International Journal of

Molecular Sciences
ReviewIs the Efficiency of RNA Silencing
Evolutionarily Regulated?
Kumiko Ui-Tei 1,2
12

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-0033, Japan; ktei@bs.s.u-tokyo.ac.jp; Tel.: +81-3-5841-3043
Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences,
The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa-shi, Chiba-ken 277-8561, Japan

Academic Editor: Y-h. Taguchi
Received: 12 April 2016; Accepted: 9 May 2016; Published: 12 May 2016

Abstract: Small interfering RNAs (siRNAs) and microRNAs (miRNAs) regulate gene expression ina sequence-specific manner. Genes with partial complementarity to siRNA/miRNA sequences intheir 31 -untranslated regions (UTRs) are suppressed by a mechanism referred to as the siRNA
off-target effect or miRNA-mediated RNA silencing. However, the determinants of such RNA
silencing efficiency are poorly understood. Previously, I and co-workers reported that the efficiency ofRNA silencing is strongly correlated with the thermodynamic stability of base pairing in the duplex
formed within an siRNA/miRNA and between the seed region and its target mRNA. In this review,
I first summarize our previous studies that identified the thermodynamic parameter to estimate thesilencing efficiency using the calculated base pairing stability: siRNAs downregulate the expressionof off-target genes depending on the stability of binding between the siRNA seed region (nucleotides2–8) and off-target mRNAs, and miRNAs downregulate target mRNA expression depending on thestability of the duplex formed between the 51 terminus of the miRNA and its target mRNA. I further
discuss the possibility that such thermodynamic features of silencing efficiency may have arisen
during evolution with increasing body temperature in various organisms.
Keywords: RNA silencing;thermodynamic stability

siRNA; miRNA; silencing efficiency;

base-pairing stability;

1. Introduction
MicroRNAs (miRNAs) are a large family of small non-coding RNAs (ncRNAs) that repress gene
expression by inhibiting translation and promoting mRNA decay. More than 2000 miRNAs have been
identified in human cells; they are predicted to regulate the activity of hundreds of protein-coding
genes to control various aspects of biological processes, including development, differentiation,
proliferation, antiviral defense, and metabolism. They also act as tumor suppressors or oncogenes
and are often dysregulated in tumors. The target genes of miRNAs can be computationally predictedbased on experimentally validated algorithms [1–3]. However, to understand the regulation of gene
expression mediated by miRNA silencing, it is essential to predict the silencing efficiency of eachmiRNA accurately in addition to its target genes.
Primary miRNAs (pri-miRNAs) are sequentially processed by the double-stranded RNA cleavage
enzymes Drosha and Dicer. Drosha cleaves pri-miRNAs to generate precursor-miRNAs (pre-miRNAs)in the nucleus [4–6]; these pre-miRNAs are further processed by Dicer to generate ~22-nucleotide (nt)miRNA duplexes in the cytoplasm [5,7,8]. The miRNA duplex is loaded onto Argonaute (AGO),
creating an RNA-induced silencing complex (RISC) [9–13], and subsequently unwound into asingle-stranded mature miRNA in the RISC [12,13]. The retained RNA strand acts as a guide toInt. J. Mol. Sci. 2016, 17, 719; doi:10.3390/ijms17050719

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a guide to recruit the RISC to the 3′-untranslated region (UTR) of a target mRNA withrecruitthe RISC to the
31 -untranslated(UTR)2–8,
of atotarget
mRNA
with complementarity
to thecomplementarity
to theseed region, region
at positions
promotetranslational
repression [14–16]
seedregion,at
positions
2–8,
topromotetranslational
repression[14–16](Figure1).
(Figure 1).

Figure 1. Schematic figure showing the pathway of small interfering RNA (siRNA)-mediatedFigure 1. Schematic figure showing the pathway of small interfering RNA (siRNA)-mediatedRNA interference (RNAi) and microRNA (miRNA)-mediated RNA silencing. The mechanisms of
RNA interference (RNAi) and microRNA (miRNA)-mediated RNA silencing. The mechanisms
downregulation of off-target genes in the RNAi pathway and that of target genes in miRNA-mediatedof downregulation of off-target genes in the RNAi pathway and that of target genes in
RNA silencing are similar: The seed region of the siRNA/mRNA recognizes the 31 -UTRs of off-target
miRNA-mediated RNA silencing are similar: The seed region of the siRNA/mRNA recognizes theor target genes and downregulates their expression. The efficiency of the off-target effect by functional
3′-UTRs of off-target or target genes and downregulates their expression. The efficiency of thesiRNAsis dependent
on the thermodynamic
stability
between stability
the seedofregion
andoff-target
effect by functional
siRNAs is dependent
onofthebinding
thermodynamic
bindingthebetween
off-target
transcript.
However,miRNA-mediatedsilencingefficiency
isdependentonthe seed region and the off-target transcript. However, miRNA-mediated silencingthe
1 -terminal miRNA duplex,
thermodynamic
stabilityonof thebindingin both thestability
seed-target
duplexand 5theefficiency is dependent
thermodynamicof binding
in bothseed-target duplex andprobably
becausethe duplex,
secondarystructureof theis different
thatof theissiRNA.
5′-terminal
miRNA
probably
becausethemiRNA
secondarystructurefromof themiRNA
different fromthat of the siRNA.

