10050–10060 Nucleic Acids Research, 2014, Vol. 42, No. 15
doi: 10.1093/nar/gku662

Published online 23 July 2014

A-to-I editing in the miRNA seed region regulates
target mRNA selection and silencing efficiency
Hideaki Kume1,2 , Kimihiro Hino1 , Josephine Galipon1 and Kumiko Ui-Tei1,*1Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan and 2 Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033,
Japan

Received May 3, 2014; Revised July 08, 2014; Accepted July 9, 2014

ABSTRACTINTRODUCTION
Adenosine-to-inosine (A-to-I) RNA editing is the sitespecific hydrolytic deamination of A-to-I by adenosine
deaminases acting on RNAs (ADARs), which occurs
within mainly double-stranded RNA regions. Because in-* To whom correspondence should be addressed. Tel: +81 3 5841 3043; Fax: +81 3 5841 3044; Email: ktei@bs.s.u-tokyo.ac.jp

Present address:
Josephine Galipon, Integrated Human Sciences Program for Cultural Diversity, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba,
Meguro-ku, Tokyo 153-8902, Japan.

C The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Downloaded from http://nar.oxfordjournals.org/ at University of Tokyo on September 8, 2014

Hydrolytic deamination of adenosine to inosine(A-to-I) by adenosine deaminases acting on RNA(ADARs) is a post-transcriptional modification which
results in a discrepancy between genomic DNA andthe transcribed RNA sequence, thus contributing to
the diversity of the transcriptome. Inosine preferentially base pairs with cytidine, meaning that A-to-I
modifications in the mRNA sequences may be observed as A-to-G substitutions by the protein-coding
machinery. Genome-wide studies have revealed thatthe majority of editing events occur in non-coding
RNA sequences, but little is known about their functional meaning. MiRNAs are small non-coding RNAs
that regulate the expression of target mRNAs withcomplementarities to their seed region. Here, we confirm that A-to-I editing in the miRNA seed duplex
globally reassigns their target mRNAs in vivo, andreveal that miRNA containing inosine in the seedregion exhibits a different degree of silencing efficiency compared to the corresponding miRNA withguanosine at the same position. The difference inbase-pairing stability, deduced by melting temperature measurements, between seed-target duplexes
containing either C:G or I:C pairs may account forthe observed silencing efficiency. These findings unequivocally show that C:G and I:C pairs are biologically different in terms of gene expression regulation
by miRNAs.

osine residues preferentially base pair with cytidines, inosine residues in the mRNA sequences are recognized as
guanosines by the translational machinery in eukaryotes,
occasionally leading to the alteration of codons. Famous
examples of editing within mRNA coding sequences include neurotransmitter receptor or ion channel mRNAs,
such as the 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)
propanoic acid (AMPA) receptor subunit, the glutamate
receptor (GluR)-B (1), the serotonine-2C receptor (2), theKv1.1 voltage-dependent potassium channel (3) and the ␥ aminobutyric acid (GABA) A receptor subunit alpha3 mRNAs (4). However, only a limited number of protein-coding
genes is subjected to A-to-I editing; indeed, the majority of
A-to-I editing sites have been identified within non-coding
regions of the transcriptome (5,6).
MicroRNAs (miRNAs) are evolutionarily conserved
single-stranded non-coding RNAs of 20–22 nucleotides(nts) in length (7). They suppress the expression of proteincoding genes via partial nucleotide sequence complementarity, and play important roles in a broad range of biological processes including development, cellular differentiation, proliferation, apoptosis and the pathogenesis of human diseases such as cancer and metabolic disorders (8).
In human, more than 1800 miRNAs have been identified
so far (9). In canonical human miRNA biogenesis, primarymiRNA transcripts (pri-miRNAs) are initially transcribed
forming stem-loop structures. Maturation of miRNAs occurs in two steps. First, pri-miRNAs are processed in the
nucleus to ∼70 nt hairpins with a 2 nt 3 overhang referred
to as precursor miRNAs (pre-miRNAs) by Drosha (10) associated with the microprocessor DGCR8 (11). In the cytoplasm, they are further cleaved by Dicer to yield approximately 22 nt-long miRNA duplexes. The miRNA duplexes
are then loaded onto the Argonaute (AGO) protein within
the RNA-induced silencing complex (RISC), and unwound
into single-stranded mature miRNAs. In this process, theRNA strand with relatively relaxed structure, such as internal bulges or mismatches, in the 5 terminal duplex is pref-Nucleic Acids Research, 2014, Vol. 42, No. 15 10051

from those containing complementary sequences with the
non-edited A-containing seed sequence to those with the
G-containing seed sequence (31). Thus, as is observed inmRNA coding sequences, the conversion from A-to-I has
been considered to be equivalent to an adenosine to guanosine change. However, it has long been known that I:C forms
a thermodynamically weaker base pair compared to G:C,and that A-to-I editing induces a change in nucleic acid geometry. Consistent with such physicochemical characteristics, our study reveals that I:C and G:C base-pairing between miRNA and their targets do not contribute equally tomiRNA-mediated downregulation of gene expression, andmelting curves showed that I:C pairs are slightly less stable
than G:C pairs. We provide evidence that edited miRNAs
with an inosine in the 5 -terminal and seed region may exhibit different degrees of in vivo silencing activity comparedto those with guanosines at the same positions.
MATERIALS AND METHODS
Preparation of chemically synthesized miRNA duplexes andseed duplexes
RNA oligonucleotides corresponding to the 5p and 3p
strands of mature miRNA duplexes (21–24-nt in length)and 7-mer oligonucleotides corresponding to the mRNAbinding sequence complementary to the active seed (nucleotides 2–8) were chemically synthesized (Sigma or
Genepharma) in accordance with the sequences registeredin the miRBase and annealed to form either 5p:3p mature
miRNA duplexes and seed:mRNA binding site duplexes(9). In addition, the same oligonucleotides containing either
inosine (I-type) or guanosine (G-type) instead of adenosine(A-type) at the possible editing sites were also synthesized.
The miRNA strand with wild-type adenosine, inosine andguanosine at the editing site are referred to as miRNA-A, -I
and -G, respectively. Both strands of miRNAs or seed duplexes were mixed to a 1:1 ratio in a solution of 10 mM NaCland 20 mM Tris–HCl (pH 7.5), and annealed by incubation at 95o C for 5 min followed by cooling down to room
temperature. The annealed miRNA duplexes containing either adenosine, inosine or guanosine at the editing sites arereferred to as A-type, I-type and G-type miRNAs, respectively. The sequence of the synthetic mature miRNAs (miR376a-2, miR-22, miR-191) and their structures are shownin Figure 1, those of the seed RNA duplexes are shown in
Figure 4. siGY441 with a sequence unrelated to the Renillaluciferase gene was used as a negative control. Duplex formation was verified by electrophoresis on a 15% polyacrylamide gel in Tris Borate EDTA (TBE) buffer.
Microarray analysis
Human HeLa cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM; Gibco BRL) supplemented with
10% heat-inactivated fetal bovine serum (FBS; Mitsubishi
Kagaku) at 37˚C. The cells inoculated in 12-well plates 3 ×
105 cells/ml were transfected with 50 nM of each miRNA
duplex using 4 ␮l of Lipofectamine 2000. At 24 h posttransfection, total RNA was purified with an RNeasy Kit(Qiagen), and RNA quality was assessed using a NanoDrop

