cytidine deaminase

blasticidin-S deaminase

U22262 L07114 U10695 L26234 P00814 P16006 L12136 P06773 P32393 P30648 P21335 P30134 P25539

U00037 U10439

U43534

P13652

L13289

L27943

P19079

JS0609

a

Gene product

GenBank a

Table 2 Gene products of apobec-1-related sequences

;

b

B. cereus

B. subtilis

human

Micopl. pirim.

E. coli

rat

C. elegans human

mouse rat rabbit human phage T2 phage T4 human yeast B. subtilis C. elegans B. subtilis E. coli E. coli

Organism

homotetramer

homotetramer

homotetramer

NA

homodimer

NA

NA monomer

homodimer homodimer homodimer homodimer homohexamer homohexamer homohexamer NA NA NA NA NA NA

a of functional units

Nakamuta et al. w40x Teng et al. w13x Yamanaka et al. w39x Lau et al. w42x Maley et al. w76x Maley et al. w77x Weiner et al. w78x McIntosh and Haynes w79x Hahn et al. w80x Sulston et al. w81x Taura et al. w82x Wilson et al. w83x Kim et al. w57x

C ™U editing in ApoB mRNA C ™U editing in ApoB mRNA C ™U editing in ApoB mRNA C ™U editing in ApoB mRNA converting dCMPrCMP to dUTPrUTP converting dCMPrCMP to dUTPrUTP converting dCMPrCMP to dUTPrUTP converting dCMPrCMP to dUTPrUTP NA NA NA NA deamination of nucleotide derivative in riboflavin biosynthesis a NA converting adenosine to inosine in double-strand RNA converting adenosine to inosine in double-strand RNA converting cytidinerdeoxycytidine to uridinerdeoxyuridine converting cytidinerdeoxycytidine to uridinerdeoxyuridine converting cytidinerdeoxycytidine to uridinerdeoxyuridine converting cytidinerdeoxycytidine to uridinerdeoxyuridine converting blasticidin-S b to deaminohydroxy derivative

Kobayashi et al. w89x

Song and Henhard w88x

Kuhn et al. w87x

Tham et al. w86x

Melcher et al. w84x Yang et al. w85x

Reference

Reaction catalyzed

20

L. Chan et al.r Biochimica et Biophysica Acta 1345 (1997) 11–26

L. Chan et al.r Biochimica et Biophysica Acta 1345 (1997) 11–26

mers or homomultimers ŽTable 2., it appears that the dimeric or multimeric nature of these closely related deaminases has been conserved through evolution. As discussed in a later section, the mechanism of action of E. coli cytidine deaminase involves, for each dimer, two active sites formed across the interface between the two monomers. The conservation of dimerization in apobec-1 may be a means for conserving the reaction mechanism during evolution. In contrast to the cytidinercytidylate deaminases, which require oligomerization for proper function, the dsRNA adenosine deaminase ŽdsRAD. appears to function in the form of a monomer w57x. Thus, despite the sequence similarities, there must be fundamental differences in the mechanism of action of these dsRNA adenosine deaminases and apobec-1 or E. coli cytidine deaminase. The presumptive protein–protein interacting leucine-rich domain of apobec-1 is unique among the cytidinercytidylate multiple gene family. Most of the other proteins are shorter and do not have a leucine-

21

rich domain in their C-terminal portion. E. coli cytidine deaminase ŽCDA. , however, is longer than apobec-1. It contains a short stretch of residues Žresidues 235–249. at its C-terminal half which shows high homology to apobec-1 at the very beginning of the leucine-rich domain as shown below: E. coli CDA Human APOBEC-1

235

PTLP PPLQGAL LILL LNL L249 PQYP PPLWMML LYAL LEL L182

169

In this 15-amino-acid stretch, 3 proline and 4 leucine residues are completely conserved between CDA and apobec-1. The significance of this homology is yet to be determined. Protein crystallography of the CDA shows that this region is not directly involved in enzyme catalysis w60x. Removal of the equivalent region in apobec-1 by deleting the Cterminal portion of the molecule abolishes apoB mRNA editing w13x. So potentially this region may be needed to maintain the correct conformation of the enzyme, or it may play a role in dimerization, a

