RNA Editing in Mammalian Cells

1
RNA Editing in Mammalian Cells
2
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 transcripts 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.
3
RNA EDITING

• In mammalian cells, RNA editing results in the
  change of a single nucleotide at a single site in
  specific mRNAs

4
RNA Editing

• Mammalian C U RNA Editing
• Mammalian A I RNA Editing

5
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.
6
The original and most fully detailed example of
C?U RNA editing is mammalian apoB mRNA, in which
a site-specific cytidine deamination introduces a
UAA stop codon into the translational reading
frame, resulting in synthesis of a truncated
protein, apoB48. C?U RNA editing of apoB occurs
within enterocytes of the mammalian small
intestine. Under physiological circumstances,
C?U editing of apoB mRNA targets a single
cytidine out of more than 14,000 nucleotides, a
process constrained by stringency in the
cis-acting elements and by the protein factors
responsible for targeted deamination.
7
APOB is a component of the plasma lipoproteins
and is crucial for the transport of cholesterol
and of triglycerides in the plasma. 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 translational stop codon..
8
C U RNA Editing APOB

• In humans, this editing event occurs in the small
  intestine but not in the liver.
• The APOB100 isoform is synthesized only in the
  liver and is used to assemble the
  very-low-density lipoprotein (VLDL) that is
  necessary for the transport of TRIGLYCERIDES and
  cholesterol. VLDL is metabolized to LOW-DENSITY
  LIPOPROTEIN (LDL).
• The carboxyl terminus of APOB100 interacts with
  the LDL receptor, and LDL is removed from the
  circulation.
• Such a functional interaction 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 CHYLOMICRONS

9
APOB Apolipoprotein B Editing

• CHYLOMICRON Large lipoprotein complex formed in
  the intestine that transports fats from the
  intestine to the liver and to adipose tissue.

10
APOB Apolipoprotein B Editing

• DEAMINATION The removal of an amino group from a
  nucleoside, thereby generating another nucleoside
  with different base-pairing properties.

11
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). 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 and requires
both trans-acting factors and cis-acting sequence
elements that surround the cytosine that is
edited. A mooring sequence of 11 nucleotides is
situated downstream of the editing site and is
separated from the target cytosine by a spacer
element that is usually four nucleotides long.
12
The site is flanked by 3' and 5' efficiency
elements (sequences that have been identified
that are required for efficient editing), and
there is an additional requirement for AU-rich
bulk RNA. The mooring sequence and the 3'
efficiency element form a double-stranded (ds)
stem that is predicted to position the edited
cytosine in a favorable configuration for
deamination.
13
Trans-acting factors C?U RNA editing is mediated
by an enzyme complex that includes the
RNA-specific cytidine deaminase, APOBEC-1 and
APOBEC-1 complementation factor, ACF, a novel
protein that serves as the RNA recognition
component of the core enzyme complex.
14

• Cytidine deamination in vitro and in vivo
  requires a multiprotein complex or editosome
• Editosome assembly requires a complex protein
  composition with an aggregate size of 27 S.
• Editing activity requires a homodimer of APOBEC-1
  (catalytic subunit for cytidine-to-uridine
  editing), and a 65 kDa RNA binding protein, ACF
  (APOBEC-1 complementation factor).
• APOBEC-1 demonstrates a weak and non-specific RNA
  binding activity to AU-rich apoB sequences and
  alone could not edit apoB mRNA.
• Editing site specificity and RNA editing activity
  are imparted upon APOBEC-1 through its
  interactions with ACF.

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The cytidine deaminase, APOBEC-1, which catalyses
the deamination reaction, and the 65-kDa
auxiliary factor ACF form a complex that is the
minimum requirement for editing of APOB in
vitro. Recombinant APOBEC1 protein cannot
deaminate the APOB mRNA in the absence of ACF.
17
APOBEC-1 belongs to a family of cytidine
deaminases (CDAs) and contains the protein motif
characteristic of a cytidine deaminase .The
carboxyl terminus is leucine-rich and is involved
in dimerization.
18
The ACF factor contains three non-identical
single-stranded (ss) RNA-recognition motifs at
the amino terminus and a putative dsRNA-binding
domain at the carboxyl terminus. The binding of
ACF to APOB mRNA is dependent on an intact
mooring sequence, indicating that the mooring
sequences assist in docking APOBEC1 to deaminate
the target cytosine. The dsRNA binding domain of
ACF might bind the stem that is formed between
the mooring sequence and the 3' efficiency
element.
19
APOB Apolipoprotein B Editing

• MOORING SEQUENCE An 11-nucleotide motif, which
  is located 3' of the cytidine that is edited in
  APOB mRNA. Both APOBEC-1 and ACF bind to this
  motif.