Downloaded from http://nar.oxfordjournals.org/ at University of Tokyo on September 8, 2014

erentially entrapped on the RISC (12). The mature miRNA
then guides RISC to target mRNA by partial sequence complementarity mainly in the seed sequence (nucleotide positions 2–8 from the 5 end of the miRNA) (13,14).
RNA interference (RNAi), which is extensively used forintended gene silencing, requires a near perfect sequence
match between the guide strand of small interfering RNA(siRNA) and target mRNA, has been used to study genefunction in a variety of organisms and it holds great promisefor therapeutic applications. The seed region (nucleotides 2–
8 from the 5 -end) of the siRNA guide strand is known to
downregulate the expression of genes outside of the canonical targets with weak interaction due to the seed sequence,
which is commonly called ‘off-target’ genes. Thus, the mechanism of the off-target effect due to siRNA is considered tobe very similar, if not identical, to that of microRNA-based
gene silencing.
Our previous study revealed that the capability of siRNAto induce off-target effects is highly correlated with its calculated melting temperature (Tm) and the free energy change(G) for formation of the protein-free seed-target duplex(15), indicating that thermodynamic stability of the RNA
duplex formed between the seed and the target is one of
the major factors in determining the degree of off-target effects: the highly stable seed-target duplex function as a positive silencing regulator but the unstable duplex is a negative regulator. However, unlike siRNA off-target effects, theefficiency of miRNA-mediated gene silencing was not simply correlated with the stability in the seed-target duplex. It
was previously shown that the efficacy of miRNA-mediatedgene silencing was partly determined by combined thermodynamic parameters of both the 5 -terminal 5-bp duplex
and the seed-target duplex (12). The stability in the 5 terminal end is known to regulate the efficiency of small RNA
loading on the RISC: an RNA strand with a thermodynamically less stable 5 terminal is preferentially entrappedon the RISC compared to the RNA strand with a stable 5terminal (16–18). The seed region (nucleotides 2–8) actually overlaps with the 5 -end (nucleotides 1–5). These tworegions were considered to function coordinately but independently, because the stability in the seed-target duplex isdefined by the nucleotide sequence, whereas the stability inthe 5 -terminal duplex is attributable to structural features
as well as nucleotide sequence (12).
The first instance of miRNA A-to-I editing was detectedby polymerase chain reaction (PCR) amplification and sequencing of the pri-miR-22 region (19). Subsequently, a systematic survey of miRNA editing focused on approximately
200 pri-miRNAs (20). In the past few years, deep sequencing of small RNAs has allowed the identification of A-to-I
editing sites in the miRNA of a variety of organisms, celllines and tissues (21–27), revealing not only pri-miRNAs
but also a number of pre-miRNAs as ADAR targets. The
editing of miRNAs has the potential to exert a profound effect on their biogenesis, such as inhibition––and sometimes
promotion––of Drosha or Dicer processing (20,28,29). Ato-I editing may induce degradation of miRNAs by the
Tudor-SN inosine-specific endonuclease (28), and was also
shown to regulate strand selection during RISC loading(30). Furthermore, when the edited site is positioned inmiR-376a-5p seed region, the target genes are shown to shift10052 Nucleic Acids Research, 2014, Vol. 42, No. 15

A miR-376a-2(i) A-type(wild type)

5’ GUAGAUUUUCCUUCUAUGGUU 3’|| ||||||||| |||||:|
3’ UGCACCUAAAAGGA-GAUACUA 5’

miR-376a-2-5p

miR-376a-2-5p(ii) I-type

5’ GUAGAUUUUCCUUCUAUGGUU 3’|| ||||||||| |:|||:|
3’ UGCACCUAAAAGGA-GIUACUA 5’

miR-376a-3p-A

miR-376a-3p-I
miR-376a-2-5p(i) A-type(wild type)

5’ AGUUCUUCAG-UGGCAAGCUUUA 3’|||||||||: ||||| ||||
3’ UGUCAAGAAGUUGACCGU-CGAA 5’

miR-22-5p(ii) I-type

5’ AGUUCUUCAG-UGGCAAGCUUUA 3’|||||||||: ||||| ||:|
3’ UGUCAAGAAGUUGACCGU-CGIA 5’

miR-22-5p

5’ AGUUCUUCAG-UGGCAAGCUUUA 3’|||||||||: ||||| ||:|
3’ UGUCAAGAAGUUGACCGU-CGGA 5’

miR-22-5p(i) A-type(wild type)

5’ CAACGGAAUCCCAAAAGCAGCUG 3’|||:||||| || |||||
3’ CCCCUGCUUUAGG-UUCGCGUCG 5’

miR-191-5p-A(ii) I-type

5’ CAICGGAAUCCCAAAAGCAGCUG 3’:||:||||| || |||||
3’ CCCCUGCUUUAGG-UUCGCGUCG 5’

miR-191-5p-I

5’ CAGCGGAAUCCCAAAAGCAGCUG 3’:||:||||| || |||||
3’ CCCCUGCUUUAGG-UUCGCGUCG 5’

miR-191-5p-G

miR-376a-3p-G

B miR-22(iii) G-typemiR-22-3p-A

miR-22-3p-I

miR-22-3p-G

C miR-191(iii) G-typemiR-191-3p

miR-191-3p

miR-191-3p

Figure 1. Structures of miRNA duplexes used in this study. (A) Three typesof miR-376a-2 duplex and its derivatives. A-type miR-376a-2 duplex is thewild-type duplex formed between miR-376a-2–5p and miR-376a-3p-A, inwhich the possible editing site of adenosine is situated at position +6 fromthe 5 end. I-type or G-type miR-376a-2 duplex is composed of miR-376a2–5p and miR-376a-3p-I or miR-376a-3p-G. (B) Three types of miR-22
duplex and its derivatives. A-type miR-22 duplex is the wild-type duplex
formed between miR-22–5p and miR-22–3p-A, in which the possible editing site of adenosine is situated at position +2. I-type or G-type miR-22
duplex is composed of miR-22–5p and miR-22–3p-I or miR-22–3p-G. (C)Three types of miR-191 duplex and its derivatives. A-type miR-191 duplex
is the wild-type duplex formed between miR-191–3p and miR-191–5p-A,
in which adenosine is situated at position +3. I-type or G-type miR-191
duplexes are composed of miR-191–3p and either miR-191–5p-I or miR191–5p-G, respectively.

2000 spectrophotometer (Thermo Scientific) and a Bioanalyzer (Agilent). cDNA was synthesized from each total
RNA sample using an Agilent One Color Spike Mix Kit(Agilent), and used for hybridization to an Agilent Whole
Human Genome Microarray (4×44 K multi-pack format)
according to the manufacturer’s protocol. Data analysis was
automated for mock, A-type, I-type and G-type miRNA
duplex transfection samples using R code as follows. First,
a filter was applied to keep only the spots that were detected with certainty in all four samples. RNA from mocktransfected cells treated with the transfection reagent in theabsence of miRNA duplex was used as a control, and the
distributions of transcript expression values were normalized across all samples by quantile normalization (32). Agilent chips tend to contain several probes per gene. Since
the filter described above selects only the spots for which
the signal is not saturated, meaning that the signals are ex-

Quantitative RT-PCR (qRT-PCR)
Total RNA purified from HeLa cells was reversetranscribed using a High Capacity cDNA Reverse
Transcription Kit (Applied Biosystems). The resultant
cDNA was subjected to real-time PCR using the PowerSYBR Green PCR Master Mix (Applied Biosystems).
PCR product levels were monitored and triplicates wereanalyzed using a StepOnePlus Real-Time PCR system (Applied Biosystems). The expression level of each transcript
was first normalized to that of lactate dehydrogenase-A(LDHA), and then to that of the mock transfection control.
The PCR primer sets used are listed in Supplementary
Table S2.
Construction of luciferase reporters with complete-matchedand seed-matched sequences
The plasmids containing reporter constructs were allderived from psiCHECK-1 (Promega). Oligonucleotides
with target sequences completely matched (CM) to eachannotated miRNA sequence were chemically synthesized with cohesive XhoI/EcoRI ends and inserted into
psiCHECK-1 at the corresponding restriction sites to generate psiCHECK reporter constructs. The wild-type A-type
miRNA target sequence carries an uridine at the position
complementary to the adenosine, and are designated as
psiCHECK-CM-U-target. psiCHECK reporter constructs
with G-type and I-type miRNA target sequences were also
constructed, which have a cytidine at the complementarysite, and were therefore designated as psiCHECK-CMC-target. Similarly, three tandem repeats of seed-matched(SM) target sequences, each of which has complementarity with just the 7 nucleotide-long seed sequence, but not

Downloaded from http://nar.oxfordjournals.org/ at University of Tokyo on September 8, 2014

5’ GUAGAUUUUCCUUCUAUGGUU 3’|| ||||||||| |:|||:|
3’ UGCACCUAAAAGGA-GGUACUA 5’(iii) G-type

pected to be within the linear range of detection, the signal
intensities of plural probes corresponding to a single gene
were averaged. Next, we used a Perl script to look up RefSeq genes with one or more regions complementary to the
5p or 3p seed regions (nucleotides 2 to 8) in their 3 UTR.
To enable direct comparison between the seed-matched output and background genes, gene references that were notfound in RefSeq were excluded from the microarray data
set. Then, the following steps were as follows, in order: (i)discarded the genes which contain a region complementary
to the dead seed, which means the seed sequence on the opposite strand of the miRNA containing the editing site; (ii)selected groups of genes with at least one seed-match for either one of four versions of the active seed, containing A, C,
G, or U at the potential A-to-I editing site; (iii) eliminatedthe genes that were included in more than one group, to end
up with a list of unique A-, C-, G- and U-seed matches; (iv)
output the corresponding lists of genes considered as background: RefSeq genes without dead-seed matches and without either one of the unique A-, C-, G- or U-seed matches,
respectively. The fold-change of gene expression level between background and seed-matched genes was obtainedby subtracting the respective average log2 of signal intensities of seed-matched target genes from that of backgroundgenes for each experiment, which provides an estimate of thearea between the red (seed-match) and black (background)curves.

