Biochimica et Biophysica Acta 1494 (2000) 1^13

www.elsevier.com/locate/bba

Review

RNA editing: cytidine to uridine conversion in apolipoprotein B

mRNA
Ann Chester a , James Scott a , Shrikant Anant b , Naveenan Navaratnam a;
*
a
MRC Molecular Medicine, Clinical Science Centre, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK
b
Department of Internal Medicine, Washington University Medical School, St. Louis, MO 63110, USA

Received 24 May 2000; received in revised form 14 August 2000; accepted 22 August 2000

Abstract

RNA editing is a post-transcriptional process that changes the informational capacity within the RNA. These processes include
alterations made by nucleotide deletion, insertion and base conversion. A to I and C to U conversion occurs in mammals and these editing
events are catalysed by RNA binding deaminases. C to U editing of apoB mRNA was the first mammalian editing event to be identified.
The minimal protein complex necessary for apoB mRNA editing has been determined and consists of APOBEC-1 and ACF. Overexpression
of APOBEC-1 in transgenic animals caused liver dysplasia and APOBEC-1 has been identified in neurofibromatosis type 1 tumours,
suggesting that RNA editing may be another mechanism for tumourigenesis. Several APOBEC-1-like proteins have been identified,
including a family of APOBEC-1-related proteins with unknown function on chromosome 22. This review summarises the different types of
RNA editing and discusses the current status of C to U apoB mRNA editing. This knowledge is very important in understanding the
structure and function of these related proteins and their role in biology. 2000 Elsevier Science B.V. All rights reserved.

Keywords : RNA editing ; Apolipoprotein B; APOBEC-1; Cancer RNA

1. Introduction DNA. RNA editing in these species can be very variable,

The concept of RNA editing was rst introduced in
1986 to describe the process of post-transcriptional inser-
tion of non-genomically encoded uridylate residues to gen-
erate mitochondrial mRNAs of kinetoplastid protozoa [1].
At this point the RNA editing event involved phospho-
diester bond cleavage and ligation. Since then many addi-
tional RNA editing events have been identied. The cur-
rent denition of RNA editing is the modication of the
RNA sequence from that of the genomic sequence, except
RNA splicing, polyadenylation and capping [2]. RNA ed-
iting is now divided into two major groups, insertion/dele-
tion editing and substitution editing.
Insertion/deletion editing was rst described in kineto-
plastid protozoa mitochondrion in which the insertion or
deletion of uridine (U) residues are specied by small
guide RNAs encoded by the minicircle DNAs [3,4]. The
pre-mRNAs are encoded by the less abundant maxicircle
* Corresponding author. Fax: +44-20-8383-2028;
E-mail : nnaveene@hgmp.mrc.ac.uk
in the case of cytochrome oxidase II only four uridines are
inserted, where as the cytochrome oxidase III mRNA of
Trypanasoma brucei has hundreds of uridines inserted and
dozens of uridines deleted. The cleavage^ligation mecha-
nism involved in this type of insertion editing requires an
RNP complex containing endonuclease, terminal uridyl
transferase, and RNA ligase. Deletion editing also requires
a U-specic 3P-exonuclease. The small guide RNAs play a
major role in specifying the editing sites [5]. Cytidine to
uridine substitution editing also occurs in the mitochon-
dria of these cells [6]. A detailed description of kinetoplas-
tid protozoa RNA editing is beyond the scope of this re-
view and the subject was reviewed recently [4,7].
Cytidine (C) insertion editing in the mitochondria of the
myoxomycete Physarum polycephalum was rst reported
by Miller and colleagues [8]. Since that time a large num-
ber of additional editing events have been identied. These
include the insertion of each of the four nucleotides and
dinucleotides CU, CG, GU, UA and AA, but no deletion
editing has been reported [9]. C to U editing in the mito-
chondrial RNAs has also been observed [10]. Thus, Physa-
rum is dierent from the norm and is capable of carrying
out substitution as well as insertion editing. The mecha-

