Editing independent effects of ADARs on the miRNA/siRNA pathways.

Abstract

Adenosine deaminases acting on RNA (ADARs) are best known for altering the coding sequences of mRNA through RNA editing, as in the GluR-B Q/R site. ADARs have also been shown to affect RNA interference (RNAi) and microRNA processing by deamination of specific adenosines to inosine. Here, we show that ADAR proteins can affect RNA processing independently of their enzymatic activity. We show that ADAR2 can modulate the processing of mir-376a2 independently of catalytic RNA editing activity. In addition, in a Drosophila assay for RNAi deaminase-inactive ADAR1 inhibits RNAi through the siRNA pathway. These results imply that ADAR1 and ADAR2 have biological functions as RNA-binding proteins that extend beyond editing per se and that even genomically encoded ADARs that are catalytically inactive may have such functions.

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Editing independent effects of ADARs
on the miRNA/siRNA pathways
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Bret SE Heale, Liam P Keegan,
Leeanne McGurk, Gracjan Michlewski,
James Brindle, Chloe M Stanton,
Javier F Caceres and Mary A O’Connell*
MRC Human Genetics Unit, Institute of Genetics and Molecular
Medicine, Western General Hospital, Edinburgh, UK
Adenosine deaminases acting on RNA (ADARs) are best
known for altering the coding sequences of mRNA
through RNA editing, as in the GluR-B Q/R site. ADARs
have also been shown to affect RNA interference (RNAi)
and microRNA processing by deamination of specific ade-
nosines to inosine. Here, we show that ADAR proteins can
affect RNA processing independently of their enzymatic
activity. We show that ADAR2 can modulate the proces-
sing of mir-376a2 independently of catalytic RNA editing
activity. In addition, in a Drosophila assay for RNAi dea-
minase-inactive ADAR1 inhibits RNAi through the siRNA
pathway. These results imply that ADAR1 and ADAR2
have biological functions as RNA-binding proteins that
extend beyond editing per se and that even genomically
encoded ADARs that are catalytically inactive may have
such functions.
The EMBO Journal (2009) 28, 3145–3156. doi:10.1038/
sj.emboj.2009.244; Published online 27 August 2009
Subject Categories: RNA; proteins
Keywords: Drosophila; DSH1; microprocessor; RNA editing;
RNA interference
Introduction
The most common RNA editing event in human mRNA is
deamination of adenosine to inosine. Proteins of the adeno-
sine deaminases acting on RNA (ADAR) family catalyse this
editing event. ADAR activity has been implicated in cancer,
development, innate immunity and RNA interference (RNAi)
(Patterson et al, 1995; George and Samuel, 1999; Beghini
et al, 2000; Maas et al, 2001; Scadden and Smith, 2001;
Hartwig et al, 2004; Luciano et al, 2004; Scadden, 2005;
Scadden and O’Connell, 2005; Yang et al, 2005, 2006; Blow
et al, 2006; Takahashi et al, 2006; Ohman, 2007; Paz et al,
2007; Kawahara et al, 2007a,b; Cenci et al, 2008). In humans,
the two most studied proteins of the ADAR family are ADAR1
and ADAR2. The ADAR1?/?mice are embryonic lethal with
liver disintegration and a deficiency in erythropoiesis (Wang
et al, 2000, 2004; Hartner et al, 2004). ADAR2 has been
shown to be necessary for the editing of the GluR-B transcript
at the critical Q/R site, which limits calcium entry through
AMPA receptors. ADAR2?/?mice die around day p20 with
seizures (Higuchi et al, 2000).
ADAR1 and ADAR2 have similar domain structures
(Figure 1D) but mainly display distinct editing site specifi-
cities—although their editing activities do overlap at some
sites. There are two major isoforms of ADAR1 because of
alternative promoter usage, the longer shuttling p150 isoform
and the shorter nuclear p110 isoform. ADAR1 p150 is upre-
gulated by interferon and can compete with PKR for double-
stranded (ds)RNA. At the amino terminus ADAR1 p150 has
two Z-DNA-binding domains and a nuclear export signal,
which allows it to shuttle in and out of the nucleus, however
it is predominantly found in the cytoplasm. Both isoforms
have three dsRNA-binding domains, the third one overlaps
with a nuclear localization signal (NLS) and the catalytic
deaminase domain is at the carboxy terminus (Strehblow
et al, 2002). ADAR1 is widely expressed throughout the body
with the exception of skeletal muscle. ADAR2, on the other
hand, is most highly expressed in the central nervous system,
has two dsRNA-binding domains and a carboxy-terminal
deaminase domain. It is localized to the nucleus by an NLS
present in the N-terminal region (Desterro et al, 2003; Wong
et al, 2003). To what extent each of the different domains of
ADAR1 and ADAR2 contributes to their biological activities is
an open question. Two other members of the ADAR family in
vertebrates are catalytically inactive, ADAR3 that is brain
specific and TENR that is expressed in the testis (Melcher
et al, 1996; Connolly et al, 2005). Drosophila has a single
Adar gene encoding a deaminase with the same domain
structure as ADAR2. Adar mutant flies are viable with severe
locomotion defects and loss of RNA editing in transcripts
expressed in the CNS (Palladino et al, 2000a, b).
MutationsinhumanADAR1
Dyschromatosis Symmetrica Hereditaria (DSH1) (Tojo et al,
2006), an autosomal dominant hyperpigmentation of the
hands and feet occurring in Chinese and Japanese families.