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22
Recognition of the edited nucleotide by the
editing enzymes. APOBEC-1 binds to APOB mRNA in
the presence of ACF and catalyses the C ? U
deamination of C6666 that is positioned at the
active site. The cis-acting sequence elements
are the MOORING SEQUENCE, and 5 and 3
efficiency elements.
23
5 and 3 efficiency elements
The mooring sequence is flanked by AU-rich
elements both 5' and 3' of an approximately
50-nucleotide editing cassette. Structural
predictions indicate that this AU-rich region
assumes significant secondary structure with the
targeted cytidine exposed at the apex of a
stem-loop bulge. RNA splicing precedes C?U RNA
editing and helps account for the specificity of
the latter processing event. ApoB RNA editing
occurs within the nucleus, and its preferred
substrate is spliced, polyadenylated RNA.
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25
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 editing also takes place
in this tissue. This broader expression of
ApoB48 in rat and mouse explains why rodents have
very low LDL levels.
26
RETROVIRAL RESTRICTION BY APOBEC PROTEINSNature
Reviews-Immunology 2004. 4868

• A novel mechanism of innate immunity has entered
  the spotlight a potent cellular defense that
  actively blocks retroviral infection.
• At least two proteins lie at the heart of this
  defense mechanism APOBEC3F and APOBEC3G.
• These cellular proteins function by hitchhiking
  with newly produced viral particles until a new
  TARGET CELL is found.
• Then, during synthesis of the first retroviral
  DNA strand (minus strand), which is an obligate
  step in the retroviral life cycle,
  APOBEC-dependent DEAMINATION of cytosine (C)
  residues results in the accumulation of excessive
  levels of uracil (U).
• This pre-mutagenic lesion leads to the demise of
  the invading RETROVIRUS on its replication,
  because uracil is recognized as thymine (T) and
  adenine (A) is incorporated into the newly
  synthesized second (plus) DNA strand rather than
  guanine (G).

27
RETROVIRAL RESTRICTION BY APOBEC PROTEINS

• To thwart this cellular defence, HIV encodes Vif,
  a small protein that mediates APOBEC degradation.
  
• Therefore, the balance between APOBECs and Vif
  might be a crucial determinant of the outcome of
  retroviral infection.
• Vertebrates have up to 11 different APOBEC
  proteins, with primates having the most

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RETROVIRAL RESTRICTION BY APOBEC PROTEINS

• APOBEC3G and Vif are key determinants of
  retroviral infectivity.
• APOBEC3G that is expressed in the PRODUCER CELL
  is incorporated into the budding virion together
  with other components of the virus, including its
  genomic RNA.
• HIV-1 Vif can reduce or eliminate APOBEC3G
  incorporation into budding virions by targeting
  it for proteasomal degradation.
• However, should APOBEC3G escape Vif, gain access
  to the virion, it can deaminate cytosine residues
  in the first retroviral DNA strand.
• The resulting uracil residues function as a
  template for the incorporation of adenine, which,
  in turn, can result in strand-specific C/G to T/A
  transition mutations that affect virus viability.

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31
RNA Editing

• Mammalian A I RNA Editing

32
Inosine has base-pairing properties like those of
guanosine. A I (G)
33
Expression of edited transcripts in the CNS. In
mammals, most of the A to I edited transcripts
are expressed in the central nervous system
(CNS).
34
The first evidence for editing came from A to G
differences among cDNA products or between cDNA
and genomic sequences during the course of
cloning. The first example of A to I editing in
an mRNA was found in the mammalian brain, in
transcripts of the gene encoding the ionotropic
glutamate receptor subunit, GluR-B. Other
examples have appeared in numerous signaling
components of the nervous systems of vertebrates
and invertebrates.
35
Glutamate-gated ion channel receptors (GluR)
36
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 eukaryotes. The enzymes
that deaminate adenosine to inosine in dsRNA are
members of a family of Adenosine Deaminases that
Act on RNA-- ADAR.
37
A ? I RNA editing

• In humans, there are three members of this family
  -ADAR1, ADAR2 and ADAR3, the names reflecting the
  order in which they were identified.
• ADAR1 and ADAR2, which are almost ubiquitously
  expressed, can convert adenosine to inosine, both
  in long dsRNA duplexes and in specific pre-mRNAs.
  