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2 A. Chester et al. / Biochimica et Biophysica Acta 1494 (2000) 1^13
nism and the components involved in this editing are not
fully understood.
The rst example of tRNA editing in a mitochondrial
system was from Acanthmoeba castellanii reported by
Lonergan and Gray in 1993 [11]. The changes consist of
single nucleotide conversions (U to A, U to G, A to G and
C to A) and they are found at the 5P end of the tRNA.
This type of editing had been observed in several other
tRNAs and the editing machinery would require at least
an endo or an exonuclease and a nucleotidyl transferase.
Studies by Gray and colleagues indicated strong evidence
for the presence of such activity in A. castellanii [11,12].
Mechanistically, this process would be more similar to
insertion/deletion editing rather than to substitution edit-
ing.
Cytidine (C) to uridine (U) substitution editing was rst
reported for the nuclear encoded mRNA of apolipopro-
tein B (apoB) in 1987 [13,14] and is discussed in detail in
this review. This example of RNA editing was the rst
type to be observed in vertebrates. C to U editing in plants
was initially reported as possible reverse transcriptase or
cloning errors [15]. However, these changes were con-
rmed as RNA editing events and are found in plant mi-
tochondria and plastid mRNA and tRNA. The plastid
genomes of higher plants consist of 120^130 kb circles
from which 20^30 cytidines are converted to uridine.
This number is signicantly higher in plants [16]. By se-
quence comparison of mRNA and genomic DNA, several
C to U editing events in Oenothera berteriana and wheat
have been identied (reviewed by Marchfelder et al. [17]).
In the mitochondria of Arabidopsis thaliana, 441 C to U
RNA editing events have been identied, most of these
editing sites alter the coding capacity of the RNA and
increase the hydrophobicity of the coded proteins [18]. It
has been suggested that nuclear encoded trans acting fac-
tors mediate plastid site-specic RNA editing [19]. The
editing site selection in plastid RNA has been shown to
be sequence dependent as in apoB mRNA editing and
dened by their distance from an essential upstream se-
quence element [20,21]. The mechanism of C to U substi-
tution editing appears to be very similar to that of apoB
mRNA editing [17], and an APOBEC-1-like cytidine de-
aminase enzyme (see Section 2.4) may be present in plants.
However, APOBEC-1 failed to edit some of the known
plant C to U editing sites in vitro (N. Navaratnam et
al., unpublished data). Complete genomic sequencing of
the plant mitochondrial and plastid genomes failed to
identify a homologous APOBEC-1-like protein. However,
in A. thaliana, at least eight cytidine deaminases has been
identied [16]. One of these cytidine deaminases, A. thali-
ana cytidine deaminase 1 (At-CDA-1), has been character-
ised but this protein shows no anity for RNA and it is
unlikely to be involved in RNA editing [22,23]. This led to
the proposal that a nuclear encoded protein may be ex-
ported to these organelles, but this enzyme is yet to be
identied.
BBAEXP 93457 30-10-00
The other most common substitution editing is the con-
version of adenosine (A) to inosine (I). It was initially
identied as RNA modication in the yeast tRNA [24].
By comparing the genomic and cDNA sequences for tran-
scripts encoding subunits of the glutamate responsive ion
channels (GluR), adenosine to guanosine (G) discrepan-
cies were identied [25]. The development of an in vitro
editing assay for A to I editing allowed the identication
of the regulatory elements within this pre mRNA [26,27].
It was shown that the A to G changes were due to the
enzymatic deamination of A to I, which is subsequently
read as guanosine (G) during translation. Several isoforms
of the enzyme responsible for this deamination have been
identied by several groups and named ADARs (adeno-
sine deaminases that acts on RNA) (reviewed by Reuter
and Emeson [28]). Mutagenic studies of ADAR1 identied
active site residues similar to those in APOBEC-1, thought
to be involved in zinc coordination [29]. ADAR1 also has
an arginine and glycine-rich double stranded RNA bind-
ing domain, which is also present in ACF (see Section 2.5)
and a Z-DNA binding domain [30,31]. Recently, the crys-
tal structure of this domain bound to the Z-DNA has been
elucidated [32].
Since the discovery of GluR editing several other sub-
strates have been identied based largely on the sequence
comparison between mRNAs and the genomic DNA.
These include viral transcripts, RNAs encoding neuronal
signalling molecules such as the serotonin 2C receptor,
sialyltransferase, Drosophila RNA-binding protein and
many others [33^37]. RNA substrate analogues for
ADAR2 have also been synthesised, allowing these to be
utilised in future structural, thermodynamic and kinetic
studies [38]. The ADAR family of RNA editing enzymes
has recently been extended by the identication of a sub-
family of tRNA-specic adenosine deaminases. They have
been identied from yeast (Tad1p), human (hADAT1),
mouse (mADAT1) and Drosophila melanogaster (dA-
DAT1) by sequence homology to the catalytic domain of
ADAR proteins [39^42]. These adenosine deaminases lack
the double stranded binding domains of ADAR1 and 2.
They have specicity for tRNA and convert adenosine to
inosine at position 37 adjacent to the anticodon loop of
eukaryotic tRNAAla .
Paul and Bass have developed a method to determine
inosine content in mRNA transcripts. The inosine con-
tent in mRNA from various tissues was found to correlate
with the ADAR mRNA expression [43]. It is most abun-
dant in the brain and estimated that 1 in 17 000 nucleo-
tides in brain mRNA is an inosine, suggesting there are
many more editing sites yet to be identied in nature. It
has also been calculated that one out of every eleven rat
brain mRNAs could contain inosine. If this results in
relevant codon changes then adenosine deamination