The majority of DSH1 mutations are associated with single
allele truncations of ADAR1 and the dominant phenotype
seems to be due to haploinsufficiency for ADAR1. The effect
may arise in migrating neural crest cells that develop into
melanocytes in the affected skin areas. The point mutant
ADAR1 G1007R is an unusual example of a DSH1-associated
variant that produces a full-length ADAR1 protein. On the
basis of the known crystal structure of the ADAR2 deaminase
domain (Macbeth et al, 2005) this mutant should not affect
deaminase domain folding and the protein may be capable of
RNA binding. However, the mutation introduces an addi-
are associatedwith
Received: 27 March 2009; accepted: 27 July 2009; published online:
27 August 2009
*Corresponding author. MRC Human Genetics Unit, Institute of Genetics
and Molecular Medicine, Western General Hospital, Crewe Road,
Edinburgh EH4 2XU, UK. Tel.: þ44 131 467 8417;
Fax: þ44 131 467 8456; E-mail: M.O’Connell@hgu.mrc.ac.uk
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tional positively charged arginine residue on the RNA-binding
face of the deaminase domain very close to the active site and
the DSH1 phenotype suggests that the ADAR1 G1007R protein
may be catalytically inactive.
dsRNA is required for both RNA editing by ADARs and
RNAi. Thus, it is not surprising that there have been several
reports of effects of ADARs in RNAi. Most of these studies
have focused on the effect of the editing base change on
processingof dsRNA or edited
(Scadden and Smith, 2001; Luciano et al, 2004; Scadden,
2005; Scadden and O’Connell, 2005; Yang et al, 2005, 2006;
Blow et al, 2006; Ohman, 2007; Kawahara et al, 2007a,b).
More recent studies focused on the downstream effects on
target selection by edited miRNAs. For example, it was
recently shown that an miRNA predicted to target the
PRPS1 transcript is edited by ADAR2 and, interestingly, levels
of PRPS1 protein are elevated in ADAR2-deficient mice
(Kawahara et al, 2007b). This result is the only demonstration
so far that miRNA modulation of protein levels is altered by
ADARs.
There are several key steps in the miRNA pathway, which
could be affected by ADARs (for review see Tomari and
Zamore, 2005; Valencia-Sanchez et al, 2006). First, primary
transcripts (pri-miRNA) are processed in the nucleus by the
microprocessor complex, which contains Drosha and its
partner DGCR8, to pre-miRNA hairpins (Gregory et al,
2004). Nuclear ADARs could therefore affect this step through
steric hindrance of Drosha/DGCR8 binding or by inhibiting
Drosha action through the alteration at the edited base in the
primary miRNA transcript. As pre-miRNA hairpins can be
bound by ADARs it is possible that their export from the
nucleus, which is mediated by Exportin 5 (Yi et al, 2003), can
be disrupted by binding. The next step in miRNA processing
is the Dicer complex-mediated cleavage of the pre-miRNA
hairpin into a 21–23 nucleotide RNA duplex in the cytoplasm.
Cytoplasmic ADARs could also influence this step through
sequestering the pre-miRNA or editing the pre-miRNA.
Ultimately, the mature miRNA guide strand guides RISC to
mRNAs that have complementarity with the guide strand,
resulting in translation inhibition or mRNA degradation.
ADAR antagonism of miRNA or siRNA production should
result in alteration in the functional activity elicited by
miRNAs or siRNAs.
In this work, we sought to determine which properties of
ADARs are responsible for influencing miRNA function.
Specifically, we have examined the ability of several trunca-
tion and point mutations of ADAR1 and ADAR2 to alter the
function of mir-376a2 in a cell culture system optimized for
analysis of the activity of a single miRNA. Our results indicate
that editing can result in retargeting of human mir-376a2 as
microRNAprecursors
Figure 1 Editing levels in the human mir-376 cluster. (A) Diagram
of the mir-376 cluster; nomenclature is from miRBASE http://
microrna.sanger.ac.uk/sequences. Numbers at bottom indicate in-
tervening lengths of sequences. Bold text regions are the mature
microRNAs. Boxes indicate major sites of editing. There is no
evidence ofediting inmir-654.
expressed from a complete mir-376 cluster. (C) Editing in mir-
376a2. Ratio G/(AþG) indicates the ratio of the guanosine signal
to the total (adenosineþguanosine) chromatogram signal. þ4 and
þ44 are positions within mir-376a2 indicated in A. (D) ADAR1 and
ADAR2 protein domains showing the positions and features used in
the construction of the hADAR expression vectors. hADAR1 p150
contains two Z-DNA-binding domains, three dsRBDs and one
deaminase domain. hADAR1 p110 begins at amino-acid position
296 of hADAR1 p150. hADAR2 has two dsRBDs and one deaminase
domain. hADAR2 DN is a deletion mutant of hADAR2 with amino
acids from positions 4–72 removed. A key glutamate residue in the
deaminase domain is amino acid 912 for ADAR1 and 319 for
ADAR2. Mutation of this residue to alanine renders the ADAR
catalytically inactive.
(B) Editing inmir-376a1
ADARs and RNAi
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shown earlier for murine mir-376 (Kawahara et al, 2007b).
Interestingly, our results also show that even in the absence
of editing, ADARs can affect the processing of miRNAs at the
Drosha cleavage level step, thereby altering the functional
activity of miRNAs. This suggests that ADARs may influence
a larger set of miRNAs than would be predicted from the
numbers that are known to be edited. We have also examined
the ability of the human ADARs to alter silencing of the
whiteþeye colour gene in Drosophila by a 300plus base-pair
hairpin, which is processed into siRNA duplexes (Vagin et al,
2006). We again find that a deaminase-inactive ADAR
can antagonize siRNA activity against whiteþ. Therefore,
RNA binding by itself is an important function of ADAR
biology.
Results
Editing of the mir-376 cluster by ADAR isoforms shows
asymmetric editing in pri-mir-376a2
The human mir-376 cluster comprises several miRNA hair-
pins encoding mir-376c, mir-376a2, mir-654, mir-376b and
mir-376a1 and others (Figure 1A). It was reported earlier that
in human medulla several of the adenosines in this cluster are
edited (Kawahara et al, 2007b). To elucidate whether parti-
cular domains of human ADAR1 or ADAR2 have prominent
roles in the effect of ADARs on miRNA function, we cotrans-
fected HEK 293T cells with constructs that express the mir-
376 cluster and with constructs that express individual
human ADAR1 or ADAR2 isoforms (Figure 1D summarizes
key features of ADAR1 and ADAR2). All mutant proteins were
expressed to the same level as the respective wild-type
protein, as detected on immunoblots probed with anti-FLAG
monoclonal antibody (data not shown). HEK 293T cells do
not express detectable levels of the endogenous cluster. RNA
editing levels were examined initially, before endeavouring to
examine the effects of these ADAR variants on precursor
processing and effects on downstream microRNA targets.