Nucleic Acids Research, 2014, Vol. 42, No. 15 10053

with the non-seed region, were also synthesized and named
as psiCHECK-SM-U-target or psiCHECK-SM-C-target.
The inserted oligonucleotide sequences are shown in Supplementary Table S1. Each of the inserted targets was expressed as part of the 3 -UTR region of Renilla luciferase
mRNA in the transfected cells.
Cell culture and RNA silencing activity assay using firefly luciferase reporter system

Measurement of Tm values
Both strands of the RNA 7-mer seed duplex were mixed
to a 1:1 ratio (final 5 ␮M) in a solution of 1 M NaCl, 0.5
mM EDTA and 5mM Na2 HPO4 (pH 7.5). Then, the absorbance of samples was monitored at 260 nm from 4 to95◦ C at a heating speed of 1◦ C/min and analyzed using a
UV-2550 spectrophotometer with the Thermal Melt Analysis System for Nucleic Acids TMSPC-8 (SHIMADZU). Tm
values were calculated by the Two Point Average method(33).
RESULTS AND DISCUSSION
Microarray analysis of global conversion of target mRNAs
by A-to-I editing in the miRNA seed region
It is well known that miRNA mainly recognizes mRNAs
with complementarity to the miRNA seed sequence (nucleotides 2–8) in their 3 -UTRs (13,14). Thus, A-to-I RNA
editing in the miRNA seed region is expected to change
the set of target mRNAs: wild-type miRNA with adenosine in the seed region recognizes mRNAs with uridines atthe complementary position, but the edited form with inosine instead of adenosine in the seed region should target
mRNAs with cytidines at the opposite sites. In fact, Kawahara et al. (31) reported that miR-376a-5p edited at the +4
site in the seed region silences a different set of genes thanits non-edited form.
To monitor the global changes in the identity of downregulated transcripts by miRNAs with or without editing inthe seed region, we used three types of miRNAs, miR-376a2, miR-22 and miR-191. MiR-376a-2 (31) and miR-22 (19)have been reported to have at least one editing site in theirrespective seed regions, and the editing site in the seed region of miR-191 was suggested by our unpublished results.
We prepared three patterns of miRNA duplexes for each of
three types of miRNAs (Figure 1): (i) an miRNA duplex

Downloaded from http://nar.oxfordjournals.org/ at University of Tokyo on September 8, 2014

HeLa cells inoculated in each well of 96-well plates at 2
× 104 cells/100 ␮l were transfected simultaneously withpsiCHECK-CM or –SM-target construct (3.3 ng), pGL3Control (33 ng; Promega) and miRNA duplex (0.05 nM,
0.5 nM, 5 nM or 50 nM) using 0.67 ␮l of lipofectamine
2000 per well (Life Technologies). An siRNA against enhanced green fluorescent protein (EGFP) was used as control. The cells were harvested 24 h post-transfection and therelative luciferase activity (Renilla luc activity/firefly luc activity) was measured using a Dual-Luciferase Reporter Assay System (Promega). The pGL3-Control encoding firefly
luciferase served as a control for the calculation of relative
luciferase activity.

was chemically generated using the non-edited wild-type
miRNA strand (miR-376a-3p-A, miR-22–3p-A, or miR191–5p-A) and the wild-type complementary strand (miR376a-2–5p, miR-22–5p, or miR-191–3p), and named as ‘Atype’ miRNA. (ii) Since adenosine is converted into inosine
by ADAR-mediated deamination, the ‘I-type’ miRNA duplex formed between the miRNA strand with inosine at thepossible editing site (miR-376a-3p -I, miR-22–3p-I, or miR191–5p-I) and its wild-type opposite miRNA strand was
prepared. (iii) Finally, since an inosine preferentially basepairs with cytidine, similarly to guanosine, the miRNA duplex formed by the miRNA strand with guanosine at theediting site (miR-376a-3p-G, miR-22–3p-G, or miR-191–5p-G) and the wild-type opposite miRNA strand was also
prepared, and referred to as ‘G-type’ miRNA. Since all of
the miRNA strands without editing (miR-376a-2–5p, miR22–5p, or miR-191–3p) in the three types of miRNA duplexes contain uridines at the positions opposite to the editing sites, A:U, I:U and G:U pairs are formed at the possible
editing positions in the wild-type (A-type), edited type (Itype) and G-type miRNA duplexes, respectively (Figure 1).
These miRNA duplexes were respectively transfected into
human HeLa cells at 50 nM, and microarray analysis was
performed using total RNA extracted 24 h after transfection.
To analyze miRNA-induced global changes in gene expression, mRNAs were divided into four groups based on
the presence in their 3 -UTRs of at least one sequence
complementary to the seed region containing either A,
U, G or C at the possible editing site, respectively. The
mRNAs that belonged to more than one of these groups,
and those with 3 -UTR complementarity to the dead seed
were eliminated to select for targets that may be downregulated by only one type of seed sequence. These separate
groups are subsequently referred to as (I) U-target, (II) Atarget, (III) C-target, (IV) G-target mRNAs (see Supplementary Figures S1–S3). Consequently, A-type miRNAs
are expected to specifically downregulate ‘U-target’ genes,
whereas the I-type and G-type miRNAs should downregulate ‘C-target’ genes. The background constitutes of all
mRNAs––excluding dead seed targets––that are outside ofeach individual group. As a result, the background differs
slightly between the four groups. The procedure for microarray data analysis is illustrated in Figure 2A. At first, wemade an MA plots showing the mean log2 of signal intensities relative to those of mock transfection (M value) andthe averaged log10 signal intensities of mock and miRNAtransfections (A value), and plotted these values as the vertical and horizontal bars, respectively (Figure 2A(1)). To
facilitate understanding, the MA plot was then converted
to the cumulative distribution (Figure 2A(2)), in which thehorizontal axis indicates the ‘M value’ and the vertical axis
is the cumulative fraction of mRNAs. The mean log2 of signal intensities relative to those of mock transfection was
calculated for each group of mRNAs and the results areshown as MA plots and cumulative distributions (Supplementary Figures S1–S3). Furthermore, to simplify the results, the ‘area’ between seed-matched mRNAs (red curve in
Figure 2A(2)) and background RNAs (black curve in Figure 2A(2)) was calculated and shown as the ‘fold-change’ inFigure 2B–G, with positive values meaning that the target

10054 Nucleic Acids Research, 2014, Vol. 42, No. 15

Downloaded from http://nar.oxfordjournals.org/ at University of Tokyo on September 8, 2014

Figure 2. Microarray and real-time PCR analysis of the expression levelsof target transcripts with seed complementary sequences. (A) Schematic
model describing the analysis workflow for microarray data shown in Supplementary Figures S1–S3. (1) MA plot: the vertical bar indicates themean log2 of signal intensities relative to those of mock transfection (Mvalue), and the horizontal bar indicates the averaged log10 signal intensities of mock and miRNA transfections (A value). The red plots show seedmatched transcripts and the black plots show background transcripts. (2)Cumulative distribution: the horizontal axis indicates the ‘M value’, andthe vertical axis is the cumulative fraction of mRNAs. The red line indicates the cumulative curve of seed-matched transcripts, and the the blackline that of background transcripts. The gray area indicates the fold-change
in the expression of seed-matched transcripts compared to those of background transcripts. Each of miR376a-2 (B) and (C), miR-22 (D) and (E)and miR-191 (F) and (G) duplexes were transfected into HeLa cells, andthe changes in expression levels of transcripts with U-target (B), (D), (F) or
C-target (C), (E), (G) sequences were analyzed by microarray. The complementarity between seed region and target mRNAs were shown in left panelin A–F. In the target sequence, N indicates any given nucleotide. In eachexperiment, three types of miRNAs (A-type, I-type, G-type) was respectively used. Fold-change indicates the difference of gene expression levels
between mean log2 of signal intensities of each miRNA targets and background genes. The expression levels of three target mRNAs of miR-376a3p (Rad23A, CKAP4 and SEL1L3) (H) or miR-22–3p (EPS15, SLC7A1
and TTYH3) (I) by mock transfection (black bar), I-type miRNA (redbar), or G-type miRNA (yellow bar) were measured by qRT-PCR, andcompared with the microarray data (each of black, red, or yellow diagonal bar). Note that the number of seed-complementary target sites ranged
from one to four. The seed regions and their complementary site are surrounded by a box, and the possible editing sites were highlighted in orange.
In (H) and (I), positions relative to the 5 end of mRNA were shown. In(H) and (I), a bar indicates the standard deviation calculated from three
independent experiments.

