A large comparative genomic sequence study has determined the extent of conservation between RNA editing sites within the mammalian evolutionary tree. See related research by Pinto et al., http://genomebiology.com/2014/15/1/R

Robert A Reenan

Yiannis A Savva

PubMed

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Savva and Reenan Genome Biology 2014, 15:103
http://genomebiology.com/2014/15/1/103RESEARCH HIGHLIGHTIdentification of evolutionarily meaningful
information within the mammalian RNA editing
landscape
Yiannis A Savva and Robert A Reenan*
See related research by Pinto et al., http://genomebiology.com/2014/15/1/R5Abstract
A large comparative genomic sequence study has
determined the extent of conservation between RNA
editing sites within the mammalian evolutionary tree.found in introns, are highly conserved (Figure 1a). It isExpansion of the RNA editing universe
Generating cellular proteomes relies on the faithful decod-
ing of genetic information. A complex network of cellular
machines transcribes DNA into matured processed
mRNA that is then translated into protein products,
which are used by the cells to carry out basic biological
functions. It is widely accepted that organismal complexity
arises via the expansion of the genetic information poten-
tial by post-transcriptional modifications, such as alterna-
tive splicing and RNA editing. The latter is mediated in
metazoans by a highly conserved protein family known as
adenosine deaminase acting on RNA (ADAR) [1]. These
enzymes hydrolytically deaminate adenosines to inosines
(A-to-I) in double-stranded RNA (dsRNA) substrates. A-
to-I RNA editing generates subtly different protein prod-
ucts by altering the primary sequence of target genes,
since upon translation the ribosomal machinery interprets
inosines as guanosines, leading to A→G substitutions
(Figure 1a). Therefore, electropherograms derived from
edited cDNAs include A/G mixed peaks, which are con-
sidered to be a hallmark of A-to-I RNA editing. A decade
ago, only few editing sites had been known to exist due to
their accidental discovery by comparison of cDNA to gen-
omic DNA sequences. However, a comparative genomics
approach in Drosophila melanogaster uncovered a phy-
logenetic signature of RNA editing [2]. Since ADAR-
mediated editing ensues through the formation of highly* Correspondence: robert_reenan@brown.edu
Department of Molecular Biology, Cell Biology and Biochemistry, Brown
University, 185 Meeting St, Providence, RI 02912, USA
© 2014 Savva and Reenan; licensee BioMed C
medium, for 12 months following its publicat
Commons Attribution License (http://creativec
reproduction in any medium, provided the orstructured and frequently complex dsRNA substrates,
necessary cis-regulatory elements should be highly con-
served across Drosophila species. Indeed, cis-elements that
promote imperfect dsRNA formation in pre-mRNA, also
known as editing complementary sequences (ECS), usually
generally thought that the nature of the structural imper-
fections in these dsRNAs lend the specificity in determin-
ing which adenosines are edited by ADAR. In addition,
sequence conservation is higher in exonic sequences near
editing sites when compared with adjacent exons due to
the functional constraints imposed by RNA structure.
Using this pattern of evolutionary conservation led to the
discovery of approximately 50 new editing sites in 16 dif-
ferent genes. Intriguingly, the conserved editing sites were
found to be present in genes encoding proteins that are
involved in electrical and chemical neurotransmission,
including synaptic release proteins and voltage-gated and
ligand-gated ion channels. To further understand the bio-
logical significance of RNA editing, contemporary studies
use deep sequencing technologies to identify novel RNA
editing sites. During the last decade, deep sequencing ana-
lysis rapidly expanded the RNA editing landscapes in vari-
ous organisms, including humans, mouse and Drosophila.