• Several isoforms of these two enzymes exist that
  vary in their editing activity.

38
Schematic diagram of human ADAR
39
Among the edited mRNAs are those that encode the
glutamate-gated ion channel receptors (GluR) the
serotonin (5-HT2C) receptor the voltage-gated
calcium and sodium channels, a glutamate-gated
chloride channel, and the voltage-gated potassium
channel pre-mRNA. In addition, mammalian ADAR2
pre-mRNAs are also edited. Editing in these
transcripts changes the coding potential of the
RNA so that different protein isoforms are
generated.
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41
Functional Importance of EDITING
Editing of the GluR-B Q/R site is a major
determinant of the Ca2 permeability of
multimeric GluR channels that incorporate the
GluR-B subunit. Editing of the GluR-B, -C and
-D R/G sites affects the rate of recovery from
desensitization of GluR channels. Editing of
the serotonin receptor 5-HT2C results in greatly
reduced efficiency of G-protein coupling in
certain edited forms.
42

• Mechanism of ADAR editing
• ADAR proteins do not require cofactors to
  deaminate adenosine to inosine through HYDROLYTIC
  DEAMINATION.
• 2. 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 intron.
• 3. The dsRNA-binding domains (dsRBDs) found in
  ADARs mediate the binding to the duplex.

43
Mechanism of ADAR editing

• The main determinant of specificity of the ADARs
  to deaminate a specific adenosine lies in the
  deaminase domain.
• Protein chimeras between ADAR1 and ADAR2, in
  which deaminase domains were exchanged, showed
  that this domain has a dominant role in defining
  substrate specificity.

44
Schematic diagram of human ADAR. The human
editing enzyme ADAR, contains an arginineglycine
(R/G)-enriched domain present in ADAR1 colored
blue.The double-stranded (ds) RNA Binding Domains
are purple. The deaminase (DM) domains are
orange. ADAR, adenosine deaminases that act on
RNA NLS, nuclear localization signal RBD,
RNA-binding domain.
45
ADARs recognize duplex RNA that is formed between
the editing site and the ECS (editing site
complementary sequence) 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.
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47
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48

• In A to I Editing
• Splicing occurs AFTER Editing because the
  double-stranded RNA must be formed between the
  upstream Exon and the downstream Intron.

49
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-B. 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. Editing at this position in the GluR-B
transcript controls the Ca2 permeability of
heteromeric a-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 receptors.
50
Calcium-permeable AMPA receptors containing
Q/R-unedited GluR2 direct human neural progenitor
cell differentiation to neuronsWhitney et al.,
FASEB Journal, doi 10.1096/fj.07-104661 April
2008

• RNA editing of the GluR2 subunit at the Q/R site
  is responsible for making most AMPA receptors
  impermeable to calcium.
• Because a single-point mutation could eliminate
  the need for editing at the Q/R site and
  Q/R-unedited GluR2 exists during embryogenesis,
  the Q/R-unedited GluR2 subunit presumably has
  some important actions early in development.
• Using calcium imaging, It was found that neural
  progenitor cells (NPCs) contain calcium-permeable
  AMPA receptors, whereas NPCs differentiated to
  neurons and astrocytes express calcium-impermeable
  AMPA receptors.

51
Calcium-permeable AMPA receptors containing
Q/R-unedited GluR2 direct human neural progenitor
cell differentiation to neurons

• NPCs contain Q/R-unedited GluR2, and
  differentiated cells contain Q/R-edited GluR2
  subunits.
• This is consistent with the observation that the
  nuclear enzyme responsible for Q/R-editing,
  adenosine deaminase (ADAR2), is increased during
  differentiation.
• Activation of calcium permeable AMPA receptors
  induces NPCs to differentiate to the neuronal
  lineage and increases dendritic arbor formation
  in NPCs differentiated to neurons.