would play a major role in the regulation of gene expres-
sion [43]. Recently, inosine-containing sequences have
Cyaan Magenta Geel Zwart
 A. Chester et al. / Biochimica et Biophysica Acta 1494 (2000) 1^13
been observed in Caenorhabditis elegans mRNA and ve
new ADAR substrates have been identied [44].
2. C to U editing of apolipoprotein B (apoB) mRNA
2.1. Physiology of the apoB-containing lipoproteins
ApoB is one of nature's largest proteins and it plays a
central role in lipid metabolism. Placental mammals use
two forms of apoB to transport cholesterol and triglycer-
ide in the blood. They are termed apoB100 and apoB48.
ApoB100 (512 kDa) consists of a lipoprotein assembly
domain and a low density lipoproteins (LDL) receptor
domain. It is synthesised in the liver and transports endo-
genously synthesised cholesterol and triglycerides on very
low density lipoproteins (VLDL). VLDL are metabolised
to intermediate density lipoproteins (IDL) and subse-
quently to LDL by the actions of lipoprotein lipase and
hepatic lipase (Fig. 1A). ApoB100 is the only apolipopro-
tein present in LDL that deliver cholesterol to all tissues of
the body by the LDL receptor pathway over a period of
several days [45]. LDL transports two-thirds of the plasma
cholesterol in humans and elevated levels of LDL choles-
terol is one of the major risk factors for coronary heart
disease (CHD).
ApoB48 (241 kDa) is identical to the amino-terminal
48% of apoB100 [14,46]. In humans apoB48 is synthesised
in the small intestine and is required for the synthesis,
assembly and secretion of triglyceride-rich chylomicrons.
These particles transport dietary lipids to the tissues and
the remaining chylomicron remnant with associated resid-
ual lipid is cleared in 2^3 h by the liver via the interaction
of apolipoprotein E (apoE) with lipoprotein receptors
(Fig. 1A). ApoB48-containing lipoproteins are cleared
more quickly than apoB100-containing lipoproteins
[45,47] and are not metabolised to LDL particles and
therefore they are not a major risk factor in CHD. The
importance of apoB100 and apoB48 have been reviewed
recently [47^49].
2.2. ApoB 48 is generated by C-U editing of apoB mRNA
ApoB100 and apoB48 are both protein products of the
same 14-kb apoB gene. C to U editing of the gene in the
intestine converts a glutamine codon (CAA), at position
2153 in exon 26, to an in-frame stop codon (UAA)
[13,14,50] (Fig. 1B). This editing event of a cytidine at
position 6666 results in the production of apoB48. Cy-
tidine6666 is edited with great precision. The mechanism
of apoB mRNA editing requires the presence of the
mRNA substrate, the catalytic subunit APOBEC-1 and
certain auxiliary proteins. It does not require any addi-
tional cofactors such as nucleotides, ATP or `guide
RNAs' as are required for insertion and deletion editing
in kinetoplastid mitochondria [5,51,52].
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3
ApoB mRNA editing is a predominantly intra-nuclear
event and is completed before the mature apoB mRNA is
exported into the cytoplasm [53]. Furthermore, editing is a
post-transcriptional event, since the nascent unspliced
apoB pre mRNA is essentially unedited [53]. We and
others have shown that APOBEC-1 localises to the nu-
cleus and the cytoplasm [54] (A. Somesekaram, N. Nav-
aratnam, unpublished data). Yang et al. used indirect im-
munouorescence microscopy to study the intracellular
distribution of APOBEC-1 and its mutants in transiently
transfected cells. They demonstrated that the N-terminal
56 amino acids were necessary for the nuclear distribution
of APOBEC-1. This region contains a bipartite nuclear
localisation signal (NLS)-like sequence but does not func-
tion as a classical NLS. They also identied a 24-amino-
acid region in the C-terminus that with the characteristics
of a cytoplasmic retention signal (CRS) or a nuclear ex-
port signal (NES). This suggests that the nuclear import of
APOBEC-1 may not be mediated by a functional NLS but
by overcoming the eects of a CRS/NES. They also re-
ported that the nuclear distribution of APOBEC-1 is cell
type dependent and is unique to cells that are competent
of editing apoB mRNA [54].
2.3. ApoB mRNA substrate
An essential rst major step towards the understanding
of the mechanism of apoB mRNA editing was the devel-
opment of the in vitro conversion assay by our group [55].
This sensitive assay together with extensive site-directed
mutagenesis has allowed the mapping of the RNA se-
quence elements that are important for in vitro editing
[56^58]. The mRNA sequences spanning the editing site
are highly conserved from marsupials to man. These se-
quences are not conserved in lower genera like avians and
apoB mRNA is not edited in these species [59^64] (Fig. 2).
We showed that 26 nucleotides (6662^6687) of apoB
mRNA are essential and sucient for in vitro editing
[56] suggesting that all the essential requirements lay with-
in this 26-nucleotide region, although other elements out-
side this region may also contribute for ecient editing
[57,65]. Site-directed mutagenesis in this region identied
an 11-nucleotide sequence located 4^5 nucleotides down-
stream of the edited C, termed the `mooring' sequence,
that is essential for editing [57]. [66]. It has been further
shown that the mooring sequence can induce RNA editing
of other cytidines in apoB mRNA other than C6666 [66^
68]. This observation has been conrmed either by trans-
ferring the conserved mammalian mooring sequence into
the normally unedited chicken sequence or by mutating
the 26 nucleotides surrounding C6666 converting them to
the mammalian sequence [60,63]. A `spacer' element of
between two and eight nucleotides between the cytidine
and the `mooring' sequence is essential for editing, with
the optimal length found to be four nucleotides [58,67] (N.
Navaratnam et al., unpublished data). In studies very sim-
Cyaan Magenta Geel Zwart
4 A. Chester et al. / Biochimica et Biophysica Acta 1494 (2000) 1^13