RNA was extracted from transfected cells, reverse tran-
scribed with random hexamers and cDNA corresponding to
individual pri-mir-376 hairpins were amplified using primers
located around a 100 nucleotides before and after each hair-
pin within the long intervening sequences between the hair-
pins. RNA editing levels at sites within the pri-mir-376
hairpins were measured from mixed sequence chromato-
grams on the amplified sequence pools for each pri-mir-376
hairpin. We found that sites in pri-mir-376 hairpins were
highly edited, as reported earlier for human medulla
(Kawahara et al, 2007b).
The different specificities of ADAR1 and ADAR2 account
for the different patterns of editing in the pri-mir-376a1
(Figure 1B), -376a2(Figure
(Kawahara et al, 2007b). For instance, ADAR2 edits pri-mir-
376a1 at the þ4 and þ44 site more efficiently than ADAR1
(Figure 1B). In contrast, the two naturally occurring isoforms
of ADAR1, p150 and p110, showed similar efficiencies at a
range of sites within pri-miR-376a1, with the nuclear ADAR1
p110 editing slightly more efficiently in all cases (Figure 1B).
The larger isoform has an extended amino terminus that
encodes Z-DNA-binding domains that can bind to dsRNA
(Oh et al, 2002; Koeris et al, 2005; Placido et al, 2007).
The editing sites that showed the strongest preferences for
editing by a particular ADAR occurred in pri-mir-376a2,
1C)and -376bhairpins
where hADAR2 preferentially edited the þ4 site of the
mature 50miRNA with 80–90% efficiency. ADAR1 edits this
site with 40% efficiency (Figure 1C). By contrast, hADAR1
edits the þ44 site within the mature 30miRNA with 80–90%
efficiency whereas ADAR2 edits this site with only 10%
efficiency. This is in contrast with the editing of miR-376a1
at the þ44 site as described above (Figure 1B). RNA editing
sites in other microRNA precursors within the cluster show
less specificity for ADARs, the þ44 site of pri-mir-376b was
edited by both hADAR1 and hADAR2 (data not shown). As
observed earlier in human medulla RNA, mir-654 was not
edited in HEK 293T cells cotransfected with ADAR.
As anticipated, cotransfection of the ADAR1 1-296 con-
struct that expresses only the first 296 residues, containing
the first Z-DNA-binding domain of ADAR or the ADAR11-442
construct containing both Z-DNA-binding domains did not
increase RNA editing. On the other hand, cotransfection with
the ADAR1 443-end construct that expresses a protein lacking
both Z DNA-binding-domains gave levels of RNA editing
similar to intact ADAR1 p110.
To determine whether editing activity is critical for
effects of ADARs on microRNA processing and/or function
we tested the effects of two ADAR1 mutant proteins.
First, a construct with a glutamate to alanine change in an
essential catalytic site residue (ADAR1 E912A) was shown to
cause only background levels of RNA editing similar to
that seen with cotransfection of the control pcDNA3.1 con-
struct. We also cotransfected a construct expressing the
naturally occurring G1007R mutation in ADAR1 that has
been linked to DSH1 with the construct expressing the
mir-376 cluster and found that this protein is incapable of
editing any sites in the microRNA precursors. This protein,
expressed in and purified from the yeast Pichia pastoris
binds long dsRNA similarly to wild-type ADAR1 as assayed
by filter binding but does not deaminate long dsRNA
(Supplementary Figure S1). ADAR2 DN is a deletion of
ADAR2 amino acids 4–72 that removes the nuclear localiza-
tion sequence and causes active ADAR2 to accumulate in the
cytoplasm (Wong et al, 2003). Surprisingly, the ADAR2 DN
isoform editedthe pri-miRNA,
found only in the nucleus (Figure 1B and C). The observed
editing by the cytoplasmic ADAR2 DN is due to editing of the
pri-miRNA transcripts that escaped to the cytoplasm without
being cleaved by the Drosha complex in the nucleus
(Supplementary Figure S2).
which isnormally
ADAR2 shows strong inhibition of miRNA activity
As the RNA editing sites in mir-376a2 show the highest levels
of preference for editing by ADAR1 or ADAR2 within
the entire miR-376 cluster, we generated a construct that
expresses pri-mir-376a2 alone. This allows the effects of
ADAR2 binding and editing at the þ4 site and ADAR1
binding and editing at the þ44 site to be studied in the
absence of other microRNAs that have strong sequence
similarities. We verified that the editing of mir-376a2 alone
was the same as editing of mir-376a2 expressed in the intact
cluster (Supplementary Figure S3A).
Processing of pre-mir-376a2 by the miRNA processing
machinery produces mature 50and 30microRNAs. To deter-
mine how the production and the specific targeting of the
mature microRNAs is affected by the presence of ADARs we
measured RNA-silencing activities with two sets of luciferase
ADARs and RNAi
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constructs containing full-length microRNA target sites in the
30UTR regions of the luciferase transcripts (Supplementary
Figure S3B). The 50miR luciferase reporter and the 30miR
luciferase reporters measure the luciferase-silencing activity
of the corresponding wild-type mature unedited microRNAs.
The use of target sites with perfect homology extending
beyond the seed match may lead to site-directed cleavage of
the target transcript as well as translational inhibition. When
ADARs edit within miRNAs they have the potential of retar-
geting miRNAs to another transcript. The RNA-silencing
activities of the mature microRNAs retargeted as a result
of editing are measured using a second pair of luciferase
reporters with single base changes in the target sites
(Supplementary Figure S3B).
We found that mir-376a2 expressed and processed in the
absence of cotransfected ADAR constructs generated substan-
tial silencing activities corresponding to the mature, unedited
50and 30miRs, as expected, (Supplementary Figure S3B),
reducing luciferase levels 10-fold to approximately 0.1 of the
value obtained in the absence of the miRNA. We optimized
the level of mir-376a2-expressing construct so that it was
sufficient to obtain this maximal level of silencing for both
microRNAs.
Expression of the mir-376a2 construct in the absence of
ADARs produced much less silencing activity directed against
the reporters for the edited microRNAs, reducing the lucifer-
ase levels by only approximately two-fold, to approximately
0.5 of the value obtained in the absence of the miRNA
(Supplementary Figure S3B). This five-fold discrimination
by the mature unedited microRNAs between the unedited
and the edited target sites is highly specific, considering that
the edited target sites in each case differ by only 1 nucleotide
in the 22 base sequences.