52
Differentiation of NPCs into neurons
53
Overexpression of ADAR2 prevents AMPA-induced
differentiation of NPCs to neurons
54
Conclusions

• NPCs contain calcium-permeable AMPA receptors,
  whereas NPCs differentiated to neurons and
  astrocytes express calcium-impermeable AMPA
  receptors
• Calcium permeable AMPA receptors containing
  Q/R-unedited GluR2 direct human neural progenitor
  cell differentiation to neurons.
• Q/R-unedited GluR2 exists during embryogenesis,
  whereas, nearly 100 of GluR2 is edited in the
  adult.
• Levels of ADAR2 increase from embryogenesis to
  adulthood and editing at Q/R increases from about
  50 in embryogenesis to nearly 100 in the adult.

55
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. RNA editing may
even influence splicing.
56
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 generated, 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, because the internal
translation initiation is relatively inefficient.
Self-editing results in a lower ADAR2
concentration, so this process can be thought of
as a negative autoregulatory mechanism whereby
rat ADAR2 can regulate protein expression by
changing a downstream splice site.
57
Coordination of editing and splicing of glutamate
receptor pre-mRNA EVA BRATT and MARIE OHMAN RNA
(2003), 9309318.
58
A system was developed to investigate if editing
and splicing of pre-mRNA are coordinated by
focusing on a selectively edited site (R/G) in
the glutamate receptor subunit B pre-mRNA. This
editing site is situated in close proximity to a
5 splice site. A GluR-B transcript (GRG 988)
containing the R/G editing site in exon 13,
intron 13, and exon 14 was assayed for in vitro
splicing.
59
GRG SS D20
The ECS was deleted
60
ADAR2 inhibited splicing of GRG SS pre-mRNA. In
vitro splicing of GRG WT or GRG SS?20 pre-mRNA
with or without recombinant rADAR2a (A2) was
analyzed at time points as indicated.
61
Model of editing and splicing of GluR-B pre-mRNA.
ADAR2 (black circle) competes with splicing
factors (SF, gray circle) for binding to the R/G
stem-loop in vitro..
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63
Editing modifies the GABAA receptor subunit a3

• J. OHLSON, J.S. PEDERSEN, D. HAUSSLER, and M.
  OHMAN
• RNA (2007), 13698703.

64
Adenosine to inosine (A-to-I) pre-mRNA editing by
the ADAR enzyme family has the potential to
increase the variety of the proteome. This
editing by adenosine deamination is essential in
mammals for a functional brain.

• To detect novel substrates for A-to-I editing an
  experimental method was developed to find
  selectively edited sites, combined with
  bioinformatic techniques that find stemloop
  structures suitable for editing.
• The first verified editing candidate detected by
  this screening procedure is Gabra-3, which codes
  for the a3 subunit of the GABAA receptor.
• Gabra-3 is a substrate for editing by both ADAR1
  and ADAR2.
• Editing of the Gabra-3 mRNA recodes an isoleucine
  to a methionine.
• The extent of editing is low at birth but
  increases with age, reaching close to 100in the
  adult brain.
• Therefore, editing of the Gabra-3 mRNA is
  important for normal brain development.

65
Method to findnovel ADAR substrates that have
been edited in a site selective manner

• The method is based on extracting intrinsic
  ADAR2RNA substrate complexes by
  coimmunoprecipitation using an anti-ADAR2
  antibody.
• A mouse genome microarray was used to detect
  enriched ADAR2RNA targets.
• In parallel, stemloop structures suitable for
  A-to-I editing in encoded sequences were detected
  by using EvoFold, a general comparative genomics
  program for identifying conserved RNA structures.

66
Subunit-specific sequences of the gabra genes.
The predicted structure of the stemloop within
exon 9 of Gabra-3. The edited A is printed in
bold and circled in gray. The differences between
Gabra-3 and other Gabra sequences are shown in
blue for Gabra-1, red for Gabra-2, and green for
Gabra-5.
67
Editing of the Gabra-3 transcript
(A) The editing of Gabra-3 was demonstrated by
DNA sequencing. The chromatogram of the genomic
DNA sequence shows an adenosine at the I/M-site,
indicated by an arrow. Total brain RNA from the
same adult mouse was reverse transcribed (cDNA)
and sequenced after PCR amplification. A
guanosine was present at the I/M site in the
cDNA. The cDNA from an ADAR2-/- adult mouse was
amplified by PCR and sequenced. A dual A and G
peak appeared with the majority of the
transcripts showing an A at the I/M site. (B)
PCR products from wild-type and ADAR2/ cDNA were
cloned and sequenced..
68
Editing in the a3 subunit of the GABAA
receptorcauses an amino acid change