Another type of small ncRNA, small interfering RNA (siRNA), is widely used as a tool ofAnother typeof small ncRNA,
small
interfering
RNA (siRNA),is widelyused astool ofthe
loss-of-function
experiments.
The siRNA
is usually
introduced
into cells
exogenously
to areduce
loss-of-function
experiments.
The siRNARNAs
is usuallycells
exogenously
to reducetheexpressionof genes
as double-stranded~21 introduced
nt in lengthinto
with
2-nt
31 overhangs.
The guide
expression
of
genes
as
double-stranded
RNAs
~21
nt
in
lengthwith
2-nt
3′overhangs.
The
guide
strand of the siRNA shares full sequence complementarity with the coding sequence of its intended
strand of the siRNA shares full sequence complementarity with the coding sequence of its intended
target mRNA and triggers enzymatic cleavage of the mRNA by AGO, the catalytic component oftarget mRNA and triggers enzymatic cleavage of the mRNA by AGO, the catalytic component of thethe RISC, between nts 10 and 11 via RNA interference (RNAi) [17–22] (Figure 1). In addition, theRISC, between nts 10 and 11 via RNA interference (RNAi) [17–22] (Figure 1). In addition, the siRNA
siRNA guide strand recognizes many mRNA 3’-UTRs with only partial complementarity to the seedguide strand recognizes many mRNA 3’-UTRs with only partial complementarity to the seed region
region and downregulates their expression. This effect is referred to as an off-target effect (Figure 1).
Such off-target effects are considered to be induced through a mechanism similar to that of target

