REVIEWS

THE MANY ROLES OF

AN RNA EDITOR
Liam P. Keegan, Angela Gallo and Mary A. OConnell
The availability of complete genome sequences has made it clear that gene number is not the
sole determinant of the complexity of the proteome. Additional complexity that is not readily
detected by genome analysis is present in the number and types of RNA transcript that can be
derived from each locus. Although alternative splicing is a well-recognized method of
generating diversity, the more subtle mechanism of RNA editing is less familiar.

CAPPING
The process by which
eukaryotic mRNA is modified
by the addition of an
m7G(5)ppp(5)N structure
at the 5 terminus. Capping is
essential for several important
steps of gene expression: for
example, mRNA stabilization,
splicing, mRNA export from
the nucleus and initiation of
translation.
KINETOPLAST
An intracellular DNA-
containing structure that is a
modified mitochondrion with
an associated centriole-like
organelle (kinetosome), which
is characteristic of the class that
includes trypanosomes.
PLASMA
A blood component that is
composed of 90% water and
transports blood cells, nutrients,
waste products, salts, antibodies,
clotting proteins, hormones and
proteins that help maintain the
fluid balance of the body.
MRC Human Genetics
Unit, Western General
Hospital, Crewe Road,
Edinburgh EH4 2XU, UK.
Correspondence to
M.A.OC. e-mail:
mary.oconnell@hgu.
mrc.ac.uk
RNA editing has been defined as a modification of RNA
that changes its coding capacity and is distinct from
RNA splicing, CAPPING or 3 processing (FIG. 1), but recent
research has challenged this narrow definition. The term
RNA editing was first coined to refer to the insertion
and deletion of uridine nucleotides in mRNAs in the
mitochondria of KINETOPLASTID protozoa1, in which RNA
editing of these transcripts is required to generate func-
tional proteins. RNA editing is of two types: it either
involves the insertion/deletion of nucleotides or their
modification. The latter is the most widespread type of
RNA editing, and is found both in plant organelles and
in the nucleus of higher eukaryotes.
This review concentrates on the best-characterized
types of RNA editing that are found in mammals, the
conversion of cytosine (C) to uracil (U) and the con-
version of adenosine (A) to inosine (I). An overview of
their mechanisms of action is provided as well as a dis-
cussion of the range of transcripts that are edited and
how this modification affects their function. We also
review the broadening role of RNA editing and the
proposed links to human disease. We would like to
raise, for the attention of readers, that official gene
nomenclature has been used throughout this review.
Synonyms that are used by the RNA community are
provided in parentheses where appropriate.
Mammalian C U RNA editing
The discovery that apolipoprotein B (APOB, also
known as apoB) mRNA is edited in mammals showed
that RNA editing could determine which protein
product was synthesized from a particular genetic
locus2,3. APOB is a component of the plasma lipopro-
teins and is crucial for the transport of cholesterol and
of triglycerides in the PLASMA4. There are two forms of
APOB: APOB100 and the shorter APOB48 isoform,
which results from the DEAMINATION of C U at
nucleotide position 6666 (C6666) in the APOB mRNA,
which causes the change of a glutamine to a transla-
tional stop codon5. Because, in humans, this editing
event occurs in the small intestine but not in the liver6,
the APOB100 isoform is synthesized only in the liver
and is used to assemble the very-low-density lipopro-
tein (VLDL) that is necessary for the transport of
endogenously synthesized TRIGLYCERIDES and cholesterol.
VLDL is metabolized to intermediate-density lipopro-
tein (IDL) and subsequently to LOW-DENSITY LIPOPROTEIN
(LDL). Owing to the interaction of the carboxyl termi-
nus of APOB100 with the LDL receptor, LDL is
removed from the circulation. Such a functional inter-
action is medically important, as high levels of LDL
cholesterol is one of the main risk factors for coronary
heart disease. Conversely, APOB48, which lacks the
carboxyl terminus of APOB100, is generated in the
small intestine and is necessary for the synthesis and
secretion of CHYLOMICRONS7.
Mechanism of C U editing. The human APOB100
locus spans 43 kb, has 29 exons and encodes one of
the largest known proteins (4,536 amino acids)7. The
editing site lies in exon 26, which, at 7,572 nucleotides,
is one of the largest known exons. Although the
mRNA is 14 kb, editing occurs with exact precision at
C6666 (REFS 2,3) and requires both trans-acting factors

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Nucleus Cytoplasm
RNA processing

(A)n 3'

Capping Editing
O CH3 Cytidine Uridine
N+ NH2 O
HN
N H2O NH3 HN
H2N N N O O O X
CH2 O P O P O P O CH2 O O N O N
O
O O O Ribose Ribose
OH OH O OH Adenosine Inosine
NH2 O
N H2O NH3 N
N NH
Splicing
N N N N
OH
Ribose Ribose

G pGU A AGp
3' end processing
Step 1
5'UTR Coding region 3'UTR
U 5'
G
p X AU CPE
AAUAAA
ACE
G OH A AGp
Subcellular
localization

Step 2 Polyadenylation
Decay
status
p + A AG OH
Translation

Figure 1 | The processing of RNA in a cell. Immediately after the RNA is transcribed in the nucleus, capping, splicing, editing and 3 polyadenylation of the
pre-mRNA occur. In mammals, RNA editing can be of two types, either the conversion of cytidine to uridine or the conversion of adenosine to inosine. Once the
mRNA is transported into the cytoplasm, additional processing of the poly(A)+ tail can occur. The elements required for this and for subcellular localization, stability
and translation are present in the 3 untranslated region (UTR). ACE, adenylation control element; CPE, cytoplasmic poly(A) element.