Having established conditions to maximize silencing activ-
ities of mature miRNAs from the transfected pri-mir-376a2
construct and to determine their target site preferences we
cotransfected the mir-376a2 reporter with constructs expres-
sing ADARs and ADAR mutations to assess how ADARs affect
the silencing activities of resulting miRNAs. Consistent with
their editing of pri-mir-376a2 at position þ44, both hADAR1
p110 and hADAR1 p150 increased the silencing activity
corresponding to the edited form of the 30miR (Figure 2B).
This change was largely editing dependent, as the deaminase
mutant ADARs (ADAR p150 E912A and ADAR p110 E912A)
did not have this effect (Figure 2B); the effect was not
dependent on the Z-DNA-binding domains of ADAR1
(ADAR1 443-end). Further, the Z-DNA-binding domains of
ADAR1 (ADAR1 1-442) were not sufficient on their own to
significantly increase the silencing activity of edited 30
miRNA.
Notably, it was ADAR2 that produced the most striking
inhibitory effects on miRNA function, particularly on the
activity of the unedited miRNAs (Figure 2A and C). ADAR2
edits the pri-mir-376a2 at position þ4 and thereby increases
the silencing activity of edited 50miR (Figure 2D), at the
expense of unedited 50mir activity (Figure 2C). As ADAR2
edits the þ4 site in pri-mir-376a2 with 90% efficiency
it is surprising that the retargeting effect was not more
complete. Silencing of the reporters for edited microRNAs
was maximized by cotransfecting equivalent amounts of
mir-376a2 constructs bearing A–G point mutations at
the RNA editing sites so that the mature miRNAs were
‘pre-edited’
Cotransfecting a pri-mir-376a2 construct pre-edited at the
þ4 site gives a silencing activity corresponding to the edited
50miRNA that was almost twice as great as seen with ADAR2
(Figure 2D). A possible explanation is that ADAR2 edits
pri-mir-376a2 very efficiently but that it also slows Drosha
processing of the pri-mir-376a2 so that less mature miRNA is
produced.
The hypothesis that ADAR2 inhibits Drosha processing of
pri-mir-376a2 is supported, first, by the fact that ADAR2
inhibits the activity of the 30miRNA even though it edits
the þ44 site within this miRNA with only 10% efficiency.
Second, the cytoplasmic protein ADAR2 DN has no effect
either on activity or on retargeting of the 30micro RNA
(Figure 2A and B). This is consistent with a nuclear inhibition
of Drosha being responsible for the ADAR2-mediated inhibi-
tion of 30miR activity.
ADAR2 DN did affect the activity of the 50miRNA and
increased the activity of the edited form. This appears to be
due to base deamination and it does not suggest further
cytoplasmic influence of ADAR2 on cytoplasmic processing
steps. ADAR2 DN, which is cytoplasmic, edits the Drosha
product, the pre-miRNA, after it is exported to the cytoplasm
(Figure 2 and Supplementary Figure S2).
to mimicthe inosine-containingproduct.
Drosha processing of pri-mir-376a2 is inhibited by
ADAR2 protein and not by the RNA editing base
changes
As mentioned earlier ADAR interactions with miRNAs can
occur at all levels of the miRNA processing pathway. We
examined the processing of pri-mir-376a2 in transfected cells
by northern analysis (Figure 3A and B; Supplementary Figure
S4). ADARs were expressed in HEK293Tcells in the presence
of the mir-376a2 expression construct and the appropriate
reporter construct. Only ADAR2 caused a significant reduc-
tion in the mature B22-nucleotide miRNA and in the B64-
nucleotide pre-miRNA, with a reduction of over 65% in both
cases. This indicates that ADAR2 activity on the pri-mir-
376a2 hairpin disrupts Drosha processing in the nucleus. In
further support of this hypothesis, ADAR2 DN, a cytoplasmi-
cally localized mutant of ADAR2, did not cause a significant
reduction in the levels of either the pre-miRNA or the mature
miRNA.
By performing in vitro pri-miRNA processing assays with
HeLa nuclear extract we found that the addition of purified
ADAR2 specifically inhibited Drosha processing of pri-mir-
376a2 but not pri-let-7a-1 (Figure 3C), though some pre-mir-
376a2 is still produced. To determine whether Drosha clea-
vage in the HeLa nuclear extract is inhibited by the RNA
editing base changes or by binding of ADAR2, in vitro
transcribed mutant pri-mir-376a2
adenosine to guanosine changes at the edited positions
were assayed. Specifically, in mir-376a2 a137g, a guanosine
replaced adenosine at the þ4 position and in mir-376a2
a177g, a guanosine replaced adenosine at the þ44 site that
is the þ6 position of the 30miR. Importantly, Drosha
cleavage was not affected by either mutation (Figure 3D),
clearly showing that the nucleotide changes mediated by
editing are not themselves sufficient to inhibit the processing
of mir-376a2. The c188t construct that removes the A-C
mismatch at the þ4 site also had no effect.
transcripts bearing
ADARs and RNAi
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Catalytically inactive ADAR2 disrupts pri-mir-376a2
processing
To confirm that inhibition of Drosha cleavage is due to
ADAR2 binding rather than because of the base change at
the edited site an active site-mutant ADAR2 E319A was
co-expressed. ADAR2 E319A retained the ability to reduce
the silencing activity of unedited 50miR but, as expected, it
was unable to retarget the silencing activity (Figure 4A).
Furthermore, ADAR2 E319A caused inhibition of the proces-
sing of pri-miRNA-376a2, as detected on small RNA north-
erns with RNA from cotransfected cells (Figure 4B). ADAR2
E319A disruption of Drosha processing verifies the conclu-
sion that ADAR2 interference with miRNA processing is
independent of editing. ADAR2 DN E319A, a deaminase
domain mutant localized to the cytoplasm, did not inhibit
30miR activity and only weakly affected 50miR activity
(Figure 4A). This shows further that it is nuclear Drosha
cleavage that is affected and not cytoplasmic Dicer activity.