genes are generally more inhibited than background. Furthermore, the Wilcoxon rank-sum test was used to assess
whether or not the target genes and background distributions are significantly different (p < 1×10−2 ).
As expected, the microarray results for transfection of
wild-type (A-type) miR-376a-2 revealed that U-target transcripts, which harbour one or more sequences complementary to the miR-376a-3p seed region UCAUAGA (underline indicating the possible editing site) in their 3 -UTRs,
were significantly downregulated. Indeed, the fold-changeof expression levels was 0.042 (p = 2.14 × 10−9 ), which
was highest and significant for U-target transcripts after
transfection with the A-type duplex, but not I-type (0.010,
p = 1.01 × 10−1 ) or G-type (-0.023, p = 3.82 × 10−2 )duplexes (Figure 2B, Supplementary Figure S1). On theother hand, both I-type and G-type miRNA duplexes ofmiR-376a-2, but not A-type, significantly downregulated
C-target genes, by 0.024 (p = 1.83 × 10−3 ), 0.096 (p =3.51 × 10−16 ) and -0.002 (p = 3.9 × 10−1 ), respectively(Figure 2C, Supplementary Figure S1). These results showthat the global inhibitory effect of I-type miR-376a-2 duplex on C-target genes was apparently weaker than that of
the G-type duplex. This result clearly suggests that the inosine in the seed region of miR-376a-3p-I (UCAUIGA) did
not exert the same extent of inhibition than its guanosinecontaining counterpart miR-376a-3p-G (UCAUGGA). Ina similar fashion, the wild-type (A-type) miR-22 and miR191 duplexes significantly repressed the expression levels of
U-target mRNAs of miR-22–3p-A and miR-191–3p-A, as
indicated by the rather high fold-changes of gene expression of 0.099 (p = 1.46 × 10−23 ) and 0.172 (p = 2.77 ×
10−24 ), respectively which was not the case of I-type andG-type miRNAs (p = 3.87∼5.46 × 10−1 , Figure 2D and E,
Supplementary Figures S2 and S3). Similarly, the C-target
mRNAs of miR-22–3p-G or miR-191–5p-G were significantly downregulated by the transfection of G-type miR-22
or miR-191 duplexes with fold-changes of gene expression
levels of 0.077 (p = 9.5 × 10−25 ) and 0.092 (p = 3.33 × 10−5 ),respectively (Figure 2E and G, Supplementary Figures S2and S3). However, the C-target mRNA silencing efficiency
exerted by I-type miRNAs differed between miRNA species
when compared to the corresponding G-type miRNAs. Although the differences were not as clearly marked as formiR-376a-2, the C-target transcripts seemed to be downregulated strongly by the G-type (0.077, p = 9.5 × 10−25 )than by the I-type (0.063, p = 1.68 × 10−12 ) in the case of
miR-22, whereas for miR-191, the effect of the I-type (0.124,
p = 1.25 × 10−8 ) appeared somewhat stronger comparedto the G-type (0.092, p = 3.33 × 10−5 ) (Figure 2F and G,
Supplemental Figures S2 and S3), but the level of significance of these smaller differences are not clear. Nevertheless, these findings suggest that the effect of inosine versus
that of guanosine is likely to depend on the miRNA duplex
involved and/or the position of the editing site and its sequence context, since nearest neighbor residues may affectthe thermodynamics of miRNAs.
The A-type miR-376a-2 and miR-191 duplexes appeared
to have little, if any, silencing activity on the predicted Ctargets of mature miR-376a-3p or miR-191–5p (Figure 2C
and G). On the other hand, the A-type miR-22 duplex induced a slight but significant downregulation of C-target

Nucleic Acids Research, 2014, Vol. 42, No. 15 10055

Reporter assay of target conversion by A-to-I editing in themiRNA seed region
Microarray experiments revealed that the expression of Ctarget transcripts were downregulated by G-type miRNAs.
Moreover, I-type miRNAs could reduce the expression levels of C-target mRNAs, but the reduction levels were notnecessarily equivalent to those of G-type miRNAs and theeffect of inosine seemed to vary between the miRNA species
tested. However, a well-known caveat of microarray experiments is that gene expression may be affected by secondary effects and/or miRNA off-target effects. Therefore,
we performed an in vivo luciferase assay in order to confirm the direct effect of each miRNA on their target mRNAs. Two types of reporter plasmids were constructed (Figures 3–5): (i) psiCHECK–SM-target, which contains threetandem repeats of seed-matched (SM) sequences with complementarity to the seed region only (Figures 3–5, upper left
panels). (ii) psiCHECK–CM-target with a single completematched (CM) sequence corresponding to the full-length
mature miRNA, as a positive control (Figures 3–5, upper
right panels). The SM or CM target sequences were inserted into the 3 -UTR of the Renilla luciferase gene in a
psiCHECK-1 vector (see Materials and Methods section,
upper panels of Figures 3–5). In both constructs, uridine orcytidine was introduced into the target sequence at the opposite position (position X in the target sequences of theupper panels in Figures 3–5) of the possible editing site ofmiRNA, and shown as SM-U-target or SM-C-target, andP

G-type miRNA

Control miRNA

Relative luciferase activity (%)

I-type miRNA

Relative luciferase activity (%)

K

SM-U-target
SM-C-target
UC
3’ C 5’3’

3’

5’check-441

140
120
100
80
60
40
20
0

F

A-type miRNA

B 5’
3’

Relative luciferase activity (%)

Relative luciferase activity (%)

Control-targetA5’
sm-wt-441

140
120
100
80
60
40
20
0
0.05 0.5

3’

5

50

A 5’
check-aG5’
3’0.05 0.5

0.05 0.5

5U|||||||

A
wr-a-sm

50
3’
5’

5

0.05 0.550

I 5’check-i

3’

5U||:||||

I
wr-i-sm

0.05 0.5

5

Q5’
3’

check-g
G 5’

3’
5’

3’

5

U

||:||||

0.05 0.5 5 50
miRNA (nM)5C|| ||||

A
ed-a-sm

0.05 0.5

3’G
wt-g-sm

3’
5’

5

C

||:||||

I
ed-i-sm

50
3’
5’

3’

3’

3’
5’

N 5’
3’

5

C

|||||||

0.05 0.5550U||||||||||||||||||||||

A
wr-a-cm3’
5’G
ed-g-sm

3’
5’

S 5’
3’

5

3’

50

U

||||||||||||||||:|||||

I
wr-i-cm3’
5’

0.05 0.5550C|||||||||||||||| |||||

A
ed-a-cm3’
5’

0.05 0.5

O5’
3’

5

50

C

||||||||||||||||:|||||

I
ed-i-cm

3’
5’

140
120
100
80
60
40
20
0
5

U

50

||||||||||||||||:|||||

G
wt-g-cm

3’
5’

140
120
100
80
60
40
20
0
0.05 0.5 5 50
miRNA (nM)

J 5’
140
120
100
80
60
40
20
0

0.05 0.5

50

3’

5’
cm-ed-441

140
120
100
80
60
40
20
0

0.05 0.5

50

140
120
100
80
60
40
20
0
0.05 0.5 5 50
miRNA (nM)

I 5’

C

E 5’
3’

5’
cm-wt-441

140
120
100
80
60
40
20
0
0.05 0.5

R 5’

CM-C-target
3’

140
120
100
80
60
40
20
0

M 5’

50

140
120
100
80
60
40
20
0

140
120
100
80
60
40
20
0

0.05 0.5

140
120
100
80
60
40
20
0
0.05 0.5

50

3’

50

140
120
100
80
60
40
20
0

140
120
100
80
60
40
20
0

H 5’

U

140
120
100
80
60
40
20
0

140
120
100
80
60
40
20
0

L 5’
3’

3’

5’
sm-ed-441

140
120
100
80
60
40
20
0

140
120
100
80
60
40
20
0

140
120
100
80
60
40
20
0

3’

CM-U-target

D 5’

0.05 0.5

T 5’
3’

5

C

50

||||||||||||||||||||||

G
ed-g-cm

3’
5’

140
120
100
80
60
40
20
0
0.05 0.5 5 50
miRNA (nM)

0.05 0.5 5 50
miRNA (nM)

Figure 3. The results of reporter assays using miR-376a-2 duplex andits derivatives. The upper panels indicate the schematic structures ofpsiCHECK-SM-target and psiCHECK-CM-target constructs containing
three tandem repeats of SM-target sequences and a CM-target sequence,
respectively, in the 3 UTR of Renilla luciferase gene of psiCHECK vector.
Position Y indicates the possible editing site in the mRNA, and position
X in the SM- or CM-target sequence indicates the position opposite to thethe possible editing site of miRNA. Control siGY441 (A)–(E), wild-type
A-type (F)–(J), I-type (K)–(O), or G-type (P)–(T) miR-376a-2 duplex wastransfected with control psiCHECK-1 (A), (F), (K), (P), psiCHECK-SMU-target (B), (G), (L), (Q), psiCHECK-SM-C-target (C), (H), (M), (R),psiCHECK-CM-U-target (D), (I), (N), (S), or psiCHECK-CM-C-target(E), (J), (O), (T), and pGL3-Control firefly luciferase expression constructwere simultaneously transfected into HeLa cells. Relative luciferase activity was measured one day after transfection. Each miRNA was transfected
at 0.05, 0.5, 5 and 50 nM, respectively. Complementarity between transfected miRNA and target construct is shown at top of each figure, and thepossible editing sites were highlighted. Bar indicates the standard deviationof triplicated samples.