DEAMINATION
The removal of an amino
group from a nucleoside,
thereby generating another
nucleoside with different
base-pairing properties.
TRIGLYCERIDE
Fat compound in the body and
in most food that is composed
of a glycerol molecule with
three fatty-acid chains attached
through their hydroxyl groups.
LOW-DENSITY LIPOPROTEIN
Insoluble lipid that is
transported in the blood and is
associated with cholesterol,
phospholipids and protein to
form a lipoprotein complex.
Low-density lipoprotein (so
defined by the type and ratio
of protein and fats that it
contains) is the main cholesterol
carrier in the blood.
and cis-acting sequence elements that surround the
cytosine that is edited (FIG. 2a). Site-directed mutagen-
esis has identified a mooring sequence of 11
nucleotides that is situated downstream of the editing
site810 and is separated from the target cytosine by a
spacer element that is usually four nucleotides long.
The site is flanked by 3 and 5 efficiency elements
(sequences that have been identified that are required
for efficient editing, the 5 one being the most impor-
tant), and there is an additional requirement for
A+U-rich bulk RNA11,12. The mooring sequence and
the 3 efficiency element form a double-stranded (ds)
stem that is predicted to position the edited cytosine
in a favourable configuration for deamination13.
The cytidine deaminase, APOBEC1 (APOB
mRNA-editing enzyme catalytic polypeptide 1, also
known as APOBEC-1)14, which catalyses the deamina-
tion reaction, and a 65-kDa auxiliary factor ACF
(APOBEC1 complementation factor; also known as
ASF, APOBEC1 stimulating factor) form a complex
that is the minimum requirement for editing of APOB
in vitro15,16. Recombinant APOBEC1 protein binds to
APOB mRNA and can also deaminate a monomeric
cytidine substrate in vitro1719, but it cannot deaminate
the APOB mRNA in the absence of ACF. APOBEC1
belongs to a family of cytidine deaminases (CDAs)20,21
and contains the protein motif characteristic of a
cytidine deaminase22 (FIG. 3). The carboxyl terminus is
leucine-rich and is probably involved in dimerization.
According to one model, the APOBEC1 dimer identi-
fies the cytosine that is to be edited by recognizing and
binding two specific bases in the APOB mRNA the
targeted cytosine at the active site and a uracil that is
positioned at the pseudo-active site23. Although
APOBEC1 occurs as a dimer in vitro, it remains to be
confirmed if it is a dimer in an active editing complex
with ACF (FIG. 2a).
Recently, two groups have independently purified
and subsequently cloned the elusive human
APOBEC1 auxiliary factor ACF15,16 (FIG. 3). The ACF
protein contains three non-identical single-stranded
(ss) RNA-recognition motif at the amino terminus
and a putative dsRNA-binding domain at the car-
boxyl terminus. In addition, there are six
arginineglycine dipeptides of unknown function
between amino acids 314 and 402 (REF. 15). The bind-
ing of ACF to APOB mRNA is dependent on an
intact mooring sequence, indicating that the latter
might contribute additional RNA binding to the edit-
ing complex and assist in docking APOBEC1 to
deaminate the target cytosine. The putative dsRNA-
binding domain of ACF might bind the stem that is
formed between the mooring sequence and the 3
efficiency element13.

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a b pre-mRNA K (KH RNA-binding domain) homology-type splicing