Fig. 1. Apolipoprotein B (apoB)-containing lipoproteins. (A) Biosynthesis of apoB-containing lipoproteins. (B) Intron/exon organisation of the apoB
gene and the origins of apoB100 and apoB48 are shown.

ilar to that performed with the chicken apoB [60,63], a
comparison of the poorly edited guinea pig apoB
mRNA with a consensus sequence from 31 dierent spe-
cies that are strongly edited identied three variations in
the 3P eciency element. Converting these changes to the
mammalian consensus sequence increased editing of guin-
ea-pig apoB mRNA to a level observed in the other spe-
cies [64].
Based on these studies, the editing motif can be divided
into three components, these being `mooring' sequence,

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 A. Chester et al. / Biochimica et Biophysica Acta 1494 (2000) 1^13 5

Fig. 2. Domain organisation and sequence alignment of mammalian apoB mRNA editing site. Sequence elements and their alignment within the apoB
mRNA that inuence the editing of C6666 are shown in colour. The editing site is highly conserved between mammals and the predicted APOBEC-1
binding site is shown. Nucleotides that are not conserved are shown in bold. Adapted from Hersberger and Innerarity [65] and Richardson et al. [61].

spacer element and enhancer region [47,66]. More distal
elements anking the editing site have also been postulated
to have a role in apoB mRNA editing including 5P and 3P
eciency elements [65]. These regions are summarised in
Fig. 2 adapted from Hersberger and Innerarity [65].
Mutagenesis of the RNA substrate and the active site of
the catalytic subunit (see Section 2.4), together with UV
cross-linking and competition studies, indicate that the
catalytic component responsible for editing preferably
binds through it's active site to an AU-rich region in the
`mooring' sequence [60,69,70]. Furthermore, additional
proteins necessary for editing (discussed in detail in Sec-
tion 2.4 below) bind the RNA in this region. In a very
recent study, overexpression of APOBEC-1 was shown to
increase the stability of c-myc RNA due to the APOBEC-
1 binding to a AU-rich sequence in the 3P untranslated
region (UTR) similar that in apoB mRNA [70]. There
are similar AU-rich sequences in the 3PUTR of interleukin
2 (IL-2), granulocyte/macrophage colony stimulating fac-
tor (GM-CSF) and tumour necrosis factor K (TNF-K)
[70]. This nding suggests that APOBEC-1 may play a
role in regulating the stability of these RNAs involved in
cell growth, proliferation and tumourigenesis [71].
Most RNA processing events require secondary struc-
ture of the RNA for substrate recognition. Computer
modelling of the apoB mRNA predicts a highly conserved
stem loop secondary structure for the apoB RNA sub-
strate with the edited C6666 within the loop [56,57]. Ribo-
nuclease probing of wild-type and a series of scrambling
mutants provided biochemical evidence for the presence of
a stem loop at the editing site [61]. A similar stem loop
structure for the RNA anking the editing site was pre-
dicted recently [70]. A double stranded stem loop structure
formed by the `mooring' sequence and the 3P eciency
element has also been proposed [64]. The secondary struc-
tures identied for the apoB mRNA substrate provide
coherent structures that would allow presentation of the
edited cytidine to the catalytic subunit and binding of the
complementing factors.
2.4. ApoB mRNA editing catalytic component 1
(APOBEC-1), the catalytic subunit for the editing
complex
A functional cloning approach was used to isolate a
cDNA that encoded a 27 kDa protein from a rat small
intestine cDNA library [72]. This protein, APOBEC-1
(apoB mRNA editing catalytic component 1), is the cata-
lytic component of the apoB mRNA editing complex that
deaminates C6666 in apoB mRNA [73]. APOBEC-1 alone
is not sucient for editing, but it confers editing when
supplemented with chicken and HeLa cell extracts that
do not have intrinsic APOBEC-1 activity [59,69]. APO-
BEC-1 is highly conserved between species, from marsu-

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6 A. Chester et al. / Biochimica et Biophysica Acta 1494 (2000) 1^13

Fig. 3. Homology model for APOBEC-1 derived from the crystal structure of ECCDA. (A) APOBEC-1 `signature' sequence. Schematic representation
of the predicted domain structure of APOBEC-1 derived from the crystal structure of ECCDA, corresponding to the amino-terminal K-helical domain,
active-site core domain, linker segment, and the carboxyl-terminal domain. The conserved active site residues and the location of the three gaps and the
insert in APOBEC-1 are shown. (B) Predicted structure of APOBEC-1. ECCDA dimer three-dimensional structure viewed along the substrate binding
channel. The APOBEC-1 model was derived by the removal of the gap peptides from the ECCDA three-dimensional structure. This model allows ac-
cess of the RNA substrate to the active site of the enzyme. Adapted from Navaratnam et al. [86].

pials to man [74]. In humans and rabbits, the APOBEC-1
gene is expressed exclusively in the small intestine, whereas
in rodents it is expressed in the liver and in various tissues
that do not express apoB, including spleen, kidney, gonads
and brain [72]. The mouse APOBEC-1 gene is located on
chromosome 6 [75], and in humans, by a gene on syntenic
chromosome 12p13.1^12p13.2 [76]. The human gene con-
sists of ve coding exons and the intron positions corre-
spond exactly to those in the mouse gene [62]. In the
mouse and rat genes there are three distinct promoters
[77^79]. Small intestinal transcript generates a short
5PUTR immediately adjacent to the APOBEC-1 open
reading frame [80]. In all other tissues, two promoters
drive transcription either located 344 nucleotides or v12
kb upstream from the APOBEC-1 translation start site
[80]. The alternative promoter usage appears to be under
endocrine and metabolic control, whereas the intestinal
promoter is under developmental control and is tissue
specic [81]. APOBEC-1 contains a putative bi-partite nu-
clear localisation-like domain at the amino terminus and a
leucine-rich domain in the carboxyl terminus [82]. In ad-
dition, APOBEC-1 contains active site sequence similarity
at to cytidine deaminases that act on monomeric sub-
strates [83,84].
The active site is composed of two clusters separated by
about 30 amino acids. The rst cluster (C/HAE) contains
a single zinc ligand (cysteine or histidine) and a crucial
glutamate residue that plays a major role in proton trans-
fer during catalysis. The second cluster (PCXXC) contains
a conserved proline and two further zinc ligands which are
always cysteine residues. Using 65 Zn binding studies and
site directed mutagenesis studies to establish that the cata-