CM-U-target or CM-C-target, respectively (Figures 3–5).
Each of these reporter constructs was co-transfected into
human HeLa cells with pGL3-Control encoding firefly luciferase gene and each miRNA, and relative luciferase activity was measured one day after transfection. The resultsof luciferase reporter assays for all combinations of targets
and miRNAs were shown in Figures 3–5, and those of thespecific combinations (SM-U-targets and A-type miRNA,
SM-C-targets and I-type miRNAs and SM-C-targets andG-type miRNAs) were picked up and compared in Figure 6.
All three A-type wild-type miRNA duplexes (miR-376a-2,
miR-22 and miR-191 duplexes) reduced the luciferase activity of their SM-U-targets in a dose-dependent manner, but

Downloaded from http://nar.oxfordjournals.org/ at University of Tokyo on September 8, 2014transcripts (0.037, p = 1.65 × 10−5 ) (Figure 2E). Since
miR-22–3p-A had the largest number of U-targets (688
genes) compared to all other miRNAs tested (152∼501
genes) (Supplemental Figures S1–S3), this may have resulted in non-negligible secondary effects. Alternatively, regions other than the seed might be involved in silencing activity (34).
To confirm the reliability of our microarray data, we selected three genes each from the C-targets of miR-376a-3p
and miR-22–3p, respectively, for which the extent of downregulation by I-type and G-type miRNAs was different, andcarried out qRT-PCR analysis (Figure 2H and I). In thecase of miR-376a-3p, the expression levels of all three mRNAs: Rad23A, Cytoskeleton-associated protein 4 (CKAP4)
and sel-1 suppressor of lin-12-like 3 (SEL1L3), appeared to
be downregulated weakly by the I-type miR-376a-2 duplex
but strongly by the G-type miR-376a-2 duplex in the microarray experiments, which was consistent with qRT-PCR
results (Figure 2H). Similarly, for miR-22–3p, the expression of epidermal growth factor receptor pathway substrate
15 (EPS15), solute carrier family 7 (cationic amino acidtransporter, y+ system) member 1 (SLC7A1) and tweety
family member 3 (TTYH3) appeared weakly repressed by
the I-type miR-22 duplex but strongly by the G-type miR22 duplex both in the microarray experiment and by qRTPCR (Figure 2I). Thus, the relative transcription levels estimated by qRT-PCR were well correlated with those obtained by the microarray analysis (Figure 2H and I). Basedon these results, we conclude that gene silencing efficiencyof I-type miRNA is not necessarily the same as that of Gtype miRNA.

10056 Nucleic Acids Research, 2014, Vol. 42, No. 15

50

5’
wr-a-smA

0.05 0.5

550

0.05 0.5

L 5’check-i
I 5’

140
120
100
80
60
40
20
0

3’

5

5

50

3’

check-g
G 5’

140
120
100
80
60
40
20
0

3’

5’

3’

5

3’

wt-g-smG

5’

3’

5

50

C 3’

||||||:

ed-i-sm I

5’50

wr-a-cm A

5’

3’

5

50

C 3’

|||||||

ed-g-smG

5’

5

3’

wr-i-cm I

5’

ed-a-cm A

3’

5

5

wt-g-cm G

5’

3’

5’

P

ed-g-cm G

0.05 0.5 5 50
miRNA (nM)

Figure 4. The results of reporter assays using miR-22 duplex and its
derivatives. Control siGY441 (A)–(E), wild-type A-type (F)–(J), I-type(K)–(O), or G-type (P)–(T) miR-22 duplex was transfected with control psiCHECK-1 (A), (F), (K), (P), psiCHECK-SM-U-target (B), (G),(L), (Q), psiCHECK-SM-C-target (C), (H), (M), (R), psiCHECK-CM-Utarget (D), (I), (N), (S), or psiCHECK-CM-C-target (E), (J), (O), (T), andpGL3-Control firefly luciferase expression construct were simultaneouslytransfected into HeLa cells. Other detailed descriptions about this figure
are same as those of Figure 3.

50

A 5’
check-a

140
120
100
80
60
40
20
0

0.05 0.5

3’

5

50

5

50

U

H 5’
3’

check-iI 5’

3’

5

50

U

|||||:|

3’

5’
wr-i-smI

140
120
100
80
60
40
20
0

0.05 0.5

5

50

3’

check-g
G 5’

140
120
100
80
60
40
20
0

3’

5

G
wt-g-sm

3’
5’

5

3’

3’

5’
ed-i-smI

5

50

U||||||||||||||||||||||

wr-a-cmA

3’
5’

0.05 0.5

N5’
3’

5

50

C

|||||||

G
ed-g-sm

3’
5’

5

3’

wr-i-cm I

3’
5’

5

50

C

||||||||||||||||||| ||ed-a-cmA

3’
5’

0.05 0.5

O5’
3’

5

50

C

|||||||||||||||||||:||

ed-i-cm I

3’
5’

140
120
100
80
60
40
20
0
5

0.05 0.5

50U|||||||||||||||||||:||

wt-g-cmG

3’
5’

140
120
100
80
60
40
20
0
0.05 0.5 5 50
miRNA (nM)

3’

50

U

|||||||||||||||||||:||

0.05 0.5

S 5’

0.05 0.5

J 5’
140
120
100
80
60
40
20
0

140
120
100
80
60
40
20
0

140
120
100
80
60
40
20
0
0.05 0.5 5 50
miRNA (nM)

3’

50

C

|||||:|

0.05 0.5

R 5’

I 5’

3’

cm-ed-441
5’

140
120
100
80
60
40
20
0

140
120
100
80
60
40
20
0
0.05 0.5

50U|||||:|

140
120
100
80
60
40
20
0

0.05 0.5 5 50
miRNA (nM)3’5’ed-a-smA

C

E5’
3’

cm-wt-441
5’0.05 0.5

50

C

140
120
100
80
60
40
20
0
0.05 0.5Q5’5||||| |

M5’
3’

CM-C-target
3’

140
120
100
80
60
40
20
0

140
120
100
80
60
40
20
0
0.05 0.5

U

D5’
3’

5’
sm-ed-441

0.05 0.5

3’

A 5’
wr-a-sm|||||||

L 5’

140
120
100
80
60
40
20
0

CM-U-target
3’

140
120
100
80
60
40
20
0

140
120
100
80
60
40
20
0

3’

50
5’

5

C

C 5’
3’

5’
sm-wt-441

G5’0.05 0.5

C 3’||||||||||||||||||||||

140
120
100
80
60
40
20
0
0.05 0.5 5 50
miRNA (nM)

0.05 0.5

K

3’

140
120
100
80
60
40
20
0

3’

50

ed-i-cm I

5

140
120
100
80
60
40
20
0

F

C 3’

||||||||||||||||||||:|

0.05 0.5

50

U 3’ T 5’||||||||||||||||||||:|

Relative luciferase activity (%)

Control miRNA
5’

3’check-4415’

SM-C-target

U

B 5’
3’

50

C 3’

140
120
100
80
60
40
20
0

140
120
100
80
60
40
20
0
0.05 0.5 5 50
miRNA (nM)5|||||||||||||||||||| |0.05 0.5

50

U 3’ O5’||||||||||||||||||||:|

0.05 0.5

S 5’

3’

140
120
100
80
60
40
20
0
0.05 0.5

N 5’

0.05 0.5

U 3’ J 5’