Int. J. Mol. Sci. 2016, 17, 719

3 of 10and downregulates their expression. This effect is referred to as an off-target effect (Figure 1). SuchInt. J. Mol. Sci. 2016, 17, 719
3 of 10
off-target effects are considered to be induced through a mechanism similar to that of target
silencing by miRNA [2,3,23]. Thus, miRNA-mediated silencing and siRNA-based off-target effects
arethought
to share[2,3,23].
similarThus,silencingmachinery throughtarget recognition
processes
silencingby miRNAmiRNA-mediatedsilencingcommonand siRNA-based
off-target effects
are
using
thetoseedregion.
thought
sharesimilar silencing machinery through common target recognition processes using theseed region.
2. The Silencing Efficiency of siRNAs on Off-Target mRNAs Is Regulated by the Stability of Base
Pairing
between thesiRNA of
Seed
Region
Off-Target
mRNA
2. The Silencing
Efficiency
siRNAsonand
Off-Target
mRNAs
Is Regulated by the Stability ofBase Pairing between the siRNA Seed Region and Off-Target mRNA
The mechanism of siRNA-induced off-target effects is similar to miRNA-mediated target
mechanismof siRNA-induced
off-target effects
is similar
to miRNA-mediatedtargetpartial
RNA
RNAThesilencing,which
regulates the expressionof many
unintended
transcripts withsilencing, which regulates
the expressionof many transcripts
unintendedaretranscripts
with partial
complementarity.
complementarity.
The 3′-UTRsof the off-target
complementary
to theguide strand
1 -UTRs of the off-target transcripts are complementary to the guide strand seed region. In the RISC,
The 3region.
seed
In the RISC, the seed nucleotides of the guide strand loaded onto AGO are present on its
the seedinnucleotidesof theform
guideloadedonto orAGO
are present
surface ina quasi-helicalsurfacea quasi-helical
tostrandserve asthe entry
nucleation
siteonforitsoff-target
mRNAs[24,25].
serve as theclarified
entry or nucleationsite foroff-target
mRNAsthe[24,25].
I and co-workers
clarified
Iform
andtoco-workers
the molecular
basis
determining
efficiencyof seed-dependent
the molecularbasis determining
the stability
efficiencyofofthe
seed-dependent
off-target
silencingin terms
off-target
silencingin terms of theprotein-free RNA
duplex[26] (Figure1).ofIntheastabilityoftheprotein-freeRNAduplex[26](Figure1).
Inaprevious
study,
weusedsiRNAswithprevious study, we used siRNAs with A or U residues at position 1 from the 5′ end of the siRNAA or Ustrand,
residues
at position
1 fromthe in51 endof the siRNA
guide
strand,three to
guide
three
to six A/Uresidues
nt positions
2–7, anda G/Cat position
19,sixas A/U
theseresidues
siRNAsinnt
positions
2–7,
and
a
G/C
at
position
19,
as
these
siRNAsarehighlyfunctional
[27].
The
are highly functional [27]. The guide strand of such highly functional siRNA is expected guideto bestrand of suchinto
highlyis expected
to be incorporated
into the RISC
veryfrom
effectively
incorporated
thefunctional
RISC verysiRNA
effectively
by unwinding
into single-strandedRNA
its 5′
1 terminal. Similarly, other groups also reported
by unwinding
into single-strandedRNAfrom its
terminal.
Similarly,
other groups also
reported
that5 thefunctional siRNAs have such asymmetry inthat thefunctional
siRNAssuch asymmetrybasesuchpairingstability of
thebe
two
[28,29].
base
pairingstabilityof thehave
two terminals[28,29].inThus,
asymmetrymay
oneterminalsof the essential
Thus, suchmay beone of theessentialfeaturesfor thefunctional
siRNAs.
However,featuresforasymmetry
the functional
siRNAs.
However,theterminal
asymmetrydid
not controlthetheterminal
asymmetrydidnotcontrolthe“efficiency”
of
the
siRNA
off-target
effect.
Therefore,to“efficiency” of the siRNA off-target effect. Therefore, to find out the determinant of the “efficiency”
find
out
the
determinantofthe“efficiency”
of
the
siRNA
off-target
effect,we
usedsiRNAssatisfyingof the siRNA off-target effect, we used siRNAs satisfying all of the highly functional siRNA
all of the highly
functional
characteristics
identified
oursiRNAs,
previouswe
report[27]. Usingthesecharacteristics
identified
in siRNA
our previous
report [27].
Using inthese
measuredthe siRNA
siRNAs,wemeasuredthesiRNA
off-target
effectsbyaluciferase
reporterassay
andmicroarray
off-target effects by a luciferase reporter assay and microarray experiments. In the luciferase reporter
experiments.
In theluciferase reporter
assay,
seedinto
complementary
sequence
was
insertedgene,intoassay,
each seed
complementary
sequence
waseachinsertedthe 3′-UTR regionof theluciferase
1 -UTR region of the luciferase gene, and the luciferase activity, the indicative of the expression
the 3theandluciferase activity, the indicative of the expression level, was measured with a luminometerlevel, thewas transfection
measured witha luminometer
afterresults
the transfectioneach
The resultsoff-target
revealed
afterof eachsiRNA. The
revealed ofthatthesiRNA.
seed-dependentthat the seed-dependent
efficiencycorrelated
was positively
correlated withefficiency
was positivelyoff-target
and negatively
with andthe negatively
melting temperature(Tm)the(rmelting= 0.74,temperature
(Tm)
(r
=
0.74,
Figure2a)andstandard
freeenergychange(∆G)(r=´0.69),
respectively,
Figure 2a) and standard free energy change (ΔG) (r = −0.69), respectively, calculated by thecalculated by the nearest-neighborprocedure[26] forof
thetheformationof the duplex
betweenseed
the siRNAnearest-neighborprocedure [26] forthe formation
duplex between
the siRNA
regionseed region2–8)(positions
and the complementary
3’-UTRin thetranscript
off-target[27].
transcript(positions
and the2–8)
complementary
3’-UTR sequence
insequence
the off-target
Thus, [27].
our
Thus,
our
results
indicated
that
the
high
base
pairingstability
shown
as
Tm
in
the
duplex
formedresults indicated that the high base pairing stability shown as Tm2–8 in the 2–8
duplex formed between
betweentheseedsiRNAseedandregionthe3’-UTRtarget 3’-UTR
strongly
reduced
off-target
transcript
expression,
the
siRNA
regiontheandtargetstrongly
reduced
off-target
transcript
expression,
but
but that
the lowstability
exerteda veryweak
effect.
that
the lowstability
exerteda veryweak
effect.