APOB mRNA
Mooring
sequence
ADAR
A
A APOBEC1 transgenic animals
3' efficiency ECS
ACF U element
C mouse strains have been generated. Surprisingly,
APOBEC1
5' efficiency
5' element 3' 5'
Exon Intron
Figure 2 | Recognition of the edited nucleotide by the editing enzymes.
a | APOBEC1 binds to APOB mRNA in the presence of ACF and catalyses the C U
deamination of C6666 that is positioned at the active site. According to one model, uracil is
positioned at the pseudoactive site. It is not certain whether APOBEC1 binds as a dimer
or monomer when it is complexed with ACF. The cis-acting sequence elements are a
MOORING SEQUENCE, and 5 and 3 efficiency elements. This is one of the many models of
the configuration of the APOB-binding site. b | ADARs recognize duplex RNA that is
formed between the editing site and the ECS that is often located in a downstream intron.
The enzymes bind to the double-stranded (ds)RNA through their dsRBDs and deaminate
a specific adenosine to inosine. ACF, APOBEC1 complementation factor; ADAR,
adenosine deaminases that act on RNA; APOB, apolipoprotein B; APOBEC1, APOB
mRNA-editing enzyme catalytic polypeptide 1; C, cytosine; ECS, editing site
complementary sequence.
The tissue specificity of APOB mRNA editing is
determined by APOBEC1 expression. In humans,
APOBEC1 is expressed in the small intestine and it is
there that the editing occurs. In some species, such as rat
and mouse, a second promoter in the Apobec1 gene
allows expression in the liver and so ApoB mRNA edit-
ing also takes place in this tissue24,25. This broader
expression of ApoB48 in rat and mouse explains why
rodents have very low LDL levels6. Interestingly, ACF is
expressed in human tissues that lack both APOBEC1
CHYLOMICRON and APOB mRNA15,16. Moreover, it is expressed in
Large lipoprotein complex chicken, which does not edit APOB mRNA, raising the
formed in the intestine that possibility that ACF interacts with catalytic subunits
transports fats from the
intestine to the liver and to
other than APOBEC1 and edits additional transcripts.
adipose tissue. Engineering mice that are null for Acf should answer
this question.
MOORING SEQUENCE Additional factors have been implicated in modulat-
An 11-nucleotide motif, which
is located 3 of the cytidine that
ing APOB mRNA editing. Recently, an ACF-related pro-
is edited in APOB mRNA. Both tein a Gly-Arg-Tyr-rich RNA-binding protein (GRY-
APOBEC1 and ACF are RBP) has been identified that is 50% homologous to
thought to bind to this motif. ACF and co-localizes with it in transfected mammalian
cells26. Because GRY-RBP can bind to APOB mRNA, to
IMMUNOGLOBULIN CLASS
SWITCHING
APOBEC1 and to ACF, it has been proposed to inhibit
A DNA recombination event RNA editing by sequestering ACF and/or by binding to
that only occurs at the APOBEC1 and to APOB mRNA. In keeping with this
immunoglobulin (Ig) heavy model, rat hepatoma cells treated with antisense
chain locus in B cells and is the
process by which IgG, IgA and
oligonucleotide to GRY-RBP had increased C U edit-
IgE antibodies can be produced ing in the endogenous ApoB mRNA. Another factor that
instead of IgM. might be involved in APOB mRNA editing is KSRP, the
regulatory protein16. When Lellek et al.16 purified
human ACF they found that it associated with KSRP
and that it could be ultraviolet-crosslinked to APOB
mRNA. However, KSRP is not an essential component
of the editing complex and its role in APOB mRNA
editing remains unclear.
To determine the phenotypic effects of editing ApoB
mRNA, Apobec1-deficient and Apobec1-overexpressing
Apobec1/ mice were healthy and fertile despite the
absence of ApoB48, and had very little alteration in
lipoprotein concentration2729. Unfortunately, mice are
probably not a very good model system to address the
3' effects of editing ApoB RNA because ApoB100 levels are
naturally low in mice; even Apobec1/ mice, which, in
the absence of editing by Apobec1 should have elevated
levels of ApoB100, still have only 510% of the levels
found in humans29.
Transgenic mice and rabbits that overexpressed rabbit
Apobec1 in their livers were generated, to determine if a
reduction of ApoB100 synthesis would lower the LDL
concentration, in the hope of using this strategy in a gene-
therapy approach to lower LDL concentration. Although
the LDL concentration was reduced, the transgenic mice
showed dysplasia and many developed hepatocellular
carcinomas30,31, probably as a result of aberrant editing of
mRNAs that are not normally edited. One such transcript
is Nat1 (novel Apobec1 target 1). The Nat1 protein is
homologous to the carboxyl terminus of the eukaryotic
translation initiation factor eIF4G and inhibits both cap-
dependent and cap-independent translation in vitro. It
has been proposed that Nat1 is a repressor of translation
and that its role in tumorigenicity arises from the aber-
rant editing of its mRNA, leading to a carboxy-terminal
truncation that could interfere with its repressor function.
The aberrant editing of Nat1 mRNA could therefore
allow the expression of potentially tumorigenic genes.
Additional C U RNA editing?
To identify other transcripts that are edited by APOBEC1,
Skuse and colleagues searched for homologous sequences
to the APOB mooring sequence and, as a result, found
that neurofibromatosis type 1 tumour suppressor (NF1)
mRNA was edited32. At present, it is not known whether
APOBEC1 is involved in editing NF1 mRNA. Originally,
it was clearly shown that editing of NF1 mRNA is not
dependent on rate-limiting quantities of APOBEC1.
However, more recently, APOBEC1 has been shown to
edit NF1 mRNA in vitro5. It will also be interesting to see
if ACF is involved in editing NF1 transcripts.
Three other human proteins have been identified
that are homologous to APOBEC1: APOBEC2 (REF.
33), phorbolin1 (REF. 34) and activation-induced cyti-
dine deaminase (AID)35. AID was identified in a
screen for factors involved in recombination that leads
to IMMUNOGLOBULIN CLASS SWITCHING. It shares 34%
amino-acid identity with APOBEC1, contains all the
amino-acid hallmarks of a cytidine deaminase and