Fig. 5. Phylogenetic tree of the apobec-1-related sequences reconstructed using neighbor-joining method w90x. Only the catalytic domain of 34 peptide residues were aligned and analyzed. The superscript denotes sequence with unknown function, and the GenBank accession numbers of these sequences are: 1, P30648, 2, P21335, 3, P30314, and 4, U00037. See Table 2 for the GenBank accession number of the other sequences. The horizontal scale at the bottom of the figure indicates the numbers of amino acid substitutions per position along the tree branches.

L. Chan et al.r Biochimica et Biophysica Acta 1345 (1997) 11–26

22

property inherent in both CDA and apobec-1, or in other protein–protein interactions required for apoB mRNA editing. It is intriguing, however, that this region is actually more variable among the apobec-1s from different mammalian species as compared, for example, to the catalytic domain. It could be that the periodicity of the prolines and leucines is important functionally and, therefore, is conserved, or, alternatively, this region could represent a part of apobec-1 that has drifted away from its ancestral function. To date, only apoB mRNA has been found to be a natural substrate for apobec-1. It is possible, however, that apobec-1 also edits other RNAs. Studies in transgenic animals suggest that overexpression of apobec-1 in the liver results in the editing of other unrelated RNA species Že.g., tyrosine kinase mRNA. that contain mooring sequence downstream of the edited site w61x. High level expression of apobec-1 is also known to give rise to promiscuous editing w62x, as well as ‘hyperediting’ Žmooring sequence-independent nonrandom editing of C’s. of apoB mRNA sequences w63x. Our phylogenetic analysis shows that apobec-1 is distantly related to the other members of the cytidinercytidylate deaminase gene family, implying that the ancestral apobec-1 gene may have had a function similar to cytidinercytidylate deaminases. How did apobec-1 evolve from a protein using a nucleoside as substrate to one that uses RNA, a polynucleotide, as substrate? In light of the fact that complementation factors seem to be widely distributed in various vertebrate tissues, apobec-1 could have evolved simply through gene duplication. However, the evolution of apobec-1 is likely to be more complicated for the E. coli rat mouse human rabbit

CDA APOBEC-1 APOBEC-1 APOBEC-1 APOBEC-1

following reasons. First, the editing of apoB mRNA involves multiple factors that require protein–protein and protein-RNA interactions. It is difficult to envision how a single gene duplication would lead to the formation of an efficient editosome complex without any co-adaptation or perhaps duplication of other genes. Second, even though the separation of apobec1 and other cytidinercytidylate deaminases is ancient, apobec-1 is present only in mammals. This implies that ancestral apobec-1 might be involved in a function other than apoB mRNA editing, and the ability to edit apoB mRNA is gained after the emergence of mammals. What is the function of the ancestral apobec-1 gene? Are there other apobec-1like genes in mammalian or non-mammalian genomes? Finding these apobec-1-related genes and deciphering their functions will help us understand how apobec-1 made a substrate transition and what structural modification was required for such a change.

10. Mechanism of action of Apobec-1 The conversion of C-6666 in apoB mRNA to a U involves the removal of an amine group. Apobec-1 has been shown to also convert free cytidine to uridine w43x, albeit at a much slower rate than E. coli cytidine deaminase. As discussed in the last section, apobec-1 shows substantial sequence similarity to E. coli cytidine deaminase ŽCDA. whose crystal structure has been solved by Betts et al. w60x. The activesite domain of the two enzymes can be readily aligned as shown below:

tvHaEqusai108 nkHvEvnfi67 snHvEvnfl67 tnHvEvnfi67 tnHvEvnfl67

Based on this sequence similarity, we infer that the mechanism of action of apobec-1 must be very similar to that of E. coli CDA. Another nucleotide deaminase, adenosine deaminase, has also been crystallized w64x, and its deduced mechanism will be used for comparison. Carter w65x notes that both CDA and adenosine

nytPCghCrqfmnE138 swsPCgeCsraitE102 swsPCgeCstaitE102 swsPCweCsqairE102 swsPCweCsmairE102

deaminase have similar chemical groups in their active sites and that both enzymes envelope their nucleoside substrates completely, a structural feature that may account for the inactivity of these enzymes on RNA substrates. The quaternary and tertiary structure of the zinc fingers in the catalytic center of apobec-1 is homologous to the corresponding region