FigureFigure 2.
2. Cont.
Cont.

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4 of 10Int. J. Mol. Sci. 2016, 17, 719

4 of 10Figure 2.
2. Relationships
Relationships between
between silencing
silencing efficiency
efficiency andand thermodynamic
thermodynamic parameters.
parameters. The
The y-axis
y-axisFiguremeans thethe silencing
silencing efficiency,
efficiency, indicating
indicating thethe value
value (%)(%) subtracted
subtracted thethe measured
measured luciferase
luciferase activity
activity
means
(%)
from
100.
(a)
The
calculated
Tm-valueoftheduplex
formedbetween
the
siRNA
seed
regionand(%) from 100. (a) The calculated Tm-value of the duplex formed between the siRNA seed region andtarget
mRNA
(Tm
2–8
)
showedapositivecorrelation
withsiRNA-mediatedsilencingefficiency;
target mRNA (Tm2–8 ) showed a positive correlation with siRNA-mediated silencing efficiency; (b) The(b) The calculated
Tm2–8
valuesa showedmarginal withcorrelation
with miRNA-mediatedsilencing
calculatedTm2–8 values
showedmarginalacorrelation
miRNA-mediatedsilencing efficiency;(c)efficiency;(c) The miRNA-mediatedsilencingshowed correlation
a strong positive
The
miRNA-mediatedsilencing efficiency
showedefficiency
a strong positive
with the correlation
parameter
1 -terminal
with the parametercomposedTm2–8 andmiRNA
5′-terminal
stability (miTm1–5), shown ascomposedof Tm2–8 andmiRNA 5ofstability(miTm
1–5 ), shown as Tm2–8 ´ 0.5 ˆ miTm1–5 .
Tm2–8 − 0.5 × miTm1–5.

The main factor regulating off-target silencing efficiency is considered to be the thermodynamic
The main factor regulating off-target silencing efficiency is considered to be the thermodynamic
stability of base pairing between the siRNA seed region and off-target transcript. However,stability of base pairing between the siRNA seed region and off-target transcript. However,considerable deviation was observed for the off-target efficiencies measured by the reporter assay
considerable deviation was observed for the off-target efficiencies measured by the reporter assay
even when the same siRNA was used. These results suggested that the non-seed region sequence may
even when the same siRNA was used. These results suggested that the non-seed region sequencealso slightly affect silencing efficiency. We recently demonstrated that there are additional auxiliary
may also slightly affect silencing efficiency. We recently demonstrated that there are additional
factors that regulate the degree of downregulation of off-target transcripts [30]; both the Tm-value
auxiliary factors that regulate the degree of downregulation of off-target transcripts [30]; both theof the siRNA non-seed region at positions 8–15 and the GC contents of its corresponding off-target
Tm-value of the siRNA non-seed region at positions 8–15 and the GC contents of its correspondingtranscripts are negatively correlated with the off-target silencing efficiency.
off-target transcripts are negatively correlated with the off-target silencing efficiency.
3. The Efficiency of Silencing of Target mRNAs by miRNAs Is Regulated by the Stability of Base
3. The Efficiencyof Silencing
TargetandmRNAsby miRNAs
Is Regulated
the Stabilityof BasePairingin the 51 Regionof the ofmiRNA
that between
the miRNA
Seed byRegionandPairingmRNAs