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SOMATIC HYPERMUTATION
The process by which point
mutations are introduced into
the V(D)J exon of
immunoglobulin genes in
B cells to create diversity.
PSORIASIS
A chronic skin disease that is
characterized by scaling and
inflammation. The exact cause
is unknown but it is probably a
disorder of the immune system.
HYDROLYTIC DEAMINATION
The process in which an amino
group is removed from an
adenosine residue and is
replaced with the oxygen from
water to produce inosine and
ammonia.
METALLOENZYME
A group of proteins that
contain metal-binding sites.
DNA METHYLTRANSFERASE
Enzyme that catalyses the
addition of a methyl group
to adenine or to cytosine.
872 | NOVEMBER 2001 | VOLUME 2
AUG 296
Z Z dsRBD NLS DM
ADAR1
RG-enriched
domain
ADAR2
ADAR3
R-enriched
domain APOBEC1
L-enriched
domain
ACF
RRM
Figure 3 | Schematic diagram of human ADAR,
APOBEC1 and ACF. The different human editing enzymes
ADAR, APOBEC1 and ACF contain similar domains.
The arginineglycine (R/G)-enriched domains present in
ADAR1 and ACF are coloured blue. The Z and Z domains
of ADAR1 are grey. The double-stranded (ds) RBDs are purple
whereas the RRMs in ACF are pink. The NLS are light green.
The deaminase (DM) domains are orange and the
pseudoactive site in APOBEC1 is yellow. The R-enriched
domain in ADAR3 is burgundy and the leucine domain in
APOBEC1 is navy blue. A methionine, from which translation
is initiated, is shown (AUG 296). ACF, APOBEC1
complementation factor; ADAR, adenosine deaminases that
act on RNA; APOBEC1, APOB mRNA-editing enzyme
catalytic polypeptide 1; NLS, nuclear localization signal;
RRM, RNA-recognition motif; RBD, RNA-binding domain.
has cytidine deaminase activity, but is unable to edit
APOB mRNA in vitro. It is located beside APOBEC1
on human chromosome 12p13, which indicates that
one of these genes might have arisen through duplica-
tion36. Null mutations in AID ablate both
immunoglobulin class-switching and SOMATIC HYPERMU-
37,38
TATION . Therefore, it is possible that AID is an RNA-
editing enzyme that edits one or more mRNAs that
encode key components of B-cell differentiation39. An
alternative hypothesis is that the substrate for AID is
DNA and not RNA. According to a model proposed
by Jacobs and Bross, AID deaminates cytosine in
DNA40; in turn, this induces base-excision repair that
often causes a single-stranded break in the DNA. If
two deamination events occurred in close proximity
on opposite strands, a double-stranded break (DSB)
would occur that could be a substrate for the error-
prone staggered DSB-repair pathway. The culmina-
tion of these events could be the introduction of
mutations. Like APOBEC1, AID probably requires a
cofactor, such as ACF, to assist it to bind to its sub-
strate. If the cofactor is homologous to ACF and con-
tains RNA-binding domains, AID is more likely to be
an RNA-editing enzyme. Conversely, finding a cofac-
tor with DNA-binding domains would strengthen the
argument that the main target of AID is DNA.
APOBEC2 which encodes a cytidine deaminase
that is homologous to APOBEC1 is expressed exclu-
sively in the heart and skeletal muscle33. Although it
2001 Macmillan Magazines Ltd
shows very low cytidine deaminase activity on
monomeric substrate in vitro, it cannot edit APOB
mRNAs, as it is probably unable to bind to the transcript.
Phorbolin1 was cloned in a screen for proteins that are
involved in PSORIASIS and it shares a 20% amino-acid
identity with APOBEC1 (REF. 34). The protein is unable to
edit or bind to APOB mRNA and its cytidine deaminase
activity has not been shown. A family of APOBEC1-like
sequences, of unknown function, have also been found
on chromosome 22 (REF. 41).
A I RNA editing
The conversion of A I, which is read by the translation
machinery as if it were guanosine, is the most widespread
type of RNA editing in higher eukaryotes42. The phenom-
enon was discovered independently by two groups, who
initially described it as a dsRNA unwinding or helicase
activity43,44. This unwinding activity is a consequence of
converting AU base pairs in the RNA duplex to an IU
mismatch, which destabilizes the duplex. The enzymes
that deaminate adenosine to inosine in dsRNA are mem-
bers of a family of adenosine deaminases that act on RNA
(ADAR)45. In humans, there are three members of this
family: ADAR1, ADAR2 and ADAR3, the names reflect-
ing the order in which they were identified46 (FIG. 3)
ADAR1 and ADAR2, which are almost ubiquitously
expressed47, can convert adenosine to inosine, both in
long dsRNA duplexes and in specific pre-mRNA tran-
scripts. Several isoforms of these two enzymes exist that
vary in their editing activity4850. ADAR3 is expressed
exclusively in the brain but it cannot deaminate adeno-
sine in dsRNA nor can it edit any known pre-mRNAs51,52.
Mechanism of ADAR editing. Unlike APOBEC1, ADAR
proteins do not require cofactors to deaminate adeno-
sine to inosine through HYDROLYTIC DEAMINATION in vitro53.
The ADARs probably evolved from the ADATs (adeno-
sine deaminases that act on tRNA)5456 and all are
members of the cytidine deaminase family. The
enzymes contain either two or three dsRNA-binding
domains57, as well as a catalytic deaminase domain that
is similar to that of APOBEC1 (REFS 58,59; FIG. 3). Support
for the hypothesis that the ADARs are METALLOENZYMES
comes from site-directed mutagenesis of the residues
thought to bind zinc at the active site, which results in a
loss of the deaminase activity60. Because ADARs have
homology to DNA METHYLTRANSFERASES it was proposed
that ADARs flip out the RNA base before deamina-
tion, similar to the base flipping mechanism of the
DNA methyltransferases61,62. Recently, Peter Beal and
colleagues63 have obtained experimental data that
support the flip-out model.
The ADARs are unique in that they recognize the
adenosine to be edited not by a surrounding consensus
sequence but by the structure of the duplex that is
formed between the editing site and an editing site
complementary sequence (ECS) that is usually located
in a downstream intron64 (FIG. 2b). The dsRNA-binding
domains (dsRBDs) found in ADARs mediate the bind-
ing to the duplex. The dsRBDs were thought to lack
sequence specificity as they interact with the RNA
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Table 1 | The known transcripts that undergo RNA editing and the functional consequence on the encoded protein
Organism Transcript Effects of editing Functional consequence References
Cytosine to uracil
Mammals ApoB Q STOP Stop codon, generation of ApoB48 isoform 2,3
Nf1 R STOP (truncated protein) 32
Adenine to inosine
Mammals GluR-B QR Decreased Ca2+ ion permeability 77,80,88
GluR-B RG Increased rate of recovery receptor desensitization 77,80,90
GluR-C RG Unknown
GluR-D RG Unknown
GluR-5 QR Unknown 77,80
GluR-6 QR Increased Ca2+ ion permeability 77,80
GluR-6 I V Modulated Ca2+ ion permeability 77,80
GluR-6 YC Unknown 77,80
5-HT2CR I V, N S, I V Reduced G-protein coupling 77,81
5-HT2CR IM Unknown
5-HT2CR ND Unknown
5-HT2CR NG Unknown
Adar2 Intronic nt changed, Generation of isoform 46,86
new splice acceptor site
Drosophila Ca2+ channel (cac) S G, I M, N S Unknown 82,83
melanogaster 1-subunit N S, S G, M V
N S, N G, N D,
RG
Na2+ channel ( para) 3 Q R, Y C, M V Unknown 84
1-subunit N D, K R, N S
K R plus two silent
changes
GluCl-1 I V, K R, N S Unknown 85
plus two silent changes
Adar SG Unknown 78
Loligo peali K+ channel Y C, I V Slow inactivation and closure of the channel 79
(squid) 10 other aa changes Unknown
Caenorhabditis 3 UTR, 5 UTR nt changes Unknown 99
elegans of several nt changes Unknown
transcripts non-coding RNA
Hepatitis HDV antigenomic STOP W Switch from replication to packaging 77,111,112
-virus RNA
5-HT2CR, 5-hydroxytryptamine (serotonin) receptor 2C; aa, amino acid; Adar, adenosine deaminase acting on RNA; ApoB, apolipoprotein B; C, cysteine; cac,
cacophany; D, aspartic acid; G, glycine; GluR, glutamate receptor; I, isoleucine; K, lysine; M, methionine; N, asparagine; Nf1, neurofibromatosis type 1; nt, nucleotide;
para, paralytic; Q, glutamine; R, arginine; S, serine; UTR, untranslated region; V, valine; W, tryptophan; Y, tyrosine.