Another pri-miRNA within the cluster is edited by ADAR2.
We wanted to determine whether binding of ADAR2 to pri-
mir-376a1 also affected processing. Similar to pri-mir-376a2,
expression of pri-mir-376a1 alone showed that its activity
was inhibited by ADAR2 E319A (Figure 4C). In addition, a
northern analysis was performed to determine the processing
of pri-mir-376a1 (Figure 4D). It was affected by the binding of
either active or inactive ADAR2 but not by ADAR1
(Figure 4D). This shows that inhibition of Drosha cleavage
by the ADAR2 deaminase-inactive mutant is not restricted to
pri-mir-376a2.
Human ADAR1 protein expressed in Drosophila inhibits
RNAi through a mechanism that is partially
independent of RNA editing activity
We generated a series of Drosophila strains that express
different human ADAR proteins using the GAL4/UAS binary
Figure 2 Effects of ADARs on functional activities of mature miRNAs processed from pri-mir-376a2. (A) Inhibition of 30miRNA activity.
A value more than 1 means that the miRNA is less active when the ADAR is present than when the pri-mir-376a2 is expressed alone.
(B) Retargeting of 30miRNA activity. A value less than 1 means that the miRNA is more active in the presence of the ADAR than when the
pri-mir-376a2 is expressed alone. Pre-edited mir-376a2 (a177g) indicates a construct expressing pri-mir-376a2 with the þ44 site (position 177)
mutated from an adenosine to a guanosine. (C) Inhibition of 50miRNA activity. (D) Retargeting of 50miRNA activity. The activity for pre-edited
mir-376a2 (a137g) is from a construct expressing pri-mir-376a2 with the þ4 site (position 137) mutated from an adenosine to a guanosine.
ADARs and RNAi
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expression system. Interactions between expressed human
ADARs and the RNAi system were examined by crossing
these UAS-ADAR-bearing strains to a fly strain in which
silencing of the wild-type whiteþgene in the eye by a
cytoplasmic white RNA hairpin construct is also directed by
the GAL4/UAS binary expression system.
Silencing of the whiteþgene in the eye is directed by
expressing a UAS-white hairpin construct under the control of
the eye-specific GMR-GAL4 driver (Kalidas and Smith, 2002).
Silencing of whiteþresults in flies with white eyes and this
has been used successfully in a screen to find genes that
antagonize the siRNA pathway (Lee and Carthew, 2003). The
Figure 3 Effects of ADARs on processing of pri-mir-376a2. (A, B) Effects of ADARs on processing in HEK 293Tcells. Small RNA northern on
RNA from cotransfected HEK 293T cells probed with antisense probes to (A) the 30miRNA and (B) the 50miRNA. Nothern blots were also
probed with a 5S rRNA-specific probe as a loading control (data not shown). ADAR2 expression reduced both the pre-miRNA and the mature
30and 50miRNA signals. The numbers below the blot indicate the amount of both pre-mir-376a2 or mature 376a2 signal in each lane relative
to that in pri-mir-376a2 expressed alone. (C) Recombinant ADAR2 inhibits Drosha cleavage in HeLa extracts in vitro. Drosha cleavage assays
performed in the presence of recombinant ADAR2 and either pri-let-7a-1 or pri-mir-376a2. A double plus in the ADAR2 table indicates that a
higher amount of ADAR2 was added. Cleavage of pri-mir-376a2 but not pri-let-7a-1 was inhibited by ADAR2, indicating that ADAR2 directly
and specifically effects Drosha processing of mir-376a2. The percentage of pre-miRNA and mature 376a2 signal relative to cleavage in the
absence of ADAR2 is indicated below the blot. (D) Lack of effect of RNA editing base changes in pri-mir-376a2 on Drosha cleavage in HeLa
extracts in vitro. Drosha cleavage was not impaired by point mutations mimicking RNA editing events in mir-376a2. a137g mimics editing of
the þ4 site. a177g mimics editing of the þ44 site. c188t closes the A–C mismatch at the þ4 site in pri-mir-376a2. The amount of pre-miRNA
signal relative to wild-type pri-mir-376a2 cleavage is indicated below the blot.
ADARs and RNAi
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white hairpin primary transcript contains an intron and has
been designed to be efficiently processed, polyadenylated and
exported to the cytoplasm. Silencing is mediated by the Dicer
processing of the resulting cytoplasmic RNA hairpin, which is
over 300-nucleotides long (Vagin et al, 2006). After Dicer
cleavage, the white hairpin-derived siRNA targets the whiteþ
mRNA for silencing by RISC.
When strains containing UAS-ADAR are crossed with the
silencing reporter strain (homozygous wþ; GMR4wIR),
ADAR is co-expressed with the white hairpin in the eye.
The interaction of ADAR with RNAi is observed as an
alteration in the silenced white eye colour. The strongest
effect of a human ADAR on white hairpin-mediated silencing
was caused by ADAR1 p150, the shuttling isoform of ADAR1
that accumulates in cytoplasm (Figure 5C). This ADAR1
isoform antagonized RNAi strongly to restore the eye colour
to red by allowing expression of the intact wild-type whiteþ
gene in the strain background. This effect on the eye colour
was not variegated but was absolutely consistent across the
eyes and between flies.
Figure 4 Deaminase-inactive ADAR2 fails to retarget miRNAs but still inhibits their processing. (A) Top panel: effects of normal versus
deaminase mutant ADAR2 on production of mature unedited and edited miRNA activities. Inactive ADAR2 E319A retained the inhibitory effect
of ADAR2 on the unedited 50miRNA activity but was unable to retarget the activity by editing. Bottom panel: effects of cytoplasmic ADAR2 DN
and ADAR2 DN E319A on the activity of mir-376a2. ADAR2 DN E319A did not affect the 50reporter, indicating a loss in retargeting. Neither
hADAR2 DN nor hADAR2 DN deaminase mutant affected the 30mature miRNA activity. (B) Effect of ADAR2 E319A on processing of mir-376a2
in cotransfected HEK 293Tcells. Small RNA northerns of RNA from cotransfected cells probed with antisense probes to the 30miRNA. Blots
were also probed with a 5S rRNA-specific probe as a loading control (data not shown). Deaminase mutant ADAR2 E319A expression reduced
both pre-miRNA and mature miRNA levels. Percentage of pre-miRNA signal relative to pri-mir-376a2 expressed alone is noted below the blot.