L. Chan et al.r Biochimica et Biophysica Acta 1345 (1997) 11–26

of the cytidine and adenosine deaminases, since apobec-1 does function as a cytidine deaminase w43x. Catalysis proceeds with the activation by zinc of a bound water molecule, presumably to hydroxide ion, which attacks the appropriate carbon to generate a tetrahedral intermediate. The detailed stereochemistry of the two resulting chiral centers is diastereoisomeric. Details of the ensuing proton transfer steps necessary to generate and release the products are also apparently different in the related enzymes. Thus, the active-site similarities are probably the result of convergent evolution. The structure of E. coli CDA complexed to the transition state analog, 5-fluoroprimidin-2-one ribonucleoside, has been solved w60x. The crystallized enzyme, which has significant homology with apobec-1, exists as a dimer. The monomer of the a 2 CDA dimer has a small N-terminal a-helical domain unrelated to the active sites, and two, larger, core domains. The two core domains have nearly identical tertiary structures and are related by approximate two-fold symmetry, but lack internal amino-acid sequence homology. The two active sites per dimer are formed across the subunit interface. The N-terminal core domain provides a pyrimidine nucleoside and zinc-binding pocket. The structurally homologous C-terminal core domain in the other monomer covers this active-site cleft, completely sequestering the ligand from solvent. The specificity for ribose depends on hydrogen bonding between the 2X hydroxyl group of the ribose moiety, Asn-89 and Glu-91 ŽFig. 6. , and the amide carbonyl of Ala-631, which hydrogen bonds to the 5X hydroxyl group. The authors note that the deeply buried zinc-binding site is formed by a novel ‘topological switch point’ at the amino termini of two a-helices in consecutive a-b-a-b-segments. The transition-state analog is bound as a covalent hydrate at C4 of the inhibitor. The hydroxyl oxygen atom of the analog interacts both with the zinc atom and the Glu-104 carboxylate group. The zinc atom is coordinated in a tetrahedral ligand field to two cysteine and one histidine ligands, plus the hydroxyl group. The conserved carboxylate side-chain, Glu104, provides all of the necessary proton transfer functions involved in generating the zinc hydroxide nucleophile, and protonating the pyrimidine ring nitrogen atom and leaving amino group. Carlow et al.

23

Fig. 6. Schematic diagram of the CDA:transition-state analog and active-site hydrogen-bonding interactions. The hydrogen bonding interactions involving Ala-103, Glu-104 and Cys-129 of CDA are shown as dashed lines. Adapted from Betts et al. w60x. The observed evolutionary conservation of amino acids in the activesite region of apobec-1, combined with site specific mutants which were devoid of activity, strongly implies that the configuration of the active site is essentially the same in apobec-1 and CDA.

w66x show that Glu-104 not only stabilizes the activated ES complex in the transition state, but also destabilizes the ES complex in the ground state. Xiang et al. w67x note that as the substrate approaches the tetrahedral transition state, the zinc-activated hydroxyl group develops maximal negative charge and forms a short hydrogen bond to the neighboring carboxylate group of Glu-104. These authors suggest that the Zn-S-g-132 bond functions throughout the reaction as a ‘ valence buffer’ that accommodates changing negative charge on the hydroxyl group as the reaction proceeds. The stereochemistry of the binding site dictates high differential affinity for the hydroxyl group compared to a hydrogen atom at C4. The K i f 3 = 10y13 to 12 = 10y13 for an activesite-directed ligand with a hydroxyl group at the site of hydrolytic deamination is about seven orders of magnitude greater than that of an homologous compound with only a hydrogen at this position w68x. The absolutely conserved residues in the first domain are the zinc-liganding histidine and glutamic acid which appears to mediate both of the obligatory protonation steps at N-3 and at the leaving amino group Ž Fig. 7.. The PCxxC sequence cluster, which constitutes the other domain of the zinc-binding site, is separated by 26 residues in CDA and 32 residues in apobec-1. The catalytically important amino acids are conserved in apobec-1, as shown by site-specific mutations of His-61, Cys-93, Cys-96 w38,39,69,70x, Glu-63, and Pro-92 w69x. The polymeric mRNA substrate of apobec-1 precludes an enzyme conformation