INTERFERON
One of a family of proteins that
occurs naturally in the body as
part of its defence mechanism.
It can be subdivided into three
distinct types: , and .
Z-DNA
A left-handed form of DNA
that can occur at stretches
of alternating G and C bases.
So named because of its
zig-zag backbone.
sugar-phosphate backbone, without making contact
with the bases65,66. Recently, however, it has been
shown that the dsRBDs in ADAR2 contribute to the
identification of the editing sites. This is probably
achieved by increasing the conformational flexibility of
nucleotides in the bases that are adjacent to the editing
site, thereby assisting the base flipping67. However, the
main determinant of specificity of the ADARs to
deaminate a specific adenosine lies in the deaminase
domain. This was confirmed when David Lazinski and
colleagues68 made protein chimaeras between ADAR1
and ADAR2, in which they exchanged the deaminase
domain, showing that this domain has a dominant
role in defining substrate specificity.
There are two forms of ADAR1 in humans: a longer,
predominantly cytoplasmic 150-kDa protein that is
induced by INTERFERON, and a shorter, nuclear 110-kDa
protein that is constitutively expressed69. In the constitu-
tive transcript, the alternative exon 1B is spliced to the
downstream exon 2, and the first methionine at which
translation is initiated is at position 296 (REFS 70,71).
Recently, the nuclear localization signal (NLS) was found
not to be at the amino terminus, as predicted by sequence
analysis, but overlapping with the third dsRBD72 (FIG. 3);
this explains why the shorter protein is in the nucleus
even though it lacks the amino terminus. Also at the
amino terminus lies a Z-DNA-binding domain that is com-
prised of Z and Z subdomains and has been proposed
to target ADAR1 to the site of transcription7376. It is puz-
zling that the shorter 110-kDa protein lacking the Z
subdomain is nuclear, whereas the larger protein, which is
mainly cytoplasmic, contains an intact Z-DNA-binding
domain. Also present in the amino terminus of ADAR1
are arginineglycine repeats of unknown function that
are also present in ACF15,59.
Expression of edited transcripts in the CNS. In mam-
mals77, Drosophila78 and squid79, most of the ADAR-
edited transcripts are expressed in the central nervous
system (CNS). One wonders whether such protein
diversity is required in the CNS or whether it is tolerat-
ed there owing to its immune-privileged status. Among