However, the extent of evolutionary conservation between
the numerous mammalian RNA editing sites across the
evolutionary tree is currently unknown.The RNA editing enigma
RNA editing sites are scattered within the human, mouse
and Drosophila transcriptomes. Geographically, editing
sites are found in both coding regions (exons) and in non-
coding regions (5′ UTRs, 3′ UTRs, introns and inter-
genic) throughout genomes. Another theme from the
deep-sequencing era is that much of the informational
content of genomes is transcribed into non-coding RNA,entral Ltd. The licensee has exclusive rights to distribute this article, in any
ion. After this time, the article is available under the terms of the Creative
ommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
iginal work is properly cited.
(a)
RNA polymeraseRecoding ADAR target mRNA
pre-mRNA
dsRNA binding 
domain
Deaminase 
domain
Exonic RNA
Intronic RNA
(Q)
CAG
(R
)
C
IG
(b)
RNA polymeraseHairpin-containing ADAR target dsRNA
ALU ALU
I
A
I
A
A
I
A
I
A
I
(c)
RNA morphospace
%
 E
di
tin
g
- 
Δ 
G
 fo
rm
at
io
n
= mRNA
= long dsRNA
= miRNA
Adaptive level
A
D
A
R
C
A
G
Figure 1 An overview of RNA editing. (a) Specific RNA editing occurring in a pre-mRNA. The nascent transcript folds into a complex dsRNA
structure, pairing coding sequences (blue) with highly conserved intronic sequences. Structural features (for example, bulges and loops) in the duplex
region focus ADAR’s deaminase activity on a few or one adenosine residue. (b) Promiscuous editing of long repeat dsRNAs. Shown here is an inverted
repeat of Alu elements whose transcription produces a long nearly perfect dsRNA. Numerous ADAR editing events can occur in such a substrate,
altering its structure, and potentially interfering with downstream processes (for example, Dicer processing into endo-siRNAs). (c) A hypothetical
scheme for conserved versus less-conserved RNA editing events. All RNA molecules can potentially form secondary structures, and thus potentially
bind and be acted upon by ADAR. Those that are minimally structured will be edited poorly or not at all. The vast majority of RNAs will fall into this
category, and be well below the action of serving as a variant incipient adaptation acted upon by natural selection (below dashed green line). Certain
RNAs will more readily serve as ADAR targets, and will as a consequence also possess more dsRNA character. Such events can be acted upon by
natural selection and serve as sources of variation in the expression of RNAs (for example, mRNAs, long non-coding dsRNA, miRNA precursors). Natural
selection will preserve certain structural features, such as those in pre-mRNA and miRNA precursors, to ensure highly evolved edited structures. Other
long dsRNAs, such as inverted repeat hairpin dsRNAs, will be edited as part of the regulation of host defenses to viruses and selfish genetic elements.
ADAR, adenosine deaminase acting on RNA; dsRNA, double-stranded RNA; miRNA, microRNA, siRNA, small interfering RNA.
Savva and Reenan Genome Biology 2014, 15:103 Page 2 of 4
http://genomebiology.com/2014/15/1/103
Savva and Reenan Genome Biology 2014, 15:103 Page 3 of 4
http://genomebiology.com/2014/15/1/103whose functions largely remain to be determined. Com-
parison of RNA editing landscapes between different gen-
etic model organisms has uncovered diverse ADAR
substrates and revealed discrepancies within RNA editing
systems. While the number of exonic editing sites that
lead to non-synonymous amino acid substitutions (gen-
omic recoding) varies significantly across a broad range of
organisms, a common theme seems to be emerging. The
majority of RNA editing sites are located in non-coding
regions within genomes. For example, in the human gen-
ome, RNA editing sites are highly over-represented in Alu
elements restricted within introns of transcribed genes [3].