(C) Effects of main isoforms of ADAR1 and ADAR2 on functional activities of 50and 30miRNAs expressed from a construct bearing mir-376a1
alone. As in the case of mir-376a2, ADAR2 was able to inhibit the activity of mir-376a1 independently of deaminase activity. (D) The ADAR2
E319A deaminase domain mutant reduced the level of pre-mir-376a1 in cotransfected HEK 293Tcells. Small RNA northern blot of RNA from
transfected cells probed with antisense probe to the 30miRNA. The percentage of pre-mir-376a1 signal relative to pri-mir-376a1 expressed alone
is listed below the blot.
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Neither nuclear ADAR1 p110 nor ADAR2 had any effect on
white hairpin-mediated silencing (Figure 5B and D). The
human ADARs are expressed in addition to the normal level
of endogenous Drosophila ADAR in the flies shown here.
Drosophila ADAR, which is also nuclear, behaved like ADAR2
in this assay: neither increasing the expression with a
UAS-dADAR construct nor deleting the single endogenous
Drosophila ADAR gene on the X chromosome affected silen-
cing (data not shown). When editing of the white hairpin over
a stretch of several hundred base pairs was analysed in these
flies there was some editing even in the absence of any
human ADAR that is presumably because of the endogenous
fly protein (Supplementary Figure S5A). Of the human pro-
teins only the cytoplasmic ADAR1 p150 showed a sub-
stantially increased level of editing. This suggests that
cytoplasmic localization is required for efficient editing and
for full inhibition of siRNA activity in this assay.
Combining UAS-Dicer-2 with UAS-ADAR1 p150 in crosses
to the homozygous wþ; GMR4wIR strain showed that
increasing DICER-2 expression above normal levels increases
the background level of RNAi in the absence of ADAR1p150
in the progeny (Figure 5, compare panels E and A) and also
partly but not completely suppresses the antagonism of RNAi
by ADAR1 (Figure 5, compare panels C and F). This suggests
that the antagonism of RNAi by ADAR1p150 is partly attri-
butable to competition with DICER-2 for long dsRNA,
although ADAR1p150 does also bind the shorter dsRNA
products of DICER cleavage (Yang et al, 2005) and changing
dosages of R2D2 and ARGONAUTE-2 might have similar
effects.
The visual assay is very consistent, especially because
groups of ADAR-expressing flies can be compared with sib-
ling progeny of the same age, which have white silencing but
lack ADAR. This gives sensitivity to the assay and testing
further ADAR1 derivatives shows that all the domains of
ADAR1, including the Z-DNA-binding domains, have percep-
tible effects on RNAi in this assay. The truncation mutant,
ADAR1 1-442, containing two Z-DNA-binding domains
showed an effect in this assay and removing the Z-DNA-
binding domains in the ADAR1 443-end construct weakened
the inhibition (data not shown).
When human ADAR1 p150EA, the deaminase-inactive
mutant, was expressed in this system it was found to still
antagonize the RNAi activity of the white hairpin (Figure 5G
and H). Again the effect was very consistent between flies.
The red eye colour was 40% as strong as ADAR1 p150, based
on quantization of red pigment in ethanol-extracted heads
and in digital images of eyes (Supplementary Figure S5B).
This result suggests that, as in the case of miRNA processing,
a significant part of the antagonism is independent of dea-
minase activity. Very surprisingly, expressing the ADAR2 DN
protein that lacks the nuclear localization signal increased the
antagonism by ADAR2 only very slightly (not shown). This
suggests that the specificity of the different ADARs is also
important for this effect.
Discussion
Earlier work on the interaction between ADARs and the
miRNA/siRNA pathways in mammals has focused on the
effects of the base changes introduced by RNA editing. This
editing was shown to affect microRNA processing or produce
retargeting (Scadden and Smith, 2001; Luciano et al, 2004;
Scadden, 2005; Yang et al, 2005, 2006; Blow et al, 2006;
Landgraf et al, 2007; Kawahara et al, 2007a,b). Our data
show, for the first time, that catalytically inactive ADAR2 can
inhibit the processing and decrease the downstream function
of miRNA, independent of base deamination. In addition, we
show that deaminase-inactive ADAR1 p150 inhibits siRNA
activity in a Drosophila test system. ADAR1 p150 also inhibits
siRNA in ADAR1?/?MEFs (Yang et al, 2005). As ADARs may
bind to a wider range of pre-miRNAs than they successfully
edit, the influence of ADARs on miRNA function may also
Figure 5 Human
RNA interference in Drosophila. Effects of human ADAR proteins
on silencing of whiteþgene expression in Drosophila eyes by
a co-expressed cytoplasmic white RNA hairpin. These flies are
female progeny of homozygous wþ; GMR-GAL4, UAS-whiteIR
(GMR4wIR) crossed to w1118; UAS construct lines. (A) wþ;
GMR4wIR alone, (B) ADAR p110 (line 19 on Chr III), (C) ADAR
p150 (line 25 on Chr III), (D) ADAR2 (line 22 on Chr II), (E) Dicer-2
(line D2 on Chr II), (F) Dicer-2 (line D2) and ADAR1 p150 (line 25),
(G) ADAR1 p150 E319A (line 34 on Chr III), (H) ADAR1 p150 E319A
(line 35 on Chr II).
ADAR1 antagonizes whitehairpin-mediated
ADARs and RNAi
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extend beyond the subset of miRNA, which are edited.
Modulation of miRNA biogenesis by binding Lin-28 protein
(Newman et al, 2008; Piskounova et al, 2008) or hnRNP A1
(Guil and Caceres, 2007; Michlewski et al, 2008) to miRNA
precursors has also been reported recently. Therefore, regula-
tion of pri-miRNA processing by accessory RNA-binding
proteins may be more common than appreciated earlier.