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Table 2 | Consequences of ADAR deficiency in animals
Organism Mutated gene Affected tissue Transcript Phenotype
Fruitfly Adar/ CNS Unknown Behaviour,
locomotion
Mouse Adar1+/ Liver Unknown Chimeric
embryos,
death by E14
Mouse Adar2/ CNS GluR-B (Q/R) Prone to
seizures,
death by P20
With the exception of the Adar2/ mutant, which is rescued by an edited form of GluR-B (GluR-BR),
the unedited transcripts that underlie the phenotype in the null animals remains unknown. Adar,
adenosine deaminases that act on RNA; CNS, central nervous system; E, embryonic day; GluR-B,
glutamate-gated ion channel receptor-B; P, post-natal day; Q, glutamine; R, arginine.
the edited mRNAs are those that encode: the gluta-
mate-gated ion channel receptors (GluR)80; the sero-
tonin (5-HT2C) receptor81; the voltage-gated calcium
and sodium channels8284, and a glutamate-gated chlo-
ride channel in Drosophila85; and the voltage-gated
potassium channel pre-mRNA in squid79 (see TABLE 1
for a more complete list). In addition, mammalian
Adar2 and Drosophila Adar pre-mRNAs are also edit-
ed78,86. Editing in these transcripts changes the coding
potential of the RNA so that different protein isoforms
are generated. The potential for diversity is perhaps
greatest in Drosophila, where there are often several
positions that can be edited for each pre-mRNA. In
transcripts of cacophony, which encodes the voltage-
gated calcium channel, editing at 10 different sites gives
the potential to generate more than 1,000 different iso-
forms by editing alone, without taking alternative splic-
ing into consideration82,83. This enormous potential for
generating diversity ought to be seen in the context of
the 13,600 predicted Drosophila genes, thereby posing
problems when deciphering the genome87.
Editing of a specific adenosine in a pre-mRNA is
usually not 100% efficient, one exception being the
glutamine/arginine (Q/R) site in GluR-B64,88.
Deamination of a specific adenosine at this site
changes the codon Q R a change that is crucial
for the correct functioning of the receptor. Work from
Peter Seeburgs group showed that editing at this posi-
tion in the GluR-B transcript controls the Ca2+ perme-
ability of heteromeric -amino-3-hydroxy-5-
methylisoxazole-4-propionate (AMPA) receptors,
which mediate fast-excitatory-synaptic transmission in
the CNS. Transgenic mice that were unable to edit only
at this position had epileptic seizures and died within
three weeks of birth, presumably due to increased Ca2+
permeability of the AMPA receptors89.
The extent of editing at a particular site can vary in
different regions of the brain, as observed in transcripts
of the serotonin 5-HT2C receptor, in which 12 principal
isoforms have region-specific expression81. Other fac-
tors can influence the extent of RNA editing; for exam-
ple, the extent of editing at the R/G site in the AMPA
receptor pre-mRNAs increases with the development
of the brain and is also affected by the splicing of the
neighbouring alternatively spliced exon90. Editing at
certain sites can require a specific ADAR; for example,
874 | NOVEMBER 2001 | VOLUME 2
2001 Macmillan Magazines Ltd
the Q/R site in the GluR-B transcript is edited by
ADAR2 (REFS 9193). Often ADAR1 and ADAR2 show
overlapping specificities in vitro and both can edit at
the same position, as for example at the R/G site in the
GluR-B pre-mRNA91,92.
The Adar null phenotype. Because of its wide distrib-
ution, Adar activity was assumed to be essential and
its removal by a null mutation was predicted to be
lethal. Therefore, it was quite surprising that
Drosophila that were deficient in Adar activity devel-
oped normally and were morphologically wild type94
(TABLE 2). However, the mutant flies did show extreme
behavioural deficits that included uncoordinated
locomotor activity, mating defects and tremors that
increase in severity with age. These phenotypes were
also accompanied by neurodegeneration. Frontal sec-
tions cut through adult heads showed large lesions in
the brain and a disorganized retina. Surprisingly,
despite such abnormalities in their brain, the flies had
a near-normal lifespan.
Mice heterozygous for Adar2 (REF. 93) were normal,
whereas homozygotes had normal embryonic develop-
ment but died during or soon after weaning. These
mice were prone to epileptic seizures and only 40% of
GluR-B mRNA molecules were edited at the Q/R site in
these animals. When a form of GluR-B (GluR-BR) in
which the arginine was genetically encoded (rather
than introduced by editing) was expressed in the
Adar2 knockout mice, the knockout phenotype was
rescued. The experiment showed very clearly that the
most important transcript that is edited by Adar2 is
that which encodes GluR-B.
Kazuko Nishikura and colleagues95 were unable to
generate Adar1+/ transgenic mice, as the chimeric
mouse embryos died before embryonic day 14 and
showed defects in the haematopoietic system. This
indicates that there might be a liver transcript, the
editing of which is sensitive to the dosage of Adar1
and cannot be compensated by the presence of
endogenous Adar2. However, a heterozygous embry-
onic-lethal phenotype is very rare and the possibility
of a dominant-negative truncated protein cannot be
eliminated. By constrast, Seeburg and colleagues have
generated viable Adar1+/ transgenic mice, but the
homozygous animals died during embryonic devel-
opment (P. Seeburg, unpublished data). It is possible
that the difference between the two results is due to
the different constructs that were used to generate the
transgenic mice.
A link between RNA editing and splicing?
Because the ECS is usually located in an intron that is
downstream of the site to be edited, editing has to
occur before splicing. In fact, RNA editing can some-
times influence splicing efficiency. For example, Adar2
null mice had an increased level of incompletely
processed GluR-B transcript that retained the intron
(intron 11) which contained the Q/R ECS. In these
mice, GluR-B mRNA levels were reduced fivefold93. The
editing frequency was 40% at the Q/R site in mature
www.nature.com/reviews/genetics