Alu elements are lineage-specific repeat genomic se-
quences, which participate in dsRNA template formation
acting as ADAR substrates (Figure 1b). Unlike mRNA tar-
gets of ADAR, such more perfect and extensive dsRNA
substrates lend themselves to more extensive deamination,
with up to 40% of adenosines in the duplex region under-
going modification. Similarly, within the mouse genome
the majority of RNA editing sites are found in B1 short in-
terspersed element repeats. RNA editing in non-coding
regions of a transcript can have several functional conse-
quences such as: creation or elimination of splicing sig-
nals, exonization of repeat elements, nuclear retention,
regulation of microRNA (miRNA) biogenesis and func-
tion, cellular defense, and regulation of RNA interference
[4]. It is thought that the collection of RNA editing sites in
an organism’s transcriptome contributes equally to the ap-
propriate functioning of the nervous system, as exempli-
fied from the generation of ADAR deficiencies in various
genetic models [5]. In Drosophila, deletion of the adar
locus results in severe neurological phenotypes, including
extreme uncoordination, seizures and neurodegeneration.
Furthermore, mice homozygous for ADAR1 null muta-
tions die during early development due to severe apop-
tosis. In addition, ADAR2 null mutant mice experience
repeated seizure episodes and die soon after birth. Inter-
estingly, the lethality phenotype of the ADAR2 null mice
is rescued by the introduction of the edited allele of a sin-
gle RNA editing site in the glutamate receptor channel,
GluR-B Q/R site [6]. This observation suggests that cer-
tain RNA editing sites are more physiologically critical
compared with others existing in the same genome. Thus,
a general enigma in ADAR-mediated editing is exactly
how to determine which, of many, RNA editing sites are
functionally important, and which may have no discern-
able function.
A unique set of RNA editing sites
In order to identify functionally important RNA editing
sites from a vast RNA editing landscape, a recent study
by Pinto et al. [7] used evolution as a key discriminator
to delineate highly conserved sites in mammalian line-
ages. Taking advantage of enormous RNA-seq datasetsof both human and mouse transcriptomes, the authors
applied a standard BLAST alignment tool to compare 40
base pairs upstream and downstream of human genomic
sequences surrounding an RNA editing site to the
mouse genome. A set of basic filters was applied to
the datasets to retain only RNA editing sites located at the
same exact position in both human and mouse genomes.
Surprisingly, this simple procedure identified 59 highly
conserved editing sites. This set of RNA editing sites,
which were termed evolutionary selected sites (ESS),
represents 0.004% of the known human editing sites to
date. Furthermore, the authors demonstrated that the
percentage of ESS sites does not increase as RNA-seq
data accumulate. Using a small fraction of the available
RNA-seq data sets from 15 different mouse strains, the
authors were able to retrieve approximately 95% of the
ESS sites in any random choice of two mouse strains.
While the ESS sites were found across all the mouse
strains tested, the non-conserved sites were not consist-
ently detected, suggesting that the ESS set is exquisitely
specific. In addition, evidence for the presence of ESS
sites can be found in RNA-seq data from an additional
four genomes within the mammalian evolutionary tree:
rat, cow, opossum and platypus. The presence of ESS
sites across large evolutionary distances suggests a func-
tionally important role in mammalian biology for these
sites, despite the fact that the set is surprisingly small.
The nature of functionally important RNA editing
sites
Due to the degeneracy of the genetic code, RNA editing
can cause both synonymous and non-synonymous
changes. In Drosophila, the majority of highly conserved
RNA editing sites lead to non-synonymous amino acid
changes in functionally important and highly conserved
residues within proteins. Similarly, the majority of the
mammalian ESS editing sites lead to amino acid recod-
ing. Specifically, 37 out of 59 ESS editing sites are found
in coding regions of the genome and 35 of them lead to
non-synonymous amino acid substitutions (94%). In
addition, most of the non-coding ESS editing sites (22/
59) occur in transcripts of genes that are also edited
elsewhere in their coding sequence. Recent studies in
Drosophila identified editing sites in ECS non-coding
genomic elements [8,9]. Not surprisingly, using struc-
tural RNA prediction software (mfold), Pinto et al.
showed that most of the ESS editing sites in non-coding
regions are located in potential ECS elements. Further-
more, two of the ESS editing sites are found in miRNAs
in agreement with the previously described regulation of
miRNA biogenesis and function through RNA editing.