140
120
100
80
60
40
20
0

140
120
100
80
60
40
20
0
0.05 0.5 5 50
miRNA (nM)5||||||||||||||||||||||

140
120
100
80
60
40
20
0

0.05 0.5

50

U 3’ R 5’||||||:

0.05 0.5

I 5’5’
ed-a-smA

140
120
100
80
60
40
20
0

140
120
100
80
60
40
20
0
0.05 0.5 5 50
miRNA (nM)

50

C 3’

0.05 0.550

wr-i-sm I

0.05 0.5Q 5’5||||||

U 3’ M 5’||||||:

140
120
100
80
60
40
20
0
0.05 0.5

3’

140
120
100
80
60
40
20
0

140
120
100
80
60
40
20
0

3’

0.05 0.5

U 3’ H 5’|||||||

140
120
100
80
60
40
20
0

A-type miRNA

5

140
120
100
80
60
40
20
0

cm-ed-441
5’

A

Relative luciferase activity (%)

A 5’check-a

140
120
100
80
60
40
20
0

3’

3’

cm-wt-441
5’SM-U-target

Control-target

C 3’

I-type miRNA

0.05 0.5

50

3’

CM-C-target

U 3’ E 5’

Relative luciferase activity (%)

G-type miRNA

A-type miRNA

Relative luciferase activity (%)

I-type miRNA

Relative luciferase activity (%)

P5sm-ed-441
5’

140
120
100
80
60
40
20
0

G 5’
3’

3’

CM-U-target

D 5’

T 5’
3’

5

50

C

||||||||||||||||||||||

ed-g-cmG

3’
5’

140
120
100
80
60
40
20
0
0.05 0.5 5 50
miRNA (nM)

0.05 0.5 5 50
miRNA (nM)

Figure 5. The results of reporter assays using miR-191 duplex and its
derivatives. Control siGY441 (A)–(E), wild-type A-type (F)–(J), I-type(K)–(O), or G-type (P)–(T) miR-191 duplex was transfected with control psiCHECK-1 (A), (F), (K), (P), psiCHECK-SM-U-target (B), (G),(L), (Q), psiCHECK-SM-C-target (C), (H), (M), (R), psiCHECK-CM-Utarget (D), (I), (N), (S), or psiCHECK-CM-C-target (E), (J), (O), (T) andpGL3-Control firefly luciferase expression construct were simultaneouslytransfected into HeLa cells. Other detailed descriptions about this figure
are same as those of Figure 3.

Combinatorial thermodynamic stability in the 5 terminal duplex and the duplex formed between miRNA seed region andtarget mRNA may regulate silencing efficiency

not that of SM-C-targets (Figures 3–6). Consistently, all of
the G-type miRNAs reduced the luciferase activity of their
SM-C-targets, but not that of SM-U-targets (Figures 3–6).
However, the effects of I-type miRNAs were different between miRNA species: the I-type miR-376a-2 duplex did
not significantly repress its SM-C-target (Figures 3 and 6A),
whereas the I-type miR-22 (Figures 4 and 6B) and miR-191
duplexes (Figures 5 and 6C) significantly reduced the luciferase activity of SM-C-target reporters, albeit at different
levels. These results were well correlated with those of the
microarray experiment (Figure 2), with the C-target silencing activity of I-type miRNAs in order of miR-191 > miR22 > miR-376a-2. Finally, we could verify the effectivenesson CM-targets of all A-type, I-type and G-type miRNA duplexes used in this study, indicating that these miRNAs arefunctional and not defective (Figures 3–5, CM-target).

The strand with relatively lower internal stability at the5 -terminus of the miRNA duplex is preferentially loaded
onto the RISC (Figure 7A, 16–18). Furthermore, miRNAcanonically recognizes target transcripts using seedcomplementary sequences to direct post-transcriptional
repression (Figure 7A, 13,14). Consistent with this knowledge, we have published a mathematical model of miRNAbase-pairing stability using known thermodynamic parameters of Watson–Crick base-pairing (35,36), and havedemonstrated that the silencing efficiency of miRNA isstrongly and positively correlated, with the correlation
score ‘miScore’ calculated as follows:
miScore = Tm2−8 − 0.5 × miTm1−5
where Tm2–8 means the melting temperature (Tm) value ofthe seed-target duplex (positions 2–8) and miTm1–5 means
Tm value of 5 -terminal miRNA duplex (positions 1–5)
(Figure 7A). Thus, the seed-target (Tm2–8 ) and 5 -terminal

Downloaded from http://nar.oxfordjournals.org/ at University of Tokyo on September 8, 2014

Relative luciferase activity (%)

0.05 0.5K

sm-wt-441
5’

140
120
100
80
60
40
20
0

140
120
100
80
60
40
20
0

F

3’

5’
check-441

C 3’

G-type miRNA

Control miRNA

Relative luciferase activity (%)

3’

SM-C-target

U 3’C 5’B 5’

Relative luciferase activity (%)

SM-U-target

Control-target

ANucleic Acids Research, 2014, Vol. 42, No. 15 10057

Relative luciferase activity (%)
0

A U-target

20

40

60

80 100 120 140

0.05
0.5

A-type

5
50
0.05
0.5

C-targetI-type

5

**

50

**
**

0.05

C-target

0.5
5G-typemiR-376a-3p

50
miRNA (nM)
Relative luciferase activity (%)
0

20

40

60

80 100 120 140

0.05
0.5
5

A-type50
0.05

C-target

0.5
5

**

I-type

50

*

0.05
0.5

**

C-target5G-typemiR-22-3p

50
miRNA (nM)
Relative luciferase activity (%)

C

0

20

40

60

80

100

120

0.05

U-target

0.5
5

A-type50
0.05

C-target0.5
5

I-type

50

*

0.5

*

0.05

C-target5G-typemiR-191-5p

50
miRNA (nM)

Figure 6. Luciferase reporter assay of silencing efficiencies of A-type, I-type and G-type miRNAs. Wild-type A-type miRNA duplexes [miR-376a-2 (A,blue bar), miR-22 (B, blue bar), or miR-191 duplex (C, blue bar)] and pGL3-Control firefly luciferase expression construct were simultaneously transfected
into HeLa cells with each psiCHECK-SM-U-target luciferase reporter construct, and the relative luciferase activity was measured. Both I-type miRNA
duplexes [miR-376a-2 (A, red bar), miR-22 (B, red bar), or miR-191 duplex (C, red bar)] and G-type miRNA duplexes [miR-376-a-2 (A, yellow bar), miR22 (B, yellow bar), or miR-191 duplex (C, yellow bar)] were respectively transfected with pGL3-Control with its psiCHECK-SM-C-target, and relative
luciferase activity was measured. Each miRNA was transfected at 0.05, 0.5, 5 and 50 nM, respectively. The seed regions and its complementary site are
surrounded by box, and the possible editing sites were highlighted in orange. S indicates cytidine or guanosine, M indicates adenosine or cytidine. (*, 0.05
≥ P > 0.01; **, 0.01 ≥ P, Student’s t-test)

5-bp duplex (miTm1–5 ) stabilities have opposite effects on
gene silencing: miRNAs comprising of an unstable 5 terminal 5-bp duplex and a stable 7-bp seed-target duplex
exhibit strong silencing activity, although the contributionof miTm1–5 might be only about half that of Tm2–8 because
the multiplier coefficient was about 0.5 (12). Therefore, both
the difference in thermodynamic stability between the 5 terminal regions of G-type and I-type miRNA duplexes,

and the difference in seed-target binding strength, are likely
to contribute to the observed difference in silencing efficiency. Thermodynamic stability in the RNA duplex can becalculated using known parameters (35–37). However, since
the thermodynamic parameters of base-pairing between inosine and cytidine have not yet been determined, we annealed both strands of chemically synthesized 5-mer and7-mer RNA oligonucleotides to form the equivalent of ei-

Downloaded from http://nar.oxfordjournals.org/ at University of Tokyo on September 8, 2014

B U-target

10058 Nucleic Acids Research, 2014, Vol. 42, No. 15

Downloaded from http://nar.oxfordjournals.org/ at University of Tokyo on September 8, 2014

Figure 7. Possible thermodynamic control of miRNA-mediated gene-silencing activity and correlations between Tm values of 7-bp seed-target duplexes
and differential fold-changes of expression levels of target transcripts determined by microarray experiments or relative luciferase activities determined byreporter assays. (A) The miRNA with low miTm1–5 value promotes miRNA unwinding into a single-stranded RNA in the RISC, and that with high Tm2–8
value promotes stable base-pairing between miRNA seed region and target mRNA. Thus, the efficacies of miRNA-mediated silencing are determinedby the combined thermodynamic parameters that might reflect their unwinding properties (miTm1–5 ) in addition to their base-pairing stabilities in theseed-target duplex (Tm2–8 ) shown as a formula, miScore = Tm2–8 − 0.5 x miTm1–5 . (B) The duplex structures formed between 7-mer seed sequence ofmiR-376a-3p containing adenosine, inosine, or guanosine in the possible editing site and target mRNA sequence with uridine or cytidine at the opposite
site of editing position, and the measured Tm values of these 7-bp duplexes. The Tm value of the duplex formed between AGAUACU and UCCAUGA
could not be measured, probably due to fairly unstable base-pairing and was shown as not determined (N.D). (C) The correlations between the 7-bp Tm
values and fold-changes in the expression levels of target mRNAs containing seed complementary sequences in their 3 -UTRs (red), or relative luciferase
activities at 50 nM of miRNA duplex (blue). The correlation coefficient (R) between Tm values and differential fold-changes was 0.82, and R between Tm
values and relative luciferase activities was -0.91.