Pre-mRNA Stop
Exon Branch site
A A
AUG A A
A A
A genomic error, such as at the Q/R site in the GluR-B
5' UTR 3' splice acceptor Intron
3' UTR
Figure 4 | Editing of adenosine bases. Adenosine bases can be edited to inosine at different
positions along an RNA molecule. These positions are found not only in the coding regions of
transcripts but also in the 5 and 3 UTR99, in introns64 and at a splicing branch site96. Editing
can also generate a 3 splice acceptor86 and relieve a stop codon111,112.
mRNA, whereas only 10% of the pre-mRNA was edit-
ed. This indicates that editing of a transcript might
facilitate splicing. Editing is often observed within
introns; if it occurs in introns that contain intronic
splicing enhancers then the function of these elements
could be compromised.
RNA editing can regulate splicing by targeting the
adenosines involved in splicing. Rat ADAR2 edits its
own pre-mRNA such that a 3 splice site is generated86,
the use of which adds 47 nucleotides to rat ADAR2 and
changes the predicted open reading frame. Internal
translation initiation then leads to the production of an
active enzyme, but lower protein levels are expressed,
possibly because the internal translation initiation is rel-
atively inefficient. As the net result of self-editing is a
lower ADAR2 concentration, this process can be
thought of as a negative autoregulatory mechanism
whereby rat ADAR2 can regulate protein expression by
changing a downstream splice site.
The adenosine at the putative splicing branch site is
thought to be the target of ADAR activity in transcripts
of haematopoietic cell phosphatase (PTPN6) from
patients with acute myeloid leukaemia96 (see FIG. 4).
Sequencing of independent cDNA clones derived from
these patients showed aberrant splicing with retention of
intron 3, which would result in the production of a non-
functional protein. What is unusual is that only one
adenosine was edited within the intron at the potential
branch site, whereas six adenosines were edited in the
downstream exon. Intriguingly, aberrant splicing is lower
in patients in remission than at the time of diagnosis,
indicating a potential link been RNA editing and
myeloid leukaemia. However, it is yet to be confirmed
that ADAR is involved in editing this pre-mRNA.
Editing or modification?
It is estimated that inosine occurs at a frequency of 1 in
17,000 nucleotides in rat brain poly(A)+ RNAs, but at
1 in 33,000 nucleotides in other tissues97. What do all
these edited transcripts encode? A screen developed by
Brenda Bass and colleagues98,99 to identify inosine-
containing mRNAs found that inosine is present in the
untranslated regions (UTRs) of some Caenorhabditis
elegans mRNAs (FIG. 4), as well as in the UTRs of mam-
PSEUDOURIDINE malian brain mRNAs (D. Morse and B. Bass, personal
One of the most abundant communications). The screen did not identify mRNAs
modified nucleosides found in
RNA; it is the result of a post-
that contained inosine in codons, possibly because their
transcriptional isomerization presence here is less abundant. RNA editing was origi-
of uridine. nally defined as a post-transcriptional process other
NATURE REVIEWS | GENETICS
2001 Macmillan Magazines Ltd
REVIEWS
than splicing or capping that changes the coding poten-
tial of an mRNA to create variability or to correct a
transcript. The presence of inosine in the UTR could
modulate other aspects of RNA processing, such as sta-
bility or export. Therefore the term RNA editing has to
have a broader connotation if it is to include these other
aspects of RNA processing. The term mRNA modifica-
tion is probably a more accurate reflection of the wider
role that these enzymes have in RNA processing.
There are 95 types of known modified nucleotide in
stable RNA100. Have other base modifications gone
undetected in mRNA? This possibility is not as far
fetched as previously imagined considering that small
nucleolar (sno) RNP-mediated modification is present
not only in rRNA and tRNA but also in small nuclear
(sn) RNAs. These modifications are guided by
snoRNAs, which contain sequence that is complemen-
tary to the sequence flanking the sites of modification.
U2 snRNA is transcribed by RNA polymerase II and
modification at its 5 terminus is essential for its func-
tion in splicing101. Modifications are also present in the
other mammalian spliceosomal snRNAs U1, U4, U5
and U6 (REFS 102,103). Together these snRNAs contain 30
2-O-methyl groups and 24 PSEUDOURIDINES.
Some orphan snoRNA-like guide RNAs have been
identified that might target mRNAs; in particular, one
brain-specific guide snoRNA that is complementary to
the serotonin 5-HT2C receptor mRNA has been
found104,105. The function of these guide snoRNAs is
unknown but, intriguingly, the putative site of 2-O-
methylation on the mRNA is one of the adenosines that
is deaminated by ADAR. Methylation can reduce the rate
of deamination by 200-fold106 so the fate of this particular
editing site might be determined by the interplay between
methylation and deamination. If this is true, the question
arises as to whether this is an isolated event or a general
method of regulating ADAR activity. In addition, if
mRNA contains 2-O-methylated nucleotides or
pseudouridines, then the RNA folding and RNAprotein
interactions of that mRNA would be affected, which
would have ramifications on the function of the mRNA.
RNA editing and disease
To assess if RNA editing is a contributory factor in dis-
ease, both the lack of and aberrant editing of a tran-
script must be considered. The conversion of cystosine
to uracil has been implicated in disease processes
because overexpression of Apobec1 leads to dysplasia
and hepatocellular carcinomas in mouse and rabbit30,31.
The carcinomas might occur as a result of aberrant edit-
ing of Nat1 transcripts, as described above. In addition,
17% of the tumour suppressor Nf1 mRNA molecules
are edited but the functional consequence of this in
tumour tissue remains to be explained32. The recent dis-
covery of Aid and its involvement in class-switch
recombination and hypermutation of immunoglobu-
lins caused much excitement37. A deficiency in Aid caus-
es an autosomal-recessive form of hyper-immunoglob-
ulin M syndrome38, but its role as an RNA-editing
enzyme remains to be shown.
VOLUME 2 | NOVEMBER 2001 | 8 7 5
REVIEWS