Further analysis revealed certain features of the ESS edit-
ing set. First, the ESS editing sites display higher levels
of expression when compared with the non-conserved
Savva and Reenan Genome Biology 2014, 15:103 Page 4 of 4
http://genomebiology.com/2014/15/1/103editing sites set. Second, the ESS editing set exhibits
higher levels of editing and, more importantly, these
levels of editing display striking conservation across 15
mice strains and between human and mouse. These ob-
servations indicate that the editing levels of the con-
served mammalian RNA editing sites are set within the
evolutionary tree and that the precise ratios of edited/
non-edited repertoires of protein products may contrib-
ute to the optimization of cellular physiology. Lastly,
similar to the highly conserved RNA editing sites reported
in Drosophila, the ESS editing sites are overrepresented in
genes that play a pivotal role in nervous system functions
such as synaptic release and ion transport. This observa-
tion suggests that natural selection acts through RNA edit-
ing to evolve RNA structures that are acted upon by
ADARs to genetically recode the proteome associated with
neurotransmission and thereby fine-tune brain physiology.
Unweaving the roles of RNA editing
Abnormalities in the RNA editing pathway are associ-
ated with multiple nervous system disorders including
schizophrenia, epilepsy, suicidal depression and amyo-
trophic lateral sclerosis [10]. The conserved RNA editing
sites within the mammalian evolutionary tree reported
by Pinto et al. [7] have the potential to contribute to a
better understanding of the link between the RNA edit-
ing process and various neurological diseases. With the
rapid advent of genetic engineering techniques, specific
RNA editing sites could be precisely examined in vivo in
various animal models, thus uncovering functions of
ESS single RNA editing events. Furthermore, the specific
characteristics of the non-conserved set of RNA editing
sites prompted Pinto et al. to propose that these events
are simply a consequence of overactivity of RNA editing
enzymes with no apparent evolutionary value. Certainly,
particular specific RNA editing events are under intense
selective pressure, yet all RNAs are structured to some
degree due to the single-stranded nature of RNA. Many
RNAs, from all classes, probably serve as poor ADAR
substrates (Figure 1c). Low-level editing of barely struc-
tured RNAs is not likely to be under intense scrutiny by
natural selection, but sequence variants that become
more structured, or obtain more stable structures under
altered environmental conditions, may be better ADAR
substrates, and then be vetted by natural selection for
their adaptive consequences. Further sequence drift,
under selective conditions, could shape RNAs into effi-
cient ADAR substrates over generations. Other targets,
for instance long dsRNA transcribed from recent dupli-
cation or transposition events, may immediately be
hyperedited, leading to intersection between RNA edit-
ing and small RNA processing.
Multiple reports suggest that the RNA editing pathway
is highly sensitive to external and internal stimuli suchas temperature and inflammation. Therefore, future
studies should aim to investigate how these two different
sets of mammalian RNA editing sites respond to alter-
ations of environmental stimuli. Non-conserved RNA
editing events within mammalian lineages could repre-
sent variations in how RNA editing reshapes a specific
transcriptome/proteome in response to external or in-
ternal changes in the environment. Such editing sites
could represent derived characters, as such, which could
then be interpreted as molecular adaptations in cellular
functions. Studies such as that by Pinto et al. open the
doorway for identifying both the conserved and, perhaps
equally interesting, non-conserved species-specific RNA
editing events that have shaped, and been shaped by,
evolution.
Abbreviations
ADAR: Adenosine deaminase acting on RNA; A-to-I: Adenosine to inosine;
cDNA: complementary DNA; dsRNA: double-stranded RNA; ECS: Editing
complementary sequence; ESS: Evolutionary selected sites; miRNA: microRNA.
Competing interests
The authors declare that they have no competing interests.
Published: 28 January 2014
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Cite this article as: Savva and Reenan: Identification of evolutionarily
meaningful information within the mammalian RNA editing landscape.
Genome Biology 2014 15:103.


Identification of evolutionarily meaningful information within the mammalian RNA editing landscape

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