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 A. Chester et al. / Biochimica et Biophysica Acta 1494 (2000) 1^13
lytic site of APOBEC-1, the catalytic site was demon-
strated to function in a manner very similar to that of
Escherichia coli cytidine deaminase (ECCDA) [69]. We
also identied phenylalanine residues across the active
site that are not present in the E. coli enzyme. These res-
idues are involved in apoB mRNA binding [60,69].
The crystal structure of ECCDA is known [85]. It is a
homodimer in nature and consists of two 31 kDa subunits.
Each monomer in the crystal structure is composed of a
small amino-terminal K-helical domain and two larger
core domains that are connected by a 37-amino-acid linker
peptide [85]. The two co-domains have no sequence ho-
mology but contain an identical tertiary structure [85]. The
rst co-domain contains the active site domain that binds
zinc and the second co-domain contains a pseudo-active
site domain. Like ECCDA, APOBEC-1 also forms homo-
dimers in nature [75]. Sequence alignment of APOBEC-1
with ECCDA revealed several amino acids that are con-
served between the two enzymes, these included the active
site region, the leucines in the carboxyl terminus and ami-
no acids that form the dimer interface in the ECCDA
crystal structure. Conserved phenylalanine residues in the
active site domain of APOBEC-1 are absent in ECCDA
and are proposed to be involved in binding the apoB
mRNA substrate [60,69]. We also identied three gaps in
the APOBEC-1 compared to the ECCDA sequence [86].
An APOBEC-1 `signature' sequence based on its homol-
ogy with ECCDA is shown in Fig. 3A [86].
Based on these observations we proposed a model for
APOBEC-1 using the ECCDA crystal structure, assuming
that both enzymes must have similar tertiary and quater-
nary structure to have similar catalytic activity [86,87]
(Fig. 3B). However, APOBEC-1 should be reshaped to
accommodate the RNA substrate in the active site. Sig-
nicant gaps are present in the APOBEC-1 sequence com-
pared to ECCDA. The combined mass of these gaps is
10 kDa and is equivalent to that the size of the minimal
RNA substrate. The APOBEC-1 three-dimensional model
was developed by removal of these gaps, allowing us to
reshape ECCDA to create a cleft to accommodate an
RNA substrate. This model is supported by extensive mu-
tagenesis of APOBEC-1 suggested by the alignment and
modelling. These mutants were examined using biochemi-
cal assays for homodimerisation, RNA binding and RNA
editing [86].
Recently, using extensive mutagenesis, Teng et al. have
studied various sequence motifs in APOBEC-1 and their
role in the enzyme catalysis and dimerisation [88]. In
contrast to our observations, amino-terminal deletion of
14 amino acids and a carboxyl-terminal deletion of 8 ami-
no acids of APOBEC-1 do not aect editing activity.
These dierences will be resolved once the crystal structure
of APOBEC-1 and the other editing components are de-
termined. The amino acids or regions of APOBEC-1 im-
portant in the catalysis of apoB mRNA editing will be
claried.
BBAEXP 93457 30-10-00
7
2.5. Auxiliary proteins
APOBEC-1 alone is not competent for editing. Addi-
tional protein factors are required in a multi-component
editing complex. This complex has been termed an `edito-
some' [86]. These factors are present in cells and tissues
that do not themselves express apoB mRNA, APOBEC-1
or both, including rat, baboon and rabbit tissues, chicken
enterocytes and several transformed cell lines
[59,69,82,89,90]. These factors are tissue-specic, heat-sen-
sitive, substrate-saturable, and sensitive to proteinase K,
but resistant to micrococcal nuclease [51,52]. APOBEC-1
binding, RNA anity and UV cross-linking studies with
apoB mRNA have been used to identify proteins that bind
to apoB mRNA from rat, baboon and chicken [61,91^96].
These proteins range in their molecular masses from 40 to
300 kDa.
Until recently, the identity of these auxiliary factors and
their role in editing was unknown. A signicant advance
was made with the molecular cloning of APOBEC-1 com-
plementation factor (ACF) [96]. Initially, a 65 kDa protein
was puried using apoB RNA and APOBEC-1 anity
chromatography from baboon kidney extract that comple-
mented editing activity [94,97]. This protein has been se-
quenced using mass spectroscopy, cloned and named
APOBEC-1 complementation factor (ACF) [96]. ACF en-
codes a novel 63.4 kDa protein that contains three non-
identical RNA recognition motifs (RRM). ACF is widely
expressed in human tissues. ACF binds to apoB mRNA
and RNA binding is dependent on an intact `mooring'
sequence, proposing that ACF functions as an RNA bind-
ing subunit and docks APOBEC-1 to deaminate the up-
stream cytidine. ACF and APOBEC-1 comprise the mini-
mum protein requirements for apoB mRNA editing in
vitro. This suggests that the simplest model for the editing
complex is composed of an APOBEC-1 dimer and ACF.
There are three variants of ACF, ACF identied by
Mehta et al. [96], an 8-amino-acid insertion termed ASP
(APOBEC-1 stimulating protein) [98] and a 55-amino-acid
deletion (N. Navaratnam et al., unpublished data). We
predict these isoforms are generated by alternative splic-
ing, although a gene duplication event cannot be ruled out
at this moment. ACF shares signicant sequence homol-
ogy to GRY-RBP, an RNA binding protein with un-
known function that is rich in glycine, arginine and tyro-
sine residues [96] (Fig. 4A). Both proteins have three
RRM motifs and belong to the hnRNP R family of
RNA binding proteins. Phylogenetic analysis of this fam-
ily of proteins has identied a `putative' RNA binding
protein from D. melanogaster as the oldest member of
this family (Fig. 4B). Both ACF and GRY-RBP bind to
APOBEC-1, apoB mRNA and to other RNAs that do not
contain the `mooring' sequence (N. Navaratnam, unpub-
lished data). GRY-RBP was found to inhibit in vitro apoB
mRNA editing (V. Blanc et al., in preparation) but its role
in vivo has not been established. Other protein factors
Cyaan Magenta Geel Zwart
8 A. Chester et al. / Biochimica et Biophysica Acta 1494 (2000) 1^13