• The edited site in the a3 subunit of the GABAA
  receptor is located in the transmembrane (TM)
  domain 3. The four TM domains of this receptor
  subunit interact with TM domains from four other
  subunits to form the channel of the receptor.
• It is possible that the isoleucine to methionine
  amino acid change in TM3 of a3 changes the
  environment in the channel, since TM3 and the
  large intracellular loop are important for gating
  and inactivation of the channel.
• A change from the branched isoleucine to
  methionine will alter the side chain to be longer
  with a more bulky sulphur atom.
• Interestingly, frog and pufferfish have a
  genomically encoded methionine at the equivalent
  position

69
Editing of the Gabra-3 transcript extracted from
mouse brain at different developmental stages.
The RT-PCR products from mice at day 2, day 7,
day 12, and adult mouse were cloned and editing
at the I/M site was analyzed by sequence
determination. The number (n) of clones analyzed
at each developmental stage is indicated. Below,
the chromatogram from the sequence at the I/M
site is based on the population derived from the
RT-PCR product.
70
Conclusions

• Gabra-3 is a substrate for editing by both ADAR1
  and ADAR2.
• Editing of the Gabra-3 mRNA recodes an isoleucine
  to a methionine.
• The extent of editing is low at birth but
  increases with age, reaching close to 100in the
  adult brain.
• Therefore, editing of the Gabra-3 mRNA is
  important for normal brain development

71
PNAS 2001, 9814687
In mammals, RNA editing by site-selective
adenosine deamination regulates key functional
properties of neurotransmitter receptors in the
CNS. Glutamate receptor subunit B is nearly 100
edited at one position (the QR-site), which is
essential for normal receptor function. In mouse
models, a slightly reduced rate of QR-site
editing is associated with early onset epilepsy
and premature death. It was found that in
tissues from malignant human brain tumors, this
editing position of glutamate receptor subunit B
is substantially under-edited compared with
control tissues. Alterations in editing and
alternative splicing of serotonin receptor 5-HT2C
transcripts were also seen. These changes
correlate with a decrease in enzymatic activity
of the editing enzyme ADAR 2, as deduced from
analysis of ADAR2 self-editing. This suggests a
role for RNA editing in tumor progression and may
provide a molecular model explaining the
occurrence of epileptic seizures in association
with malignant gliomas.
72
Five adenosines within the coding sequence of the
serotonin 2C receptor (5-HT2C) pre-mRNA are
converted to inosines by RNA editing (named A, B,
C (E),C, and D sites). In human prefrontal cortex
(PFC), the most abundant 5-HT2C mRNA sequences
result from editing at the A site, or from the
editing combinations ACC, ABCD, and ABD. In
suicide victims with a history of major
depression, C site editing is significantly
increased, D site editing is significantly
decreased. Treatment of mice with the
antidepressant drug fluoxetine (Prozac) causes
changes in C and D site editing that are exactly
opposite to those seen in suicide victims. Thus,
one outcome of fluoxetine treatment may be to
reverse the abnormalities in 5-HT2C pre-mRNA
editing seen in depressed suicide victims.
73
Why bother with RNA editing? Why not just change
a nucleotide through mutation? One intrinsic
advantage of editing a nucleotide over having
change hard-wired into the genome through
mutation is the regulation of the degree to which
a coding position is modified within mRNAs.
Certain pre-mRNA editing sites vary greatly in
the frequency with which their editing is
detected in vivo, ranging from a few percent to
nearly100. Thus, editing introduces levels of
expression intermediate to the usual genetic
variation (i.e. 0, 1 or 2 copies), possibly
conferring selective advantage. In addition,
because the known ADAR target mRNAs are commonly
edited at multiple positions independently the
combinatorial effect of editing greatly increases
the number of protein products that can be
generated from an edited gene.
74

• Another difference between editing and normal
  genetic variation is the potential for spatial
  and temporal regulation.
• Because an ADAR enzyme introduces modifications
  into mRNAs, editing is dependent on the amount
  and location of that protein.
• Developmental regulation of pre-mRNA editing
  occurs in organisms as diverse as fruit fly and
  rat. Editing of transcripts increases during the
  course of development.
• In the mammalian brain, RNA editing of GluRs and
  5HT2C is sub-region specific.
• ADARs themselves have different tissue expression
  profiles.