Nucleic Acids Research, 2014, Vol. 42, No. 15 10059

report (15). This may be caused by the structural perturbation due to quite different glycosidic bond angles formedby G:U wobble base pair compared to Watson–Crick basepairing (39), which may make it difficult to form stable duplexes between the miRNA seed region and its target mRNAs on the surface of Ago protein as shown in our previous
paper (15). Finally, our results have implications beyond themiRNA field, as the thermodynamic profiles of C:G and
I:C may also be important for RNA folding, binding affinity with double-stranded RNA-binding proteins, or otherbase-pairing-associated phenomena.
CONCLUSION
On the one hand, inosine behaves like guanosine when interpreted by the translational machinery. On the other hand,inosine has different thermodynamic base-pairing characteristics compared to guanosine: guanosine strongly basepairs with cytidine, while inosine base pairs weakly withcytidine. However, the biological significance of this difference in physico-chemical properties had not been wellunderstood so far. Our microarray analysis reveals that Ato-I editing in the seed region of an miRNA may assign
it to different target mRNAs genome-wide. Indeed, wildtype miRNA downregulates target mRNAs with an uridine
at the complementary opposite site of editing position, but
edited miRNA downregulates target mRNAs with guanosine at that same position. Furthermore, our results effectively demonstrate that the silencing efficiency of miRNAs
with inosine differs from that with guanosine, and that thismay be due to the difference in base-pairing stability between I:C and G:C pairs. In conclusion, our results unambiguously demonstrate the biological significance of thedifference in thermodynamic profiles between inosine andguanosine in the context of RNA duplex formation.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
ACKNOWLEDGEMENT
We thank Mr. Rintaro Ishii for helpful information andcomments for selecting miRNAs used in this study, Dr.
Kenji Nishi for technical advice on the plasmid construction
and microarray experiment, Dr. Yuki Naito for providing
the perl script for looking up seed-matched genes, and Drs.
Gosuke Hayashi and Akimitsu Okamoto for their support
in conducting measurements of the melting temperatures ofRNA duplexes.
Author contributions: H. K. and K. H. performed experiments, J. G. analyzed the microarray data and revised themanuscript, K. U.-T. designed the study and wrote the paper.
FUNDING
Ministry of Education, Culture, Sports, Science and Technology of Japan; Cell Innovation Program (MEXT); Core
Research Project for Private University [to K.U.-T.]. Funding for open access charge: Research grant form Ministryof Education,Culture, Sports, Science and Technology ofJapan [21115004 to K.U.-T.].

Downloaded from http://nar.oxfordjournals.org/ at University of Tokyo on September 8, 2014

ther the 5 -terminal 5-bp duplex and the seed-duplex, respectively, and measured their Tm values using a UV spectrophotometer. In our sequence contexts, sufficient melting curves were obtained only with 7-bp RNA duplexes
but not with 5-bp duplexes in 1M NaCl. We selected miR376a-2 to examine the relationship between silencing efficiency and miScore. Since the editing site is positioned atnucleotide +6 counting from the 5 end in miR-376a-2, themiTm1–5 value of miR-376a-2 is the same for A-type, I-type
and G-type miR-376a-2 duplexes, meaning that we can use
the Tm2–8 value alone instead of miScore. As shown in Figure 7, the measured Tm2–8 value for the A-type seed-duplex(formed by the wild-type 7-mer RNA oligonucleotide seedsequence of miR-376a-3p-A and the wild type U-target 7mer RNA) was 26.6◦ C, whereas that of the G-type seedduplex (formed by the C-target 7-mer and the miR-376a-3pG seed sequence) was the highest at 39.0◦ C. This is conceivable, as G:C pairs are widely known to be more stable than
A:U pairs. Strikingly, the Tm2–8 value of the I-type seedduplex (formed by the C-target 7-mer and the miR-376a-3pI seed sequence) was 23.3◦ C, which is low compared to thatof the G-type duplex and similar to A:U base-pairing. Furthermore, G:U wobble base-pairing showed even weakerstability at 19.3◦ C, and I:U pairing showed the lowest Tm2–8
value at 9.3◦ C. The Tm2–8 value of the duplex formed between AGAUACU and UCCAUGA (A:C pair) could not
be measured, probably due to fairly unstable base-pairing.
The Tm2–8 values and their silencing activities determinedby microarray experiments showed a strong positive correlation (R = 0.82). Consistently, the Tm2–8 value also showeda strong negative correlation with relative luciferase activity
measured at 50 nM of miRNA duplex by reporter assays(R = −0.91), suggesting that at least the Tm2–8 value behaves like a major determinant for miRNA-mediated genesilencing efficiency for a fixed miTm1–5 value. As for miR-22
and miR-191, the editing sites are positioned at +2 and +3from the 5 , respectively, indicating that the accurate miScores could not be determined, since the miTm1–5 values
were not successfully measured. In fact, in the case of themiR-22 duplex, the Tm2–8 values alone were correlated neither with their silencing activities determined by microarrayexperiments (R = 0.36), nor with luciferase activity (R =−0.49) (Supplemental Figure S4). This result suggests thatthe 5 -terminal 5-bp duplex might also be responsible for
miRNA silencing efficacy in the case of inosine-mediatedbase-pairing. Thus, we propose that inosine in the miRNA5 -terminal and seed region has the potential to regulate
miRNA silencing efficiency by regulating base-pairing stability within RNA duplexes.
The relative variability represented by R observed in
Figure 7C for the microarray data may be partly due tothe different set of non-seed sequences in target mRNAs,
which was demonstrated in our and others’ previous reports(15,38). Furthermore, in the case of miR-376a-2, although
the Tm2–8 value of the 7-mer duplex with G:U wobble basepairing was similar with A:U base-pairing and higher thanwith I:U pairing, the results of fold-change of microarrayexperiment as well as relative luciferase activity of reporter
assay revealed that G:U base-pairing exhibits no substantial silencing effect (Figure 7), consistent with our previous

10060 Nucleic Acids Research, 2014, Vol. 42, No. 15

Conflict of interest statement. None declared.
REFERENCES

Downloaded from http://nar.oxfordjournals.org/ at University of Tokyo on September 8, 2014