may play a role in apoB mRNA editing, and are discussed ocyte S100 extracts that can be UV cross-linked to apoB
below. mRNA [60,61,91,99]. We have shown that the 60 kDa
We and others have identied proteins of v 60 kDa and protein can be cross-linked to the `mooring' sequence
v 40 kDa from partially puried rat and chicken enter- and is specic for a four nucleotide region (6671^6678)

BBAEXP 93457 30-10-00 Cyaan Magenta Geel Zwart

 A. Chester et al. / Biochimica et Biophysica Acta 1494 (2000) 1^13 9

Fig. 4. Homology between GRY-RBP and ACF. (A) Sequence alignment of GRY-RBP and ACF. Identical amino acid residues are boxed and con-
served residues are shaded. The three RRM motifs are shown in colour. Sequences are human GRY-RBP (AAC12926), human ACF (AF209192), ACF
with 8-amino-acid insertion (ASP) (AJ227078) and ACF with 55-amino-acid deletion (N. Navaratnam et al., unpublished data). (B) Phylogenetic tree of
the hnRNP R family of proteins constructed using neighbourhood joining method in the PAUP phylogenetic programme [126]. The sequences used are
human GRY-RBP (AAC12926), mouse GRY-RBP (AAC62511), mouse SYNCRIP (BAA88342), human hnRNP R (AAC39540), human ACF
(AF209192) and human (BAA91086), human (BAA91049), C. familiaris (CAB46854), C. elegans (CAB70238) and D. melanogaster (AAF55805) proteins
identied by homology searches with either GRY-RBP or ACF.

[57]. This protein may be one of the variants of ACF that
have been identied. Partial characterisation of auxiliary
factors from McArdle cells by APOBEC-1 anity chro-
matography identied 100 and 55 kDa proteins that cross-
link to apoB mRNA [100]. Lau et al. utilised the yeast
two-hybrid system to identify proteins that interact with
APOBEC-1 and identied APOBEC-1-binding protein 1
(ABBP-1) [93]. ABBP-1 is identical to a previously re-
ported human A/B hnRNP except for a 47-residue inser-
tion at its C-terminal region. HnRNP C1 and hnRNP F
proteins were also found to interact with APOBEC-1 [95]
(N. Navaratnam et al., unpublished data). Recombinant
hnRNP C1 was found to inhibit apoB mRNA editing and
proposed to have a regulatory role in this process [95].
Therefore, some hnRNPs do appear to interact with APO-
BEC-1 and apoB mRNA, but their role in editing is un-
clear.
Smith and colleagues have demonstrated that a 27S ed-
iting complex assembles on the apoB RNA surrounding
the edited site [101]. Monoclonal antibodies generated
against this complex were used to identify a protein of
240 kDa termed AUX240 [92] Antibody depletion of the
protein from rat hepatoma cell line extracts resulted in
impaired editing that could be restored by the supplemen-
tation with AUX240. Several proteins ranging in molecu-
lar mass from 150^45 kDa co-immunopurify with
AUX240. AUX240 does not bind to APOBEC-1 or
apoB mRNA but it was proposed this protein is required
for assembly of the editosome and ecient RNA editing.
The same group has also identied editing of apoB
mRNA in the yeast strain CL51 by co-expressing the
mammalian APOBEC-1 and apoB mRNA. This suggests
that the auxiliary proteins necessary for editing complex
formation are also expressed in yeast [102].
The true identity of the complete in vivo apoB mRNA
editing complex is yet to be elucidated but the identica-
tion of ACF has brought about a great advance in this
area and it has been clearly shown that APOBEC-1 and
ACF form the minimal in vitro apoB mRNA editing com-
plex [96]. ApoB mRNA editing is also regulated by devel-
opmental, hormonal and dietary factors (reviewed by
Chan et al. [48]) and this adds another dimension to the
problem.
2.6. C to U RNA editing in gene therapy and cancer
In humans, apoB mRNA editing occurs exclusively in
the intestine, whereas in rat and mice, editing also occurs
in the liver [103]. Interestingly, rats are less susceptible to
coronary heart disease, suggesting that editing in the liver
may protect them against atherosclerosis. Many groups
have made attempts to overexpress APOBEC-1 mRNA
in liver to determine the feasibility of using APOBEC-1
as a means of gene therapy in the ght against atheroscle-
rosis and to reduce plasma LDL cholesterol levels.
Adenovirus-mediated gene transfer was rst used to in-