in the 5′ Region of the miRNA and that between the miRNA Seed Region andTargetTarget mRNAs
Target mRNAs are recognized by miRNAs via a mechanism similar to the siRNA off-target effect,
TargetmRNAs
mRNAs complementary
are recognized bytomiRNAs
via a seedmechanismto thedownregulatedsiRNA off-target
effect,and target
the miRNA
region similar
are mainly[2,3,23].
andtargetmRNAs
complementary
to
the
miRNA
seed
regionaremainlydownregulated[2,3,23].
However, unlike siRNA off-target silencing, Hibio et al. previously showed that the efficiency ofHowever, unlike siRNA
off-target
Hibio et withal. previously
showedthatthe efficiency
ofmiRNA-mediatedsilencing
was notsilencing,
simply correlated
the Tm-valueof theseed-target
duplexmiRNA-mediated
was not differences
simply correlated
the Tm-valueof the seed-target
duplex(Figure2b) [31]. Thesilencing
most prominent
betweenwith
siRNAsand most miRNAs
are the structural(Figure of2b)the[31].
The most prominent
differences
betweensiRNAs duplexes,
and mostwhile
miRNAsare thefeaturesduplex—siRNAs
usually form
completely
complementary
most miRNA
structuralhave
featuresof theduplex—siRNAs
usuallyform
complementary
duplexes,
while
duplexes
internal
bulgesor mismatches.
Hibio
et al.completely[31] evaluatedthe involvementof miRNA
most miRNA
have
internal
bulges orthemismatches.
[31] evaluatedtheduplexes
formedduplexesin any given
regionby quantifyingbase pairing Hibio
stabilityetbyal.calculating
Tm-values
involvement of
duplexes
formedbulges
in anygivenmismatches
region by quantifyingbase pairing
incorporating
themiRNAparametersof internal
and/orassigned as thethe Tm-valueinstability
by duplex
calculating
Tm-values
incorporating
thewereparametersof tointernalbulgesand/orthe
miRNA(miTm).
Luciferase
reporter assays
performed
determine
the optimalmismatches
assignedthetheTm-valueinefficiency.
the miRNA
duplex(miTm).
Luciferase
assayswere
miRNA
duplexregionasfor
silencing
Our
results
indicatedthat thereporter
silencingefficiency
performed
to isdeterminethedetermined
optimal miRNAregion forthe silencing efficiency.
Ourofan miRNA
principallyby theduplex
combinatorial
thermodynamic
parametersof results
Tm2–8
indicated thatthe silencing
efficiency
of an miRNAis principallyby the combinatorial(Tm-value
betweenthe miRNAseed region
at positions
2–8 and itsdetermined
perfectly complementarytargetthermodynamic
parametersof Tmof
2–8the(Tm-value
betweenmiRNA
seed
at positions 2–8
sequence)
and miTm
miRNA duplex
at thepositions
1–5),
andregionthat miRNA-mediated
1–5 (Tm-value
and its perfectly
complementarytarget withsequence)
and miTm
1–5 (Tm-valuetheˆmiRNAatsilencingefficiency
is strongly correlated
the following
formula:Tm2–8 ´of0.5
miTm1–5duplex(r = 0.74,
positionsandmiRNA-mediatedis strongly
theFigure2c) 1–5),[31]. In
thisthatformula,Tm2–8 reflectssilencing
the base efficiency
pairing stability
in thecorrelated
seed-targetwith
duplex;following
formula:2–8 − 0.5
× miTm
1–5 (r
= 0.74,[31]. silencing
In this formula,Tm2–8
reflects 1–5
thestrong(weak)
base Tmpairingstabilitymay
resultin Figurestrong 2c)(weak)
efficiency.
Positionsbase pairing stability in the seed-target duplex; strong (weak) base pairing stability may result instrong (weak) silencing efficiency. Positions 1–5 correspond to the 5′-terminal end of the miRNA,Int. J. Mol. Sci. 2016, 17, 719