There is specificity in how different ADARs affect different
RNAi phenomena as only ADAR2 robustly affects the proces-
sing of pri-mir-376a2. Despite overexpressing other ADAR
proteins with dsRNA-binding domains, they did not radically
inhibit processing of this pri-miRNA by Drosha. What is
really surprising is that ADAR1 binds to and edits the þ4
position in mir-376a2 to between 40 and 50% yet its affect on
Drosha processing is not as strong as ADAR2 (Figure 3). The
þ4 editing site is a mere three base pairs from the Drosha
cleavage site.
The specificity of ADAR effects is also clear in relation to
Dicer processing. It was recently shown that pre-mir-151
could be edited by ADAR1 p110 and ADAR p150 in vitro,
and that this editing led to disruption of Dicer processing
(Kawahara et al, 2007a). However, cytoplasmic ADAR2 DN
did not result in disruption of Dicer processing of the pre-mir-
376a2 in this study. Neither was there degradation of miRNA
duplexes by Tudor-SN but instead there was retargeting of 50
miRNA activity by ADAR2 DN because of editing. In addition,
ADAR1 p150 did not have a major impact on mir-376a2
processing. Therefore, not all editing events in pre-miRNA
in the cytoplasm necessarily disrupt Dicer processing just as
not all editing events in pri-miRNA in the nucleus affect
Drosha cleavage.
We found that single nucleotide changes in miRNA target
sites impair the ability of a particular miRNA to target the
mRNA. Several publications have documented the decrease
in the ability of miRNA to silence an mRNA when there are
nucleotide changes in the miRNA responsive site (reviewed
in Tomari and Zamore, 2005). It seems that siRNA activity is
even more sensitive to nucleotide changes. Therefore, editing
may have a more dramatic effect on silencing of a transcript if
it occurs within a miRNA responsive site in the 30UTRs of
specific mRNAs rather than if the miRNA itself is edited.
This may be important in some species like Caenorhabditis
elegans where there is significant editing of 30UTRs.
We have shown here that one DSH1-associated variant of
ADAR1 (Miyamura et al, 2003; Zhang et al, 2004, 2008; Liu
et al, 2006) produces an ADAR1 protein that binds dsRNA
with a similar Kdto wild type but is catalytically inactive
(Supplementary Figure S1). Members of the family having
this variant show the same dermatological symptoms of
DSH1, which are associated with protein truncations of
ADAR1 and that probably reflect decreased RNA editing
activity in other families. However, affected members of the
Japanese family having the ADAR1 G1009R allele also show
additional symptoms including progressive neuromotor de-
fects and calcium accumulation in basal ganglia. On the basis
of our demonstration of efficient RNA binding by this protein
we suggest that these additional symptoms arise because
ADAR1 G1007R partially antagonizes normal RNA editing.
Inhibition of mir-376 cluster processing in the brain may be
relevant but the mutant protein would also probably bind
RNA editing target sites such as the Q/R editing site in the
GluR-B transcript and reduce editing by ADAR2 and ADAR1 at
this site. This would lead to calcium-permeable AMPA recep-
tors and possibly contribute to the brain calcium accumula-
tions and to the slowly progressing neurological symptoms in
this family.
In conclusion, we report a number of different and very
selective effects of ADARs on RNAi processes that appear to
depend significantly on the RNA-binding activities of ADARs.
It has been shown earlier that ADARs can bind to many
transcripts without editing them (Klaue et al, 2003). A
biological function can now be ascribed to this binding
activity. Binding of ADAR2 to pri-mir-376a2 is sufficient to
inhibit processing by Drosha, an inactive form of ADAR1
p150 is sufficient to inhibit RNAi in a Drosophila assay, and
disease symptoms in a Japanese family are probably ex-
plained by production of an ADAR1 protein that binds RNA
but lacks catalytic activity. This implies a broader functional
role for ADARs as RNA-binding proteins in different cellular
compartments interacting with a range of dsRNA-based
phenomena.
Materials and methods
Expression plasmids
miRNA-expressing plasmids contained mir-376 cluster genomic
DNAwere amplified with PCR primers and cloned using BamHI and
HindIII restriction enzyme sites in the primers into the pcDNA3. 1
vector. All constructs had 76–100 nucleotides of genomic sequence
on both the 50and 30sides of the encoded miRs depicted in
Figure 1A (see Supplementary Table 1 for sequences of primers).
Site-directed mutagenesis was performed to incorporate the a137g,
a177g and c188t mutations into pri-mir-376a2.
Human ADAR-expressing plasmids, containing ADAR coding
sequences with N-terminal Flag and C-terminal His epitope tags,
were constructed by Gateway Cloning (Invitrogen) with pc3D as the
destination vector. pc3D was derived by blunt-end ligation of the
gateway cloning cassette-B from Invitrogen into pcDNA3.1. There-
fore, the vector for all expression constructs was pcDNA3.1.
miRNA reporter plasmids
Target sites for each miRNA were subcloned into the 30UTR of
Renilla luciferase in psicheck 2.0 (Promega) using the XhoI and
NotI restriction enzyme sites. As a transfection control the vector
also contains humanized Firefly luciferase under its own promoter.
This is also a control for any general effects on translation. (see
Supplementary Figure 3 for sequences of target sites).
Editing assays
RNA was purified from transiently transfected cell cultures and
subjected to reverse transcription with random hexamers (Invitro-
gen) and MMLV-RnaseH (Invitrogen). Editing was determined by
direct sequencing of PCR product pools with primers located
substantially downstream and upstream of the relevant mir-hairpin,
as performed by Blow et al (2006) and Kawahara et al (2007b).
Primers used were originally described in Kawahara et al (2007b)
and their sequences are listed in Supplementary Table 1. The
primers are designed to amplify a sequence from the pri-miRNA
before Drosha cleavage. Our notation in Figure 1 for the þ4 and
þ44 site follows Kawahara et al, although new information in
miRBASE suggests that the Drosha cleavage site is one base closer
to the editing site.
In vitro RNA binding and editing
In vitro RNA-binding assay was performed as described by Ohman
(Ohman et al, 2000). In vitro RNA editing assays was performed as
described by O’Connell (O’Connell and Keller, 1994).
Cell culture
HEK 293T cells were cultured in DMEM supplemented with
10% FCS.