PREFRONTAL CORTEX
Region of the brain that is
important for mood states and
might harbour abnormalities in
patients with unipolar and
bipolar depression.
STRIATUM
Region of the brain that receives
excitatory input from the
cortex, thalamus and midbrain.
It has a pivotal role in
modulating motor activity and
higher cognitive function.
POLYOMA VIRUS
DNA virus that causes
subclinical infections early in
childhood. It leads to viral
latency within the kidney but
reactivation occurs in
transplant recipients as a result
of immunosuppressive therapy.
Because most of the pre-mRNAs that are edited by
the Adar enzymes are expressed in the CNS, defects
in Adar activity result in neurological phenotypes.
Drosophila that are null for Adar activity show severe
behavioural defects 94, whereas Adar2 / transgenic
mice are prone to seizures and die soon after birth93.
This lethality of Adar2/ mice is due to the inability
to edit the Q/R site in GluR-B because early onset
epilepsy and premature death is also observed in
mice with the GluR-B ECS allele that contains an
uneditable Q/R site89.
According to one study, reduced editing of the sero-
tonin 5-HT2C receptor transcripts occurs in schizophre-
nia patients107. Another study contradicts this and
instead found elevated levels of editing in subjects who
have committed suicide108. An alteration in the level
of editing at the Q/R site in GLUR-B in the PREFRONTAL
CORTEX and the STRIATUM of patients with Alzheimer dis-
ease, Huntington disease and schizophrenia has also
been reported109, but more research is required to sub-
stantiate these findings.
The hepatitis -virus (HDV) is often found associ-
ated with hepatitis B virus in patients with severe liver
disease110. ADAR-mediated editing of HDV RNA at a
stop codon converts it into a trp codon, thereby
extending the open reading frame by 19 amino acids.
This results in the synthesis of HDAg-p27, which is
required for viral packaging111,112. Hyper-editing of
viral transcripts has also been implicated in the deaths
of patients with subacute sclerosing panencephalitis
a rare disease that occurs 510 years after acute
measles infection and also in measles inclusion
body encephalitis113,114. Hypermutations in the cDNAs
that encode both the matrix gene and the other four
major measles viral proteins were found in infected
brains. The mutations in the measles virus are U C
transitions in the positive-strand antigenome and are
thought to arise from adenosine to inosine editing of
the negative-strand genomic RNA115. After hyper-edit-
ing of the transcripts, the measles matrix protein can-
not be detected, indicating that the virus is unable to
bud, so causing a persistent infection. As previously
mentioned, editing in intron 3 and exon 4 of PTPN6
transcripts from patients with acute myeloid
leukaemia is predicted to produce a non-functional
protein96. Furthermore, G A and C U changes
have been found in HIV-1 (human immunodeficiency
virus type 1) transcripts116, but it is still unclear
whether this is a result of RNA editing or error-prone
reverse transcription117.
Hyper-editing is also found in early-strand transcripts
of POLYOMA VIRUSES, in which almost 50% of adenosines can
be edited118. Hyper-editing of polyoma transcripts pre-
vents them from being transported to the cytoplasm. A
complex containing p54nrb, the splicing factor PSF and
the inner nuclear matrix structural protein binds to these
hyper-edited transcripts and favours nuclear retention
over export119,120. Edited transcripts do get translated but
the maximum number of inosines per transcript that
allows export from the nucleus is unknown. Nuclear
retention of edited transcripts might act as a safeguard to
prevent translation of hyper-edited transcripts and
expression of mutant proteins. Indefinite accumulation
of hyper-edited transcripts would be problematic for the
cell so there must be a nuclease that specifically cleaves
deaminated mRNA. Such an activity has been found not
in the nucleus but in the cytoplasm121. This nuclease is
specific for inosine-containing dsRNAs and cleaves them
at a specific site that consists of alternating IU and UI
base pairs, leaving ss and dsRNA intact. This endonucle-
ase could also safeguard against translation of any hyper-
edited transcripts that have escaped from the nucleus.
Conclusions
RNA editing is a process that generates diversity in pro-
teins that are encoded by a single locus. This process is
essential in some cases and the transcripts that are being
edited range from CNS receptors to proteins that are
involved in lipid transport in the blood. With the race to
identify all the proteins encoded in the various genomes,
it is important to know how much diversity in proteins is
generated by RNA editing. In the 5-HT2C receptor, edit-
ing at 5 different positions generates 12 major isoforms,
some having reduced sensitivity to LSD (d-lysergic acid
diethylamide) and atypical antipsychotic drugs108.
Therefore, it is important for drug therapy to know that
the receptor that is being targeted does not have other
isoforms. Also, as the rate of editing at a position can
change during development90, the ratio of different iso-
forms of a receptor in a young child could vary signifi-
cantly to that found in an adult. This again would have
implications in drug therapy.
The RNA-editing machinery does not discriminate
between coding and non-coding sequences. So what
might the purpose of editing non-coding regions be? This
editing might affect splicing, stability or transport of a
transcript. It has become obvious in recent years that the
pathways of RNA processing are inter-related and this is
probably another example. The challenge in the future will
be to uncover all the roles of RNA editing within the cell.

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Acknowledgements
We are grateful to N. Gray and D. Scadden for their helpful com-
ments and to S. Bruce for drawing the original figures. In addition,
we are grateful to B. Bass, D. Morse and P. Seeburg for sharing
unpublished work, and to W. Keller and R. Reenan for their many
discussions. We apologize to colleagues whose work could not be
cited due to space constraints. Work in the authors laboratory is
supported by the MRC and a grant from the British Heart
Foundation.
Online links
DATABASES
The following terms in this article are linked online to:
Locuslink: http://www.ncbi.nlm.nih.gov/LocusLink/
ACF | ADAR1 | ADAR2 | ADAR3 | AID | APOB | APOBEC1 |
APOBEC2 | GRY-RBP | NF1 | PTPN6
OMIM: http://www.ncbi.nlm.nih.gov/Omim/
acute myeloid leukaemia | Alzheimer disease | Huntingdon
disease | hyper-immunoglobulin M | measles inclusion body
encephalitis | schizophrenia | subacute sclerosing
panencephalitis
SWISS-PROT: http://www.expasy.ch/
5-HT2C | dsRBD | eIF4G | KSRP
Access to this interactive links box is free online.
www.nature.com/reviews/genetics