24

L. Chan et al.r Biochimica et Biophysica Acta 1345 (1997) 11–26

Although the strong sequence homology between the catalytic center sequences of E. coli CDA and apobec-1 suggests similarities in the mechanism for deamination of the cytidine in their respective substrates, the polynucleotide nature of the natural substrate, apoB mRNA, for apobec-1 indicates that there are some important differences between the two enzymes. Apobec-1 by itself is a relatively weak RNAbinding protein w70,71x; the major RNA-binding component of the editing enzyme complex appears to reside in another complementation Žor auxiliary. proteinŽs. that also interact with apobec-1. How and when the mRNA substrate fits in the apobec-1 or apobec-1-complementation factor complex must await future structural analysis using the purified components.

Fig. 7. Schematic diagram of the implied mechanistic steps for CDA. ŽA. The Michaelis complex, E:H 2 O:S. The rearrangements that lead to the transition state include a nucleophilic attack by the zinc hydroxide group on C-4 of cytidine and protonation of N-3 by OE1 of Glu-104. ŽB. The first tetrahedral intermediate, derived from the transition-state analog complex. ŽC. The second tetrahedral intermediate formed by proton transfer to the leaving group from the hydroxyl via OE2 of Glu-104. ŽD. The enzymeproduct complex, EP. ŽE. The ligand-free enzyme after entry of a new substrate water molecule, which is deprotonated by the carboxylate group of Glu-104. ŽF. Entry of a new substrate cytidine. Apobec-1 Glu-63 is the functional equivalent of Glu-104 in CDA, serving successively as a proton donor ŽStep A., acceptor ŽStep B. and donor ŽStep C. during the catalytic deamination of the cytidine residue in mRNA. The carboxylate and the zinc hydroxide nucleophile of the enzyme is regenerated ŽStep E. by the disproportionation of a water molecule. Adapted from Betts et al. w60x.

in which the substrate is buried and not accessible to solvent, but those still to be identified specific structural features apparently have little effect on the catalytic mechanism. MacGinnitie et al. w69x report that mutation of the first four leucines within the heptad repeat of the leucine-rich region of apobec-1 reduced RNA editing activity without affecting cytidine deaminase activity. Mutation of His-61 abolished RNA binding, while the Glu-63 and Cys-96 mutant proteins showed wild-type levels of RNA binding. The His-61 and Glu-63 mutants acted as dominant negative inhibitors, reflecting the fact that apobec-1 exists as a spontaneous homodimer w42x.

11. Concluding remarks In this article, we have reviewed our current knowledge of apoB mRNA editing with special emphasis on the structure and function of apobec-1. We expect that progress in this area will be rapid and, before long, all the components of the apoB mRNA editing complex will be purified or cloned, at which time we will have a clear understanding of how they are assembled into an editosome complex. The coordinate regulation of the individual components, including apobec-1, will determine the efficiency of apoB mRNA editing in the cell. Since its initial description about a decade ago w6,7x, apoB mRNA is still considered by some to be a quirk of nature. However, other examples of RNA editing are beginning to be described with increasing frequency in vertebrates Ž e.g. Refs. w72–75x.. It is likely that in the next decade RNA editing will be recognized as an important, or perhaps even a common, mechanism for the post-transcriptional regulation of gene expression in the eukaryote.

Acknowledgements The work described performed in the authors’ laboratories was supported by U.S. National Institutes of Health grants HL-27341 and HL-56668. We thank

L. Chan et al.r Biochimica et Biophysica Acta 1345 (1997) 11–26

Ms. Irene A. Harrison for her expert secretarial assistance in the preparation of this manuscript.

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