5 of 10correspond
5117,-terminalend of the miRNA, which is essential to determine the direction of5 ofmiRNA
Int. J. Mol. to
Sci.the
2016,
719
10
unwinding. An miRNA molecule with low stability can be easily unwound into single-stranded RNA
essential andto determine
the direction
miRNA
unwinding.
An miRNA
with low
fromwhichthe 51isterminus
incorporated
into the ofRISC[27–29].
Thus, miRNAs
withmolecule
low miTm
1–5 values
stabilitycanbeeasilyunwoundinto
single-stranded
RNA
from
the
5′
terminusandincorporated
may enhance target RNA silencing according to the ease of unwinding, while those with a highinto the RISC [27–29]. Thus, miRNAs with low miTm1–5 values may enhance target RNA silencing
miTm1–5 may repress target RNA silencing according to the difficulty of unwinding. Thus, althoughaccording to the ease of unwinding, while those with a high miTm1–5 may repress target RNA
positions 2–5 overlap between the seed region and 51 -terminal region (1–5), the results of Hibio et al.
silencing according to the difficulty of unwinding. Thus, although positions 2–5 overlap between theindicated that the miTm1–5 and Tm2–8 values have opposite effects. In addition, it was necessary toseed region and 5′-terminal region (1–5), the results of Hibio et al. indicated that the miTm1–5 andsubtractvalue
of miTmfrom thatof Tm2–8ittowas
measurethe tosilencinga high
1–5 effects.
Tm2–8thevalueshave
opposite
In addition,necessary
subtractefficiency;
the value of
miTmTm
1–5 2´8
value
and
low
miTm
value
will
increasethesilencing
activity,
while
low
Tm
and
high
miTm
1–5
2–8
from that of Tm2–8 to measure the silencing efficiency; a high Tm2−8 value and low miTm1–5 value will 1–5
valuesdecreasesilencing
activity.
Furthermore,
the highfactormiTm
0.5 forindicates
the effectincrease
the thesilencing
activity,
while
low Tm2–8 and1–5 miTm
values1–5
decrease
thethatsilencingof Tm
efficiency
is about
two-foldstrongerthanthat
miTmof1–5Tm. 2–8 on silencing
activity.
Furthermore,
the factor
0.5 formiTm1–5
indicatesthat
theofeffect
2–8 on silencing
efficiency
is about
two-foldonline:
strongerthan that of miTm1–5.
In miRBase(Available
http://www.mirbase.org/),
two miRNAs originating from the
In miRBase(Available
online:http://www.mirbase.org/),
miRNAs originatingfromthe issame predictedprecursor
are often
annotated.
Among them, thetwo
predominantly
expressed
miRNA
same
predicted
precursor
areoften
annotated.
Among
them,
the
predominantly
expressed
miRNA
referred to as miR-xxx, and the opposite arm of the precursor is designated as miR-xxx*, as shownis referred
to as miR-xxx,
the oppositeof theprecursor
is values
designated
miR-xxx*,shown
in Figure
3. Hibio
et al. [31]and
calculated
the armTm2–8
andmiTm1–5
for aseach
side ofasthe
miRNA
in Figure 3. Hibio et al. [31] calculated the Tm2–8 and miTm1–5 values for each side of the miRNA fromfrom 16 sets of miRNA duplexes (Figure 3). Interestingly, most of the predominant miRNAs showed
16 sets of miRNA duplexes (Figure 3). Interestingly, most of the predominant miRNAs showed
higher Tm2–8 values (14/16, 88%) and lower miTm1–5 values (11/16, 69%) compared to the opposite
higher Tm2–8 values (14/16, 88%) and lower miTm1–5 values (11/16, 69%) compared to the opposite
miRNAs, and the resultant Tm2–8 ´ 0.5 ˆ miTm1–5 values increased more (13/16, 81%) than themiRNAs, and the resultant Tm2–8 − 0.5 × miTm1–5 values increased more (13/16, 81%) than the
opposite
miRNAs.
Thus,althoughseedpositions
positions2–8
2–8andandthethe 51
opposite
miRNAs.
Thus,althoughpositions
positions2–5
2–5overlappedoverlapped between
between seedterminus(positions
1–5),1–5),
the predominant
miRNA
may
toreduce
reducethethe 515′ terminus(positions
the predominantmiRNA
mayform
formaasecondary
secondary structure
structure toterminal
Tm-valuein theinmiRNA
duplex,even even
if theifTm-valueof theofperfectcomplementary
duplex5′ terminal
Tm-valuethe miRNA
duplex,the Tm-value
the perfectcomplementary
formedbetweenseed region
andthe targetmRNA
high. isThus,
may regulatesilencingduplex
formeditsbetween
its seedregionand thetargetismRNA
high.miRNAs
Thus, miRNAs
may regulatesilencingefficiency theby changing
secondarystructure
their toown
duplexreducethe base
efficiency
by changing
secondarythe
structureof theirown ofduplexreducethetobasepairingstability,
1
pairingstability,
especiallyinthe5′-terminal
region.
especially in the 5 -terminal region.