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10 A. Chester et al. / Biochimica et Biophysica Acta 1494 (2000) 1^13
duce transient hepatic overexpression of APOBEC-1 in
mice [104]. The results showed that hepatic transduction
of the rat APOBEC-1 gene led to marked overexpression
of APOBEC-1 mRNA and protein. APOBEC-1 expres-
sion in the liver was found to virtually abolish both
apoB100 and LDL production. Similar results were seen
with the transduction of the human APOBEC-1 gene in to
mice and rabbits [105,106]. The APOBEC-1 transfer has
been repeated in the LDL receptor knockout mouse [107]
which is an animal model for familial hypercholesterolae-
mia (FH), an autosomal dominant disorder in humans
characterised by LDL receptor deciency, marked hyper-
cholesterolaemia and premature coronary heart disease
[108]. Hepatic overexpression in these LDL receptor de-
cient mice resulted in marked lowering of plasma LDL
levels and reductions in apoB100-containing lipoproteins,
VLDL and LDL [107]. The gene transduction led to in-
creased apoB mRNA editing activity in the liver. In LDL
receptor decient rabbits, the reduction in plasma
apoB100 was accompanied by a transient reduction in
VLDL and LDL [109]. Hence, APOBEC-1 seemed to be
a good candidate for gene therapy for the treatment of
elevated VLDL and LDL that is associated with LDL
receptor deciency and hyperlipoproteinaemia.
Yamanaka and colleagues overexpressed APOBEC-1 in
mice and rabbits, using the strong hepatic apoE promoter.
They found the transgenic animals had liver dysplasia and
many developed hepatocellular carcinomas [110]. As an-
ticipated, the plasma LDL levels in these animals were
lowered, however the tumourigenesis associated with the
hepatic overexpression of APOBEC-1 compromised this
method as a means of gene therapy to lower LDL levels
and hence prevent atherosclerosis. This observation was
probably caused by the aberrant editing of growth-related
or dierentiation-related genes. A novel translational re-
pressor mRNA was recently found to be extensively edited
in the tumourigenic livers [111]. This mRNA has been
called novel APOBEC-1 target 1 (NAT1). The NAT1
mRNA is ubiquitously expressed and is highly conserved
between species. NAT1 shares sequence homology to the
C-terminal portion of the eukaryotic translation initiation
factor eIF4G which inhibits cap-dependent and cap-inde-
pendent translation in vitro. Editing of this mRNA was
suggested to interfere with its repressor function and was
proposed to contribute to the tumourigenicity caused by
the overexpression of APOBEC-1 [111]. In a similar study
in rat hepatic cell lines, overexpression of APOBEC-1 was
found to increase the eciency of apoB mRNA editing of
C6666 . `Promiscuous' editing of additional cytidines within
the apoB mRNA was also observed [112]. This observa-
tion dampened the enthusiasm for the potential use of
APOBEC-1 as a gene therapy target. The recent observa-
tion that overexpression of APOBEC-1 leads to stabilisa-
tion of c-myc mRNA further lend support to the idea of
APOBEC-1 being considered a proto-oncogene [70].
It is interesting to note that low to moderate levels of
BBAEXP 93457 30-10-00
APOBEC-1 expression (up to about ten times its endoge-
nous level in mice) in the liver was not associated with any
liver dysplasia or tumourigenesis [110]. Expression at these
levels still caused a signicant reduction in plasma IDL
and LDL in transgenic rabbits. Therefore, there is still
scope to use APOBEC-1 as a therapeutic gene for the
treatment of hypercholesterolaemia by utilising low-level
regulatable expression of the APOBEC-1 gene in the liver
[113]. Recent work by Wang et al. [114] investigated the
possibility of reducing apoB100 production using a target-
specic hammerhead ribozyme to cleave the apoB mRNA.
Ribozymes targeted at apoB mRNA sequences anking
the edited base, C6666 , were designed. HepG2 cells were
infected with adenovirus expressing these hammerhead ri-
bozymes and the apoB mRNA was cleaved at the expected
target site, resulting in reduced apoB mRNA levels and
truncated apoB of the expected size. Therefore, ribozyme
cleavage could be also be utilised as a therapeutic ap-
proach to lowering plasma levels of apoB100 and LDL
[114].
A sequence similar to the apoB mRNA mooring se-
quence has been identied in human neurobromatosis
type 1 (NF1) mRNA, and it has been observed that C
to U deamination of the appropriately spaced cytidine
does occur [115,116]. Editing of the NF1 mRNA modies
a cytidine at nucleotide 2914, converting an arginine co-
don (CGA) to an in-frame stop codon (UGA). The NF1
gene codes for a tumour suppressor that acts through a
GTPase-activating domain to suppress mitogenic signal-
ling. Editing of NF1 is predicted to cause a truncation
of the protein such that the GTPase-activating domain is
lost and presumably abolishes the ability of the NF1 pro-
tein to act as a tumour suppressor. Recent results have
shown that malignant tumours edit NF1 more eciently
than benign tumours suggesting that the editing of NF1
mRNA plays a role in the tumourigenesis of NF1 [117].
Unlike apoB mRNA editing, NF1 editing is not dependent
on rate-limiting quantities of APOBEC-1, suggesting that
dierent trans-acting factors, including the catalytic com-
ponent, may be involved in these two processes [115].
More recently, APOBEC-1 has been shown to edit NF1
mRNA in the in vitro conversion assay ([116]; A. Chester,
N. Navaratnam, unpublished data). The alternatively
spliced form containing exon 23A was preferentially edited
in the tumours [116]. Moreover, these tumours demon-
strated the presence of APOBEC-1, the rst case of extra-
intestinal expression of APOBEC-1 in humans [116]. In an
earlier study, Greeve et al. failed to identify hyperediting