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Transient transfections
Transient transfections were performed in 24-well plates seeded 1
day before transfection with 0.14?106cells, with 1.005mg DNA and
1.01ml Lipofectamine 2000. The amounts of miRNA-expressing
plasmid, ADAR-expressing plasmid and luciferase reporter target
construct plasmid were 500, 500 and 5ng, respectively. Cells were
collected on the third day after the transfection.
Luciferase assays
The miRNA target plasmid, derived from psicheck 2.0, contains
humanized Firefly and Renilla luciferase. The miRNA responsive
site is in the 30UTR of Renilla luciferase. Assays for Firefly and
Renilla luciferase were performed with the Dual-Glo luciferase kit
from Promega. Luciferase was measured on a Perkin-Elmer
Victorlite. To ascertain the activity of mir-376a2, pri-mir-376a2
was co-expressed with the luciferase reporters so that the amount of
DNA was constant. The resulting luciferase level was compared to
that obtained in a cotransfection of pcDNA3.1 and the luciferase
reporter without the pri-mir-376a2 expression vector. Fold Luc
activity is the activity remaining in the presence of the miRNA.
Fold Luc¼(luciferase activity with the pri-mir-376a2 construct)/
(luciferase activity without the pri-mir-376a2 construct).
Assays on ADARs were performed by co-expressing the ADAR,
pri-mir-376a2 and the appropriate luciferase reporter. The resulting
luciferase level was compared to co-expression of ADAR, pcDNA3.1
and the appropriate reporter without pri-mir-376a2. For the ADAR
constructs.
Fold Luc¼(luciferase level with pri-mir-376a2 and ADAR
construct)/(luciferase level without pri-mir-376a2 and with the
relevant ADAR construct).
The activity was then normalized to the ratio obtained with
pcDNA3.1 and the pri-miRNA expression construct alone. Fold Luc
over pri-mir-376a2 alone (labelled on the y-axis in Figure 2) is the
fold change in luciferase activity compared with expression of the
pri-miRNA alone. This indicates how the ability of the microRNAs
to direct silencing of luciferase activity from the different reporters
has been affected by the presence of ADAR proteins. Avalue greater
than 1 indicates inhibition of the miRNA activity, a value less than 1
indicates increased miRNA activity.
Small RNA northerns
RNA samples were electrophoresed on a denaturing 12% PAGE 8M
urea gel. The RNA was then transferred to a Nylonþmembrane
(Amersham) and hybridized with the appropriate probe. Radio-
active probes that were internally labelled were made with the
Mirvana probe construction kit (Ambion) with the primers listed in
Supplementary Table 1. The probes used in the northerns were
antisense to either mature mir-376a2 50miR, mature mir-376a2 30
miR or to the 5S rRNA. The sizes of the resulting bands were
verified by electrophoresis next to the Ambion decade marker. The
northerns were visualized with a phosphoimager and their density
was measured. All experiments were performed in triplicate. Probe
for the loop specific primer, used in Supplementary Figure S4, is
GTTACGTGTTTGATGGTTAATCCTGTCTC.
In vitro Drosha cleavage assays
Drosha cleavage assays were performed as described earlier (Guil
and Caceres, 2007; Michlewski et al, 2008). pri-miRNA-376a2,
which was transcribed in vitro, was added to HeLa extracts
competent for Drosha cleavage. After incubation at 371C, reactions
were stopped and electrophoresed on a denaturing PAGE gel.
For Drosha cleavage assays in the presence of hADAR2, the
recombinant hADAR2 had been expressed and purified from
P. pastoris (Ring et al, 2004).
Drosophila transgenic lines
ADAR coding sequences were cloned in the pUAST plasmid bearing
GAL4-binding sites. Constructs were injected in Drosophila eggs and
transgenic lines were obtained and balanced by standard methods.
Female flies bearing UAS-ADAR constructs on chromosomes II or III
were crossed to males homozygous for both the GMR-GAL4 and
UAS-whiteIR constructs (wþ/Y; ; GMR-GAL4, UAS-wIR/ GMR-GAL4,
UAS-wIR). Levels of red pigment in the eyes of female progeny flies
in the RNAi assay were quantitated by ethanol extraction of batches
of 50 heads followed by optical absorption measurements (Pal-
Bhadra et al, 2004). The maximal level of red pigment in the
presence of UAS-ADAR1 p150 is 12% of the wild-type level.
The images in Figure 5 were made with an imaging system
comprising a Nikon AZ100 macroscope with 2? plan fluor
objective at 3? zoom setting (Nikon UK Ltd, Kingston-on-Thames,
UK), a Qimaging Micropublisher 5 cooled colour camera (Qima-
ging, Burnaby, BC, Canada). Image capture and control of the
filterwheel and image analysis were performed using IPLab
Spectrum (Scanalytics Corp, Fairfax, VA). White balance for the
illumination level used was set on a white background, and all
images were captured in a single session using identical capture
settings.
The data for Supplementary Figure 5B were imaged on a Leica
MZFLIII stereo-microscope with 0.5?, 0.63?, 1? and 1.6?
objectives. Images were captured with a Hamamatsu Orca AG CCD
camera (Hamamatsu Photonics (UK) Ltd, Welwyn Gardens City,
UK) and CRI liquid crystal RGB filter (Cambridge Research &
Instrumentation, Woburn, MA). Image capture and analysis were
performed with in-house scripts written for IPLab Spectrum
(Scanalytics Corp). The pixel intensity was measured on both eyes
from 10 different flies from each genotype.
Supplementary data
Supplementary data are available at The EMBO Journal Online
(http://www.embojournal.org).
Acknowledgements
We thank David W Lazinski for the gift of an ADAR2 DN expression
construct, David Finnegan, Dean Smith, and the Bloomington
Drosophila Stock Center for fly strains. In addition, we thank
Roberto Marcucci for recombinant ADAR2, Marion Hogg for dis-
cussion, Anne Leroy for sequencing white hairpin, Paul Perry and
Matthew Pearson for assistance with photography and Craig Nicol
for assistance with figures. MO’C is supported by core funding from
the Medical Research Council (U.1275.01.005.00001.01). BH is
supported by a Fellowship from the Marie Curie Foundation (PIIF-
GA-2008-220317).
Conflict of interest
The authors declare that they have no conflict of interest.
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