Figure 3. Calculated Tm2–8, miTm1–5, and Tm2–8 − 0.5 × miTm1–5 values for the miRNA families let-7,

Figure 3. Calculated Tm2–8 , miTm1–5 , and Tm2–8 ´ 0.5 ˆ miTm1–5 values for the miRNA families let-7,
miR-125, and miR-34.
miR-125, and miR-34.

Int. J. Mol. Sci. 2016, 17, 719

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In addition to positions 1–5, a weak but significant correlation between silencing efficiency andthe thermodynamic profile of the miRNA duplex was also observed at positions 13–17 [30]. Thus,
weak stability at non-seed positions 8–15 may increase the miRNA-mediated silencing efficiency.
4. The Base Pairing Stabilities of miRNAs in Various Species are Strongly Correlated with
Body Temperature
RNA silencing mediated by miRNAs is a conserved phenomenon in a broad range ofmetazoans [32]. We found that the silencing efficiency of miRNAs could be determined by thethermodynamic properties of protein-free RNA duplexes, Tm2–8 and miTm1–5 . As the state ofnucleotide base pairing is regulated by thermodynamic properties, growth temperature is important toregulate the silencing efficiency of miRNAs. An RNA duplex with low thermodynamic stability is notformed in the cells of an organism growing at a high temperature, but such a duplex could be formed ata low growth temperature. In contrast, an RNA duplex with high thermodynamic stability would not
be unwound into single-stranded RNAs at low temperatures, although it could be unwound at hightemperatures. Therefore, it is likely that organisms growing at high temperatures will have miRNAs
with strong base pairing stability, while those with low growth temperatures will have miRNAs withweaker base pairing.
We calculated the thermodynamic parameters of miRNAs from 16 different organisms registeredin miRBase: Tm2–8 , miTm1–5 , and Tm2–8 ´ 0.5 ˆ miTm1–5 . The average Tm2–8 values varied from 30 to38 ˝ C in these organisms (Figure 4a). The planarian Schmidtea mediterranea and ascidian Ciona intestinalis
are heterothermic animals, but they are usually maintained at temperatures of approximately 10–15 ˝ C.
The African frog Xenopus tropicalis, the nematode Caenorhabditis elegans, the silkworm Bombyx mori,Drosophila melanogaster, Drosophila pseudoobscura, the lamprey Petromyzon marinus, and the zebrafish
Danio rerio are reared at 23–27 ˝ C. Homothermic animals such as humans (Homo sapiens), mice(Mus musculus), dogs (Canis familiaris), horses (Equus caballus), orangutans (Pongo pygmaeus), and pigs
(Sus scrofa) have body temperatures of about 37 ˝ C. The body temperature of chicken (Gallus gallus) isthe highest at about 42 ˝ C. Our results clearly showed that the average Tm2–8 values of animals grown
at a low temperature were low, while those grown at higher temperatures were high (Figure 4a). The
correlation coefficient was high at r = 0.83.
The average miTm1–5 values for the miRNAs of 16 different organisms were also calculated. The
values varied from ´25 to ´9 ˝ C (Figure 4b). With the exception of B. mori (miTm1–5 = ´23.0 ˝ C) andC. intestinalis (miTm1–5 = ´22.2 ˝ C), which showed significantly low values, the miTm1–5 values ofthe organisms were low and similar (around ´15 to ´10 ˝ C). The correlation coefficient between themiTm1–5 values and growth temperatures was very low at r = 0.44 (Figure 4b).
The thermodynamic parameters of miRNA silencing efficiency, Tm2–8 – 0.5 ˆ miTm1–5 values,
were calculated using miRNAs from 16 organisms. The values varied less from 38 to 44 ˝ C (Figure 4c).
Although the values for B. mori (Tm2–8 – 0.5 ˆ miTm1–5 = 44.2 ˝ C) and C. intestinalis (Tm2–8 ´ 0.5 ˆ
miTm1–5 = 43.2 ˝ C) were high compared to those organisms with similar body temperatures, otherorganisms showed strong correlations, with a correlation coefficient for the 16 organisms of r = 0.60
(Figure 4c).
As a control, the average Tm-values of all 7-mer duplexes in tRNA sequences were calculated,
and almost no correlation was observed (r = 0.12, Figure 4d).
These observations clearly supported the idea that the organisms with high growth temperature
have miRNAs with strong base pairing stability, while those with low growth temperature havemiRNAs with weaker base pairing.
Some miRNAs are conserved across species [33–37]. However, miRNAs with the same sequenceconserved in different organisms with different body temperatures are unlikely to show the same
silencing activities, as base pairing stability is strongly affected by temperature.

Int. J. Mol. Sci. 2016, 17, 719
Int. J. Mol. Sci. 2016, 17, 719

7 of 10
7 of 10Figure 4. Strong correlations between the silencing parameters Tm2–8 and miTm1–5, and the growth
Figure 4. Strong correlations between the silencing parameters Tm2–8 and miTm1–5 , and the growthtemperatures of various organisms. (a) A strong correlation was found between Tm2–8 and growthtemperatures of various organisms. (a) A strong correlation was found between Tm2–8 and growthtemperature; (b) A marginal correlation was found between miTm1–5 and growth temperature;temperature; (b) A marginal correlation was found between miTm1–5 and growth temperature;(c) A strong correlation was found between Tm2–8 − 0.5 × miTm1–5 and growth temperature;(c) (d)A strong
waswasfound
betweenTm´ 0.5 ˆmiTm1–5forandtemperature;
2–8average
Almostcorrelation
no correlation
found
betweentheTm-values
allgrowth7-mer tRNAsequences(d)Almostnocorrelation
was
found
betweentheaverageTm-values
for
all
7-mer
tRNA
sequencesandand growth temperature.
growth temperature.