1. Higuchi,M., Single,F.N., Köhler,M., Sommer,B., Sprengel,R. andSeeburg,P.H. (1993) RNA editing of AMPA receptor subunit
GluR-B: a base-paired intron-exon structure determines position andefficiency. Cell, 75, 1361–1370.
2. Burns,C.M., Chu,H., Rueter,S.M., Hutchinson,L.K., Canton,H.,
Sanders-Bush,E. and Emeson,R.B. (1997) Regulation of serotonin-2C
receptor G-protein coupling by RNA editing. Nature, 387, 303–308.
3. Hoopengardner,B., Bhalla,T., Staber,C. and Reenan,R. (2003)
Nervous system targets of RNA editing identified by comparative
genomics. Science, 310, 832–836.
4. Ohlson,J., Pedersen,J.S., Haussler,D. and Ohman,M. (2007) Editing
modifies the GABA(A) receptor subunit alpha3. RNA, 13, 698–703.
5. Li,J.B., Levanon,E.Y., Yoon,J.K., Aach,J., Xie,B., Leproust,E.,
Zhang,K., Gao,Y. and Church,G.M. (2009) Genome-wide
identification of human RNA editing sites by parallel DNA capturing
and sequencing. Science, 324, 1210–1213.
6. Nishikura,K. (2010) Functions and regulation of RNA editing by
ADAR deaminases. Annu. Rev. Biochem. 79, 321–349.
7. Filipowicz,W. (2005) RNAi: the nuts and bolts of the RISC machine.
Cell, 122, 17–20.
8. Bashati,N. and Cohen,S.M. (2007) microRNA functions. Annu. Rev.
Cell Dev. Biol., 23, 175–205.
9. Kozomara,A. and Griffiths-Jones,S. (2014) miRBase: annotatinghigh confidence microRNAs using deep sequencing data. Nucleic
Acids Res., 42, D68–D73.
10. Lee,Y., Ahn,C., Han,J., Choi,H., Kim,J., Yim,J., Lee,J., Provost,P.,Rådmark,O., Kim,S. et al. (2003) The nuclear RNase III Drosha
initiates microRNA processing. Nature, 425, 415–419.
11. Han,J., Lee,Y., Yeom,K.H., Kim,Y.K., Jin,H. and Kim,V.N.(2004)
The Drosha-DGCR8 complex in primary microRNA processing.
Genes Dev., 18, 3016–3027.
12. Hibio,N., Hino,K., Shimizu,E., Nagata,Y. and Ui-Tei,K. (2012)
Stability of miRNA 5’ terminal and seed regions is correlated withexperimentally observed miRNA-mediated silencing efficacy. Sci.
Rep., 2, 996.
13. Lim,L.P., Lau,N.C., Garrett-Engele,P., Grimson,A., Schelter,J.M.,
Castle,J., Bartel,D.P., Linsley,P.S. and Johnson,J.M. (2005)
Microarray analysis shows that some microRNAs downregulate large
numbers of target mRNAs. Nature, 433, 769–773.
14. Lewis,B.P., Burge,C.B. and Bartel,D.P. (2005) Conserved seedpairing, often flanked by adenosines, indicates that thousands ofhuman genes are microRNA targets. Cell, 14, 15–20.
15. Ui-Tei,K., Naito,Y., Nishi,K., Juni,A. and Saigo,K. (2008)
Thermodynamic stability and Watson-Crick base pairing in the seed
duplex are major determinants of the efficiency of the siRNA-based
off-target effect. Nucleic Acids Res., 36, 7100–7109.
16. Schwarz,D.S., Hutvágner,G., Du,T., Xu,Z., Aronin,N. andZamore,P.D. (2003) Asymmetry in the assembly of the RNAi enzyme
complex. Cell, 115, 199–208.
17. Khvorova,A., Reynolds,A. and Jayasena,S.D. (2003) Functional
siRNAs and miRNAs exhibit strand bias. Cell, 115, 209–216.
18. Ui-Tei,K., Naito,Y., Takahashi,F., Haraguchi,T., Ohki-Hamazaki,H.,
Juni,A., Ueda,R. and Saigo,K. (2004) Guidelines for the selection ofhighly effective siRNA sequences for mammalian and chick RNAinterference. Nucleic Acids Res., 32, 936–948.
19. Luciano,D.J., Mirsky,H., Vendetti,N.J. and Maas,S. (2004) RNA
editing of a miRNA precursor. RNA, 10, 1174–1177.
20. Kawahara,Y., Megraw,M., Kreider,E., Iizasa,H., Valente,L.,
Hatzigeorgiou,A.G. and Nishikura,K. (2008) Frequency and fate of
microRNA editing in human brain. Nucleic Acids Res., 36,
5270–5280.
21. Chiang,H.R., Schoenfeld,L.W., Ruby,J.G., Auyeung,V.C., Spies,N.,
Baek,D., Johnston,W.K., Russ,C., Luo,S., Babiarz,J.E. et al. (2010)
Mammalian microRNAs: experimental evaluation of novel andpreviously annotated genes. Genes Dev., 24, 992–1009.

22. Linsen,S.E., de Wit,E., de Bruijn,E. and Cuppen,E. (2010) Small
RNA expression and strain specificity in the rat. BMC Genomics, 11,249.
23. Schulte,J.H., Marschall,T., Martin,M., Rosenstiel,P., Mestdagh,P.,
Schlierf,S., Thor,T., Vandesompele,J., Eggert,A., Schreiber,S. et al.
(2010) Deep sequencing reveals differential expression of microRNAs
in favorable versus unfavorable neuroblastoma. Nucleic Acids Res.,38, 5919–5928.
24. Berezikov,E., Robine,N., Samsonova,A., Westholm,J.O., Naqvi,A.,
Hung,J.H., Okamura,K., Dai,Q., Bortolamiol-Becet,D., Martin,R.
et al. (2011) Deep annotation of Drosophila melanogastermicroRNAs yields insights into their processing, modification, andemergence. Genome Res., 21, 203–215.
25. Wulff,B.E. and Nishikura,K. (2012) Modulation of microRNA
expression and function by ADARs. Curr. Top. Microbiol. Immunol.,353, 91–109.
26. Ekdahl,Y., Farahani,H.S., Behm,M., Lagergren,J. and Öhman,M.
(2012) A-to-I editing of microRNAs in the mammalian brainincreases during development. Genome Res., 22, 1477–1487.
27. Yao,M.J., Chen,G., Zhao,P.P., Lu,M.H., Jian,J., Liu,M.F. and
Yuan,X.B. (2012) Transcriptome analysis of microRNAs indeveloping cerebral cortex of rat. BMC Genomics, 13, 232.
28. Yang,W., Chendrimada,T.P., Wang,Q., Higuchi,M., Seeburg,P.H.,
Shiekhattar,R. and Nishikura,K. (2006) Modulation of microRNA
processing and expression through RNA editing by ADAR
deaminases. Nat. Struct. Mol. Biol., 13, 13–21.
29. Kawahara,Y., Zinshteyn,B., Chendrimada,T.P., Shiekhattar,R. andNishikura,K. (2007) RNA editing of the microRNA-151 precursor
blocks cleavage by the Dicer-TRBP complex. EMBO Rep., 8,
763–769.
30. Iizasa,H., Wulff,B.E., Alla,N.R., Maragkakis,M., Megraw,M.,
Hatzigeorgiou,A., Iwakiri,D., Takada,K., Wiedmer,A., Showe,L.
et al. (2010) Editing of Epstein-Barr virus-encoded BART6
microRNAs controls their dicer targeting and consequently affects
viral latency. J. Biol. Chem., 285, 33358–33370.
31. Kawahara,Y., Zinshteyn,B., Sethupathy,P., Iizasa,H.,
Hatzigeorgiou,A.G. and Nishikura,K. (2007) Redirection of silencing
targets by adenosine-to-inosine editing of miRNAs. Science, 315,
1137–1140.
32. Bolstad,B.M., Irizarry,R.A., Gautier,L. andWu,Z. (2005) Preprocessing high-density oligonucleotide arrays. In:
Gentleman,R., Carey,V., Huber,W., Irizarry,R. and Dudoit,S. (eds.),
Bioinformatics and Computational Biology Solutions Using R and
Bioconductor. Springer, New York, pp. 13–32.
33. Hagihara,S., Kumasawa,H., Goto,Y., Hayashi,G., Kobori,A., Saito,I.
and Nakatani,K. (2004) Detection of guanine-adenine mismatches by
surface plasmon resonance sensor carrying
naphthyridine-azaquinolone hybrid on the surface. Nucleic Acids
Res., 32, 278–286.
34. Grimson,A., Farh,K.K., Johnston,W.K., Garrett-Engele,P., Lim,L.P.
and Bartel,D.P. (2007) MicroRNA targeting specificity in mammals:
determinants beyond seed pairing. Mol. Cell, 27, 91–105.
35. Xia,T., SantaLucia,J. Jr, Burkard,M.E., Kierzek,R., Schroeder,S.J.,
Jiao,X., Cox,C. and Turner,D.H. (1998) Thermodynamic parameters
for an expanded nearest-neighbor model for formation of RNA
duplexes with Watson-Crick base pairs. Biochemistry, 37,
14719–14735.
36. Mathews,D.H., Sabina,J., Zuker,M. and Turner,D.H. (1999)
Expanded sequence dependence of thermodynamic parameters
improves prediction of RNA secondary structure. J. Mol. Biol., 21,
911–940.
37. Daniel,J.W., Jamie,L.R., Dawn,M.Y. and Brent,M.Z. (2007) Nearest
neighbor parameters for inosine-uridine pairs in RNA duplexes.
Biochemistry, 46, 4625–4634.
38. Jackson,A.L., Bartz,S.R., Schelter,J., Kobayashi,S.V., Burchard,J.,
Mao,M., Li,B., Cavet,G. and Linsley,P.S. (2003) Expression profilingreveals off-target gene regulation by RNAi. Nat. Biotech, 21, 635–637.
39. Varani,G. and McClain,W.H. (2000) The G:U wobble base pair.
EMBO J., 1, 18–23.