of apoB, NAT1 or NF1 mRNA in any of the 28 resected
tumour specimens studied. However, low level expression
of APOBEC-1 was detected in colorectal and gastric car-
cinomas [118].
The APOBEC-1 mRNA undergoes alternative splicing,
APOBEC-T arises when exon 2 is spliced [119,120]. This
splicing event creates a frameshift that introduces a pre-
mature termination codon into the reading frame. The
Cyaan Magenta Geel Zwart
 A. Chester et al. / Biochimica et Biophysica Acta 1494 (2000) 1^13
Fig. 5. Phylogenetic analysis of recently identied APOBEC-1-related
proteins. Active site sequences from APOBEC-1-related proteins (34
amino acids) were aligned with CLUSTAL W (DNA STAR). The phy-
logenetic tree was generated using the neighbourhood joining method in
the PAUP phylogenetic programme [126]. Sequences are human APO-
BEC-1 (L26234), human APOBEC-2 (AAD45360), mouse AID
(AAD41793), E. coli cytidine deaminase (P13652). Other sequences are
located on bacmids 150C, 494G and 742C on chromosome 22 [125].
resulting 36-amino-acid protein shares only its rst six
amino acids with APOBEC-1 and does not have any of
the functional motifs necessary for RNA binding or RNA
editing. APOBEC-T is expressed in normal, adenomatous
and cancerous gastrointestinal tissues and levels of the
mRNA encoding this peptide are signicantly increased
in colon cancer. This spliced form is also the dominant
form detected during foetal intestinal and colonic develop-
ment, suggesting that APOBEC-T may play a role during
gastrointestinal cellular growth. Expression of APOBEC-T
appears to have a signicant decrease in cell growth and it
may represent a novel class of inhibitory peptides whose
expression is limited to the human intestinal tract [119].
2.7. Other APOBEC-1-like proteins in nature
These studies suggest that RNA editing events utilising
APOBEC-1-like proteins may be involved in tumourigen-
esis. This led to the search for other APOBEC-1-like pro-
teins. So far, three proteins have been identied in mam-
mals that are related to APOBEC-1, Phorbolin-1,
APOBEC-2 and activation-induced deaminase (AID)
[121^123] (N. Navaratnam, unpublished data). Phorbo-
lin-1 is highly expressed in psoratic lesions and is encoded
by a gene on chromosome 22q13. Phorbolin-1 shares se-
quence identity and similarity to APOBEC-1 however it
does not exhibit cytidine deaminase activity and it does
not show editing of apoB mRNA or binding of an AU-
rich RNA template [121]. APOBEC-2 is a member of the
BBAEXP 93457 30-10-00
11
cytidine deaminase superfamily expressed in human heart
and skeletal muscle and the gene maps to chromosome 6
[122] (N. Navaratnam et al., unpublished data). APOBEC-
2 does not bind or edit apoB mRNA [122]. Since the name
APOBEC-2 is a misnomer, we suggest that these family of
proteins should be called APOBEC-1-related proteins or
ARP until their function is known. In such a case, APO-
BEC-2 should be renamed ARP-1 [124]. AID expression is
induced in murine B lymphocytes on stimulation with IL-
4, TGF-L and CD40L. AID is homologous to APOBEC-1
and has cytidine deaminase activity but like ARP-1, it
does not demonstrate C to U editing of apoB mRNA.
AID is a new member of the cytidine deaminase super-
family and may play a role in the regulatory steps in class
switch recombination unique to the germinal centre [123].
The gene structure of APOBEC-1 is not conserved be-
tween ARP-1 and AID (N. Navaratnam et al., unpub-
lished data) [123]. Using the APOBEC-1 active site `signa-
ture' [86] (Fig. 3A), similarity searches of various
databases have identied a family of APOBEC-1-like se-
quences on chromosome 22 [125]. A phylogenetic tree
based on the active site sequence homology of APO-
BEC-1, ECCDA and recently identied proteins (Phorbo-
lin-1, AID, ARP-1 and those on chromosome 22) is shown
in Fig. 5. These novel proteins contain the conserved mo-
tifs required for RNA binding and editing as in APOBEC-
1, and may be involved in novel RNA editing/processing
events yet to be identied.
2.8. Concluding remarks and future prospects
In this article, we have reviewed the current opinions of
apoB mRNA editing. Although much is known about the
mRNA substrate and the catalytic subunit of the editing
complex, APOBEC-1, very little is known about the iden-
tity of the complementing factors. Many candidate pro-
teins were isolated and characterised but their role in apoB
mRNA editing was uncertain. The recent cloning and
identication of the auxiliary factor, ACF, has provided
a welcome boost to this area. This may not be the only
additional factor required for editing activity but it opens
up the area for future studies. Characterisation of the
apoB mRNA editing can now be undertaken using the
minimal holoenzyme comprising of APOBEC-1 and
ACF, leading to a greater understanding of the mecha-
nisms involved. The expression of ACF in response to
nutritional and hormonal stimuli can be studied and this
may account for the diering levels of apoB mRNA edit-
ing observed under these conditions.
The identication of a family of new APOBEC-1-related
proteins has strengthened the concept that other mamma-
lian C to U mRNA editing enzymes and mRNA targets
may exist in Nature. They may play a role in RNA stabil-
ity and/or possibly in tumourigenesis. Finding a function
for these proteins will be the new challenge in the eld of
RNA editing.
Cyaan Magenta Geel Zwart
12 A. Chester et al. / Biochimica et Biophysica Acta 1494 (2000) 1^13
Acknowledgements
We apologise to those whose relevant publications could
not be cited due to space limitations. A.C. and N.N. were
supported by the Medical Research Council, and we ac-
knowledge the other members of the MRC Molecular
Medicine Editing Group.
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