5. Perspectives
5. Perspectives
Biological functions are regulated by the environmental temperature, which influences the
Biological reactions
functionsinside
are regulated
the environmental
influences it
thebiochemical
the cell. Aby
specialcharacteristic of temperature,temperature iswhich
its pervasiveness;biochemical
reactionsallinside
the cell. Ainspecial
ofmorphological,
temperature isphysiological,
its pervasiveness;
can affect virtuallymacromolecules
the cell.characteristic
As behavioral,
and
it canaffectvirtually
all macromoleculesin the bycell.temperature,
As behavioral,
morphological,
physiological,
and
other
variablefunctionscan be regulated
it waspredicted thatevolutionary
adaptationthese phenomena
would bybe temperature,observed.
othervariable offunctionscan be regulated
it was predicted that evolutionary adaptation
temperature
is abekey
regulator of base pairing stability, it was also expected that theof theseAs
phenomenawouldobserved.
nucleotide
composition
inthegenomes
ofpairingorganisms
grown
at highertemperaturesbe
As temperature is a key regulatorof basestability,
it wasalso expectedthat thewould
nucleotide
selectedforahigher
proportionofG+C
contents
thanA+T
contents
becausethenumberofcomposition in the genomes of organisms grown at higher temperatures would be selected for a higherhydrogenofbonds
G thanand CAnucleotides(threehydrogenbonds) isofhigher
compared
tobetweenthat ofproportion
G + Cbetween
contents+ T contents
becausethe numberhydrogen
bonds
A
and
T
nucleotides(twohydrogenbonds)
andincreases
thethermodynamic
stability.
G and C nucleotides (three hydrogen bonds) is higher compared to that of A and T nucleotides (twoInt. J. Mol. Sci. 2016, 17, 719

8 of 10hydrogen bonds) and increases the thermodynamic stability. Unexpectedly, however, recent extensive
sequencing of entire genomes revealed no obvious correlation between the genomic G + C content andthe optimal growth temperature [38–40]. The thermostability of the double-stranded structure of DNA
is conserved by reverse gyrase [41] or selection for certain dinucleotides [42] in thermophiles.
As there is no correlation between the G + C content of genomic DNA and growth temperature
at least within prokaryotic genomes, it was considered that there would also be no such correlation
at the RNA level. In bacteria, most genomic DNA is protein coding, so no such correlation has beenfound for protein-coding mRNAs either [38]. However, there is a correlation between the G + C
content of structured RNAs (e.g., ribosomal and tRNAs) and growth temperature [38]. The G + C
enrichment in structured RNAs represents striking evidence for selection in order to increase thethermostability of the corresponding regions by changing the nucleotide composition; as such, natural
selection increasing the G + C content is observed in double-stranded regions of rRNA molecules,
while the G + C content is decreased in single-stranded regions to maintain the single-stranded state inprokaryotes [39]. Thus, the correlation between G + C content and growth temperature is considered
to be obvious for structured RNA molecules but not DNA duplexes. One possible reason is that asingle nucleotide mutation would have a much greater effect on the thermodynamic stability of an
RNA molecule than genomic DNA. Although the thermodynamic properties of 7-mer tRNA sequencesin various species with different growth temperatures calculated by the nearest-neighbor procedure
did not show an obvious correlation with growth temperature in our analysis, miRNA seed regions
showed strong correlations with growth temperature [31], suggesting that evolutionary pressure isalso added to miRNAs with secondary structures. However, RNA silencing efficiency is more strongly
affected by the seed region than the 51 -terminal region, suggesting that evolutionary pressure on themiRNA 51 -terminal region is weak. Furthermore, our results cannot exclude other factor(s), suchas the biogenesis procedure of siRNA/miRNA or the hydrolytic activity of nucleotide, as the other
determinant(s) of the efficiency of RNA silencing.
Acknowledgments: This review was written based on our previous study, mainly performed by Naoki Hibio,
Yoshiro Nagata, Eigo Shimizu, and Kumiko Ui-Tei. This work was supported by Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan to Kumiko Ui-Tei The
funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in thewriting of the manuscript; or in the decision to publish the results.
Conflicts of Interest: The author declares no conflict of interest.

Abbreviations
siRNA
miRNA
UTR
ncRNA
pri-miRNA
pre-miRNA
AGO
RISC
CDS
RNAi
Tm

small interfering RNA
microRNA
untranslated region
non-coding RNA
primary miRNA
precursor miRNA
Argonaute
RNA-induced silencing complex
coding sequence
RNA interference
melting temperature

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