MicroRNA-repressed mRNAs contain 40S but not
60S components
Bingbing Wang*, Adrienne Yanez*, and Carl D. Novina*†‡
*Department of Cancer Immunology and AIDS, Dana–Farber Cancer Institute and Department of Pathology, Harvard Medical School, Boston, MA 02115;
and †Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA 02141
Communicated by Phillip A. Sharp, Massachusetts Institute of Technology, Cambridge, MA, February 5, 2008 (received for review June 4, 2007)
argonaute 兩 translation 兩 ribosome
M
icroRNAs (miRNAs) are endogenous small RNAs that may
regulate large networks of genes in several species. miRNAs
bind to target mRNAs with imperfect sequence complementarity
and repress translation through mechanisms that are incompletely
understood (reviewed in ref. 1). Intensive efforts have focused on
determining the precise stage of translation repressed by miRNAs.
Early observations suggested that lin-4, the first miRNA described
in animals, represses translation of its target mRNAs lin-14 or lin-28
after initiation, because the distribution of target mRNAs in
polysomes is similar to that of untargeted mRNAs (2, 3). Similar to
these early studies in worms, a recent report in mammals also
indicated that miRNA-targeted mRNAs are found in the same
polysomal fractions as their untargeted counterparts (4). In contrast, another report in mammals indicated that polysome profiles
of miRNA-targeted mRNAs shift toward monosomes, indicating a
translation initiation block (5). The causes and consequences of
these discrepancies in the mechanism of miRNA-dependent translation repression have not been resolved (1).
Recently, several cell-free miRNA-dependent translation repression reactions have been described. We reported the first cell-free
translation repression reactions (6), which faithfully recapitulate
important properties of miRNA function in cells including requirements for 5⬘ phosphates on miRNAs (7, 8) and perfect seed region
complementarity between miRNAs and target mRNAs (9–11).
Importantly, translation is repressed without reduction in target
mRNA levels. However, significant reduction in target mRNA
levels is observed when perfectly complementary siRNAs are
added to these reactions. Additionally, these translation repression
reactions directly demonstrated a dependence on a 7-methyl
guanosine cap (5, 12) and a polyA tail (12) on target mRNAs for
translational repression as observed in cells. Other cell-free translation repression reactions have been described recently. These
reactions also demonstrate a requirement for 7-methyl guanosine
capped target mRNAs for translational repression in mouse (13),
human (14), and fly (15) extracts, further supporting a model of
miRNA repression of translation initiation. Still, the precise mechanisms of miRNA function are unknown. We define a mechanism
of miRNA-directed repression of translation initiation by decreased
60S ribosome recruitment to target mRNAs using cell-free monocistronic and bicistronic miRNA reporter assays, ribosome-binding
assays, precipitation of miRNA-targeted mRNAs followed by
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0801102105
Northern blot analysis, Western blot analysis, and toeprinting
analyses of miRNA-targeted mRNAs.
Results
miRNA-Targeted mRNAs Contain Reduced Amounts of 60S Ribosome
Components. To investigate the mechanism of repressed translation
initiation, we tested the ability of the 40S and 60S ribosome subunits
to physically associate with miRNA-targeted mRNAs in the cellfree reactions described first in Wang et al. (6). To enable recovery
of miRNA-targeted mRNAs, firefly luciferase reporter mRNAs
were subjected to polyadenylation such that, on average, two
biotinylated adenosines were incorporated into a polyA tail consisting of ⬇200 adenosines. These reporter mRNAs contained six
imperfectly complementary binding sites to the CXCR4 siRNA
(FL6X). Consistent with previous observations (6), the FL6X
reporter mRNA demonstrated translation repression upon addition of CXCR4 siRNAs when normalized to an untargeted renilla
luciferase reporter mRNA lacking CXCR4 siRNA-binding sites
(RL0X, Fig. 1A).
Upon completion of the translation repression reactions, reporter mRNAs were precipitated with streptavidin beads, and
precipitates were subjected to Northern blot analysis for ribosomal
RNAs (Fig. 1B). In all analyses in Fig. 1B, a fixed amount of RNA
was loaded in each lane, and Northern blot analysis signals were
normalized by calculating the ratio of 60S rRNA:40S rRNA and
tRNAi-Met:40S rRNA. Northern blot analysis probes directed
against 18S rRNA, a component of the 40S ribosome subunit,
detected negligible differences in 40S content between reactions
containing or lacking siRNAs. However, Northern blot analysis
probes directed against 28S, 5.8S, and 5S rRNAs of the 60S
ribosome subunit detected significant reductions in 60S ribosome
components relative to the 18S rRNA in reactions containing
mRNAs targeted by miRNAs (Fig. 1B). Additionally, the reduction
in 28S, 5.8S, and 5S rRNAs associated with FL6X was approximately the same as the degree of translational repression observed
in Fig. 1 A (⬇60%). These results indicate that miRNAs promote
reduced 60S ribosome subunit loading on target mRNAs. Conversely, Northern blot analysis probes directed against tRNAi-Met
detected no change relative to the 18S rRNA in reactions containing mRNAs targeted by miRNA compared with untargeted mRNAs. This result indicates that miRNAs permit 43S ribosome
subunit loading on target mRNAs.
Several control reactions confirm the specificity of reduced 60S
ribosome recruitment to miRNA-targeted mRNAs: (i) There was
no significant change in the 40S or 60S ribosomes in Northern blots
of total lysates without precipitation of miRNA-targeted mRNAs
from translation repression reactions, suggesting that the ribosome
subunits are not being lost because of degradation. Relative to
Author contributions: B.W., A.Y., and C.D.N. designed research; B.W. and A.Y. performed
research; B.W., A.Y., and C.D.N. analyzed data; and C.D.N. wrote the paper.
The authors declare no conflict of interest.
‡To
whom correspondence should be addressed. E-mail: carl㛭[email protected].
This article contains supporting information online at www.pnas.org/cgi/content/full/
0801102105/DCSupplemental.
© 2008 by The National Academy of Sciences of the USA
PNAS 兩 April 8, 2008 兩 vol. 105 兩 no. 14 兩 5343–5348
BIOCHEMISTRY
MicroRNAs (miRNAs) are small noncoding RNAs that may target
more than one-third of human genes, yet the mechanisms used
by miRNAs to repress translation of target mRNAs are obscure.
Using a recently described cell-free assay of miRNA function, we
observe that miRNA-targeted mRNAs are enriched for 40S but
not 60S ribosome components. Additionally, toeprinting analysis of miRNA-targeted mRNAs demonstrates that ⬇18 nt 3ⴕ
relative to the initiating AUG are protected, consistent with 40S
ribosome subunits positioned at the AUG codon. Our results
suggest that miRNAs repress translation initiation by preventing
60S subunit joining to miRNA-targeted mRNAs.
A
-
C
+ siCXCR4
T
S
-
FL
RL
+
-
P
+
-
+
siCXCR4
RPL18 (60S)
RPS7 (40S)
73 ± 2 % Reduction FL
B
siCXCR4
T
- +
S
- +
5’ seed mut. FL6X
5’ unphospho. siCXCR4
P
T
S
P
T
- +
- +
- +
- +
- +
S
- +
P
- + siRNA
28S rRNA
60S
5.8S rRNA
5S rRNA
18S rRNA
40S
tRNAi-Met
Fold Change
T
S
P
T
S
2
1
0
2
1
0
2
1
0
2
1
0
P
T
S
P
28S rRNA
5.8S rRNA
5S rRNA
tRNAi-Met
Fig. 1. miRNA-targeted mRNAs have reduced 60S ribosome components. (A) Translation repression reactions using FL6X and RL0X mRNAs without (⫺) and with
(⫹) CXCR4 siRNAs (siCXCR4). Both mRNAs possessed 7-methyl-guanosine caps and polyA tails. The FL6X mRNA contained biotinyl-adenosines (99:1 adenosines:
biotinyl-adenosines). Translation repression of firefly luciferase (FL, % reduction FL) is normalized by renilla luciferase (RL) and calculated by the equation
[1⫺FL/RL(⫹siCXCR4):FL/RL(⫺siCXCR4)] x 100%. (B) Northern blot analysis for 40S and 60S rRNAs in total (T) translation repression reactions, supernatants (S), and
precipitates (P) after streptavidin precipitation of biotinylated mRNA reporters from total translation repression reactions. Streptavidin-precipitated FL6X mRNAs
from reactions with (⫹) CXCR4 siRNA demonstrate reduced amounts of 60S rRNAs relative to reactions lacking (⫺) CXCR4 siRNA (Left). Streptavidin-precipitated
FL6X mRNAs from reactions with (⫹) and without (⫺) unphosphorylated CXCR4 siRNA (5⬘ unphospho. siCXCR4, Middle). Streptavidin-precipitated FL6X mRNAs
with point mutations in the 5⬘ seed region (5⬘ seed mut. FL6X) from reactions with (⫹) and without (⫺) CXCR4 siRNA (Right). Signal intensities were normalized
by calculating 60S (5S, 5.8S, and 28S):40S (18S) rRNAs and initiator methionine tRNA (tRNAi-Met):40S rRNA. The fold changes in signal intensities between
reactions containing and lacking siRNAs were calculated by the equation [(60S/40S⫹siCXCR4):(60S/40S⫺siCXCR4)] or [(tRNAi⫺Met/40S⫹siCXCR4):(tRNAi⫺Met/
40S⫺siCXCR4)]. Results are presented as bar graphs below each image as an average of n ⫽ 3 trials (Left), n ⫽ 2 trials (Middle), and n ⫽ 2 trials (Right). (C) Western
blot analysis for 40S- and 60S-associated proteins in T, S, and P after streptavidin precipitation of biotinylated mRNA reporters from total translation repression
reactions. The precipitate from reactions with (⫹) CXCR4 siRNA demonstrates a slightly increased amount of 40S-associated protein (RPS7) and a strongly
decreased amount of 60S-associated protein (RPL18). In corresponding lanes between reactions without (⫺) or with (⫹) CXCR4 siRNAs in T, S, and P, identical
total protein amounts were loaded.
reactions with siRNAs, there was no change in the amounts of 60S
ribosome subunits between reactions using (ii) FL6X mRNAs with
unphosphorylated CXCR4 siRNAs (Fig. 1B Middle), (iii) FL6X
mRNAs containing point mutations in the 5⬘ seed region of the
miRNA-binding sites in the 3⬘ UTR (Fig. 1B Right), (iv) FL6X
mRNAs with nonspecific control siRNAs [supporting information
(SI) Fig. S1C], and (v) FL0X mRNAs with CXCR4 siRNAs (Fig.
S1C). The absence of significant changes in 40S and 60S ribosomal
RNA content in these control reactions is consistent with the
absence of translational repression in these control reactions (Fig.
1 A and Fig. S1B) and indicates that the reduction in 60S ribosome
RNAs is specific to miRNA-repressed mRNAs.
To independently confirm these observations, precipitated reporter mRNAs were subjected to Western blot analysis for 40S- and
60S-associated proteins (Fig. 1B). Whereas slightly increased
5344 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0801102105
amounts of 40S-associated proteins were detected on miRNArepressed mRNAs, strongly decreased amounts of 60S-associated
proteins were detected on these same mRNAs. Together, these
observations demonstrate that miRNA-targeted mRNAs have
steady-state levels of 40S ribosome components but reduced levels
of 60S ribosome components relative to untargeted mRNAs.
Chemical Inhibitors Identify High Molecular Mass Complex Formation
on miRNA-Targeted mRNAs That Depends on 40S but Not 60S Ribosomes. To define the stage of translation initiation affected by
miRNAs more precisely, we used ribosome-binding assays to
analyze the sedimentation profiles of radiolabeled miRNAtargeted mRNAs from translation repression reactions (Fig. 2).
Similar to all reports of polysome profiling of miRNA targeted
mRNAs (2–5, 15, 16), ribosome-binding assays require chemicals to
Wang et al.
0.12
0.10
0.10
0.08
0.08
0.06
0.06
0.04
0.04
0.02
0.02
0.00
0.00
0.18
0.18
GMP-PNP
-siRNA
+siRNA
0.14
E
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0.18
Cycloheximide
0.16
-siRNA
+siRNA
0.14
F
5’ seed mut. FL6X
-siRNA
+siRNA
0.14
-
+
siCXCR4
FL6X, Cycloheximide
T
S
P
- + - + - + siCXCR4
28S rRNA
60S
5.8S rRNA
5S rRNA
0.02
0.00
18S rRNA 40S
0.18
Uncapped FL6X
0.16
0.06
0.04
0.04
0.02
0.02
tRNAi-Met
-siRNA
+siRNA
0.14
0.06
0.00
10 12 14 16 18 20 22
25
0.04
0.08
8
50
0.06
0.08
6
75
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4
100
0.10
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H
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G
0
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0.00
-siRNA
+siRNA
0.14
0.12
0.16
5’ unphospho. siRNA
0.16
T
0
2
4
Fraction
6
8
S
P
2
1
0
28S rRNA
2
1
0
5.8S rRNA
2
1
0
5S rRNA
2
1
0
tRNAi-Met
10 12 14 16 18 20 22
Fraction
Fig. 2. A high molecular mass complex containing 40S but lacking 60S ribosome subunits forms on miRNA-repressed mRNAs. (A–F) Ribosome-binding assays
using 7-methyl-guanosine capped or uncapped FL6X mRNAs containing 32P-labeled polyA tails. (A) Ribosome-binding analysis of translation repression reactions
containing hippuristanol identifies a peak (fraction 4) corresponding to unbound mRNAs in reactions without (⫺, dotted line) and with (⫹, solid line) CXCR4
siRNAs. (B) Ribosome-binding analysis of translation repression reactions containing GMP-PNP identifies a 48S complex (fraction 8) in reactions without (⫺,
dotted line) and a high molecular mass complex in reactions with (⫹, solid line) CXCR4 siRNAs. (C–F) Ribosome-binding assays of translation repression reactions
containing cycloheximide identify an 80S complex (fraction 12) in all reactions without CXCR4 siRNAs (⫺, dotted line). (C) A high molecular mass complex is formed
on FL6X mRNAs in reactions with CXCR4 siRNAs (⫹, solid line). (D) An 80S complex is formed on FL6X mRNAs in reactions with unphosphorylated CXCR4 siRNAs
(⫹, solid line, 5⬘ unphospho. siRNA). (E) An 80S complex is formed on FL6X mRNAs with three point mutations in the 5⬘ seed region (5⬘ seed mut. FL6X) in reactions
with CXCR4 siRNAs (⫹, solid line). (F) An 80S complex is formed on uncapped FL6X mRNAs in reactions with CXCR4 siRNAs (⫹, solid line). Horizontal axis indicates
fraction number. Vertical axis indicates the cpm (c.p.m) of each fraction as a percent of total counts recovered (% of total c.p.m.). In B–F, the upward pointing
arrows indicate peak fractions of mRNA sedimentation in glycerol gradients. (G) Dual luciferase assay using FL6X mRNAs containing 32P-labeled polyA tails in
the presence of CXCR4 siRNAs. Firefly luciferase (FL) measurements were normalized to renilla luciferase expressed from RL0X mRNAs with an unlabeled cap and
a polyA. Bars indicate percentage translation of FL (%,y axis). (H) Northern blot analysis of translation repression reactions in the presence of cycloheximide using
FL6X mRNAs with 7-methyl-guanosine caps and biotinylated polyA tails from reactions without (⫺) or with (⫹) CXCR4 siRNA. Total translation repression
reactions (T), supernatants (S), and precipitates (P) from total translation repression reactions after streptavidin precipitation of biotinylated FL6X mRNA were
Northern blotted for 40S and 60S rRNAs, and initiator methionine tRNA (tRNAi-Met) as in Fig. 1B. Results are presented as a bar graphs with error bars indicating
the standard deviation of n ⫽ 2 trials.
stabilize intermediates of monosome (80S) assembly. In ribosomebinding assays, addition of CXCR4 siRNAs to translation repression reactions without chemical inhibitors is not sufficient to
capture complexes at specific stages of ribosome assembly during
translational repression (data not shown). Complexes stabilized at
specific stages of ribosome assembly on radiolabeled mRNAs are
sedimented through a glycerol gradient and detected by Cerenkov
scintillation counting of individual glycerol gradient fractions (reviewed in ref. 17).
First, translation repression reactions were performed in the
presence of the eIF4A inhibitor hippuristanol (18). Hippuristanol
blocks 43S recruitment to mRNAs, resulting in the majority of the
mRNA migrating at the top of the glycerol gradient as unbound
mRNA (fraction 4) in ribosome-binding assays. Addition of
CXCR4 siRNA to translation repression reactions did not alter the
sedimentation of mRNAs in the presence of hippuristanol (Fig.
2 A), indicating that any complexes that form on repressed mRNAs
require 43S recruitment. Next, translation repression reactions were
Wang et al.
performed in the presence of the nonhydrolyzable GTP analog
GMP-PNP, which blocks 60S ribosome recruitment, resulting in the
capture of 48S initiation complexes (fraction 8) in ribosome-binding
assays. When CXCR4 siRNAs were added to translation repression
reactions containing GMP-PNP, the mRNA sedimentation peak
was shifted from fraction 8 to fraction 14, indicating the formation
of a high molecular mass complex (Fig. 2B). These results suggest
that this high molecular mass complex is formed on miRNAtargeted mRNA after 43S recruitment but before 60S recruitment.
Then, translation repression reactions were performed in the
presence of the translation elongation inhibitor cycloheximide,
which traps fully assembled 80S monosomes at the initiation codon
of mRNAs (fraction 12). Reactions without CXCR4 siRNA led to
the expected mRNA sedimentation, consistent with captured 80S
complexes (Fig. 2 C–F). Addition of CXCR4 siRNAs to translation
repression reactions containing cycloheximide, however, generated
an mRNA sedimentation profile identical to the profile observed
with GMP-PNP (Fig. 2C). This observation indicates that 80S
PNAS 兩 April 8, 2008 兩 vol. 105 兩 no. 14 兩 5345
BIOCHEMISTRY
C
32P labeled FL6X (% of t ot al c.p.m.)
0.14
B
0.18
D
-siRNA
+siRNA
Translat ion of FL (%)
Hippuristanol
0.16
Fold Change
0.18
32P labeled FL6X (% of t ot al c.p.m.)
A
48S Complexes Are Positioned at AUG on miRNA-Repressed miRNAs.
To identify the position of 40S ribosomes assembled on miRNArepressed mRNAs, primer extension analysis was performed (Fig.
3A). In this assay, a radiolabeled primer hybridizing to sequences 3⬘
relative to the AUG codon of miRNA-targeted mRNAs was used
to initiate reverse transcription without extraction from associated
proteins. A ‘‘toeprint’’ of bound protein complexes is generated
when steric hindrance prevents reverse transcriptase from transcribing cDNA from regions of the mRNA. Translation repression
reactions with CXCR4 siRNAs generated bands at 18 nt 3⬘ relative
to the AUG codon (compare lanes 5 and 6). This toeprint was
identical to translation repression reactions containing GMP-PNP
(lanes 10–12), which marks 40S ribosomes positioned at the start
codon after completion of scanning. Consistent with the ability of
hippuristanol to block formation of the high molecular mass
complex in ribosome-binding assays (Fig. 2 A), the CXCR4 siRNAinduced toeprint was blocked in the presence of hippuristanol
(compare lanes 6 and 9).
Toeprinting was quantified by using the ratio of the 3⬘ (specific)
band protected in toeprinting relative to the 5⬘ (nonspecific) band
relative to AUG. RNA secondary structure causes MMLV reverse
transcriptase to dropoff of its template, thus generating a nonspecific 5⬘ band that can be used to quantify specific miRNA toeprint
formation. By this measure, the CXCR4 siRNA-induced toeprint
ratio was 2.3 (lane 6), the GMP-PNP-induced toeprint ratio was 6.9
(lanes 10 and 11), and the combined ratio was 7.1 (lane 12), 3-fold
more than the toeprint ratio induced by CXCR4 siRNA alone.
These data indicate that GMP-PNP more strongly stabilizes 40S
complexes positioned at AUG compared with miRNAs alone and
may help explain why the addition of miRNAs alone is not sufficient
to capture complexes in ribosome-binding assays.
5346 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0801102105
A
A U G C -
18 nt
monosomes do not form in these reactions, and that the repression
occurs at an earlier step of translation initiation.
Formation of the high molecular mass complex was specific to
the CXCR4 siRNA and its interaction with its target site. Inclusion
of unphosphorylated CXCR4 siRNAs (Fig. 2D), a triple point
mutant in the 5⬘ seed region of the miRNA-binding site in the FL6X
mRNA 3⬘ UTR (Fig. 2E), nonspecific control siRNA (Fig. S2C) or
fully phosphorylated CXCR4 siRNAs and the FL0X mRNA (Fig.
S2D) all resulted in the formation of an 80S complex in the presence
of cycloheximide and had no effect in translation repression reactions (Fig. 2G and Fig. S1B). Consistent with the lack of translational repression of uncapped mRNAs (ref. 6; Fig. S3), FL6X
lacking a 7-methyl guanosine cap did not result in formation of the
high molecular mass complex in reactions containing CXCR4
siRNAs but instead resulted in formation of an 80S complex (Fig.
2F). These results demonstrate that the high molecular mass
complex forms only on translationally repressed mRNAs and
possesses the 40S ribosome subunit but lacks the 60S ribosome
subunit, further supporting a model that miRNAs repress translation by preventing 60S ribosome subunit recruitment to target
mRNAs. Additionally, the ability of the eIF4A inhibitor, hippuristanol, to prevent formation of the high molecular mass complex
suggests that miRNA-directed repression of translation occurs after
cap-facilitated, 40S ribosome subunit recruitment.
To rule out any effect of cycloheximide in the reactions in Fig.
2, we performed Northern blot analysis on precipitated complexes
from translation repression reactions containing cycloheximide
(Fig. 2H). Consistent with data presented in Fig. 1B, Northern blot
analysis of precipitated FL6X demonstrated similar levels of 18S
ribosomal RNA but reduced 28S, 5.8S, and 5S ribosomal RNAs
(but no change in tRNAi-met) associated with target mRNAs in
reactions with CXCR4 siRNA compared with reactions without
CXCR4 siRNAs. Also consistent with data presented in Fig. 1B,
60S ribosome association was reduced to a similar degree (⬇70%)
as translational repression observed in Figs. 1 A and 2G.
B
- + + + - - - Hipp.
- - - - + + + GMP-PNP
+ - - + - - + siCXCR4
A
U
G
C
1 2 3 4 5 6 7 8 9 10 11 12
4
8
12
14
siCXCR4
+
eIF2α
+
eIF3g
+
eIF4E
+
eIF4A
+
Ago2
+
RPS7
+
RPL18
Fig. 3. miRNAs repress translation after 48S scanning but before 60S subunit
joining. (A) Toeprinting assay of FL6X mRNAs with a 7-methyl-guanosine cap
and polyA tail. Compared with reactions without (⫺) CXCR4 siRNAs (siCXCR4,
lane 5), reactions with (⫹) CXCR4 siRNA (lane 6) demonstrate a strong toeprint
that protects a region encompassing the initiating AUG codon. Addition of
hippuristanol (hipp.) to translation repression reactions does not lead to a
toeprint (lanes 7 and 8) and blocks the CXCR4 siRNA-induced toeprint (lane 9).
Addition of GMP-PNP to translation repression reactions indicates the 40S
toeprint (lanes 10 and 11) and augments the CXCR4 siRNA-induced toeprint
(lane 12). A labeled ladder (lanes 1– 4) indicates nucleotide positions relative
to AUG. (B) Western blot analysis using antibodies to eIF2␣, eIF3g, eIF4E,
eIF4A, Ago2, RPS7 (40S-associated protein), and RPL18 (60S-associated protein) detects proteins that coprecipitate with FL6X mRNAs from glycerol
gradient fractions 4 (free mRNA), 8 (48S peak), 12 (80S peak), and 14 (high
molecular mass complex peak) in ribosome-binding assays. In corresponding
lanes between reactions without (⫺) or with (⫹) CXCR4 siRNAs, identical total
protein amounts were loaded. Although images are presented one above the
other, for each antibody, data were obtained from the same blot.
eIF2 and eIF3 Are Associated with miRNA-Targeted mRNAs. To investigate the complement of translation initiation factors associated
with mRNAs in fraction 4 (free mRNA), fraction 8 (48S complexes), fraction 12 (80S complexes), and fraction 14 (high molecular mass complex), mRNA precipitates from glycerol gradient
fractions in ribosome-binding assays were subjected to Western blot
analysis. Because many translation initiation and miRNAinteracting factors are highly conserved through evolution, antibodies against these human and mouse proteins crossreact with
their rabbit homologs.
The translation initiation factors eIF2 and eIF3 are recruited to
43S ribosome complexes before joining mRNAs and dissociate
from 48S ribosome complexes just before (or concomitant with)
60S ribosome subunit joining mRNAs (reviewed in ref. 19). Therefore, we probed ribosome-binding assay fractions for these factors
known to assemble with the 40S subunit during translation initiation (Fig. 3B). The eIF2 subunit, eIF2␣, and the eIF3 subunit,
eIF3g, were significantly enriched in fractions 12 and 14 from
reactions with CXCR4 siRNAs compared with reactions without
CXCR4 siRNAs. Together with toeprinting analysis, Western blot
analyses support a model in which miRNAs block translation after
43S subunit joining and scanning but before eIF2 and eIF3 release
and 60S ribosome subunit joining.
To interrogate the cap dependency of miRNA-directed translational repression and high molecular mass complex formation, we
performed Western blot analysis of glycerol gradient fractions from
ribosome-binding assays for the cap-binding protein (eIF4E) and
Wang et al.
4E
m 7G
4G
4A
40S
2
PABP
miRNP
Ago2
Fig. 4. A model of miRNA-directed repression of translation initiation.
Several translation initiation factors may interact with a recruited Ago protein
to repress translation including the cap-binding factor, eIF4E; the protein
associated with the polyA tail, PABP; the bridging protein between cap
structures and the polyA tails, eIF4G; the RNA helicase that unwinds local
mRNA secondary structure, eIF4A; and the multicomponent proteins associated with the 40S ribosome, eIF3 and eIF2.
the RNA helicase that facilitates 40S recruitment (eIF4A). Increases in both of these factors were observed in reactions containing CXCR4 siRNAs relative to those lacking CXCR4 siRNAs
(compare fractions 12 and 14 without CXCR4 siRNAs vs. with
CXCR4 siRNAs). Together, these observations indicate that eIF4E
and eIF4A are still bound to translationally repressed mRNAs after
40S subunit joining and suggest that interaction of these proteins
with the cap is important for translational repression by miRNAs.
In all species capable of small RNA-directed gene silencing,
microribonucleoprotein (miRNP) complexes possess a member of
the Ago family of proteins (reviewed in ref. 20). To determine
whether Ago proteins are recruited to miRNA-targeted mRNAs in
the reactions reported here, Western blot analysis was performed
with antibodies against Ago2 (Fig. 3B). Consistent with the notion
that the high molecular mass complex formed on miRNA-repressed
mRNAs is a bona fide miRNP, fractions 12 and 14 were significantly
enriched for Ago2 in reactions containing CXCR4 siRNAs relative
to reactions lacking CXCR4 siRNAs. To demonstrate that Ago
proteins recruited to miRNAs preannealed to mRNAs are functional, Ago2-dependent RNA cleavage assays were performed. Our
data indicate that Ago2-mediated cleavage of target RNAs in vitro
maps to the exact position as reported for Ago2-dependent cleavage
in cells (Fig. S4 and SI Materials and Methods). These data indicate
that these reaction conditions permit formation of functional
miRNP/RISC on miRNA-repressed mRNAs.
Discussion
The process of translation initiation is typically regulated at one of
two steps: either at the 43S preinitiation complex formation or at
the ribosome recruitment phase (19). However, more specialized
mechanisms of translational control have been reported. The
mechanism for miRNA-directed translation repression proposed
here is analogous to a previously identified 3⬘ UTR regulatory
ribonucleoprotein complex that represses translation by inhibiting
60S subunit joining with the 40S subunit positioned at the AUG
codon of lipooxygenase mRNA (21). Because miRNAs may regulate large networks of genes, the mechanism of blocked 60S
recruitment may be far more prevalent than originally anticipated.
A model integrating the observations reported here is presented
in Fig. 4. It is important to note that this model makes no
conclusions about whether the 7-methyl guanosine cap-associated
eIF4F components or Ago2 are part of the miRNA-dependent high
molecular mass complex. Indeed, it was recently shown that eIF4E
(13) and Ago2 (22) bind to 7-methylguanosine caps to mediate
miRNA-directed repression of translation. Recently, two other
groups reported miRNA repression consistent with reduced 60S
Wang et al.
Materials and Methods
Translation Repression Reactions. All mRNA reporters used in these studies were
prepared and used as described in ref. 6. Plasmids expressing all of these mRNA
reporters are available from (www.addgene.org). The sequences of the CXCR4
siRNA were 5⬘P-GUUUUCACUCCAGCUAACACA-3 (sense strand) and 5⬘PUGUUAGCUGGAGUGAAAACUU-3⬘ (antisense strand). The sequences of the GFP
siRNA were 5⬘P-GGCUACGUCCAGGAGCGCACC-3⬘ (sense strand) and 5⬘PUGCGCUCCUGGACGUAGCCUU-3⬘ (antisense strand). The control mRNA reporter
used in Supporting Online Fig. 3 was human CD3 (kind gift of Chenqi Xu,
Dana-Farber Cancer Institute).
Translation repression reactions were performed as described in ref. 6. Briefly,
preannealed mRNA reporter (0.025 pmol) and CXCR4 siRNA (0.15 pmol) were
incubated with a master mix containing 7 ␮l of nuclease-treated rabbit reticulocyte lysate (RRL, Promega), 4 – 8 units RNase Out (Invitrogen), 20 ␮M amino acid
mixture (complete or minus methionine and cysteine, Promega), and 0.4 ␮l (5.7
␮Ci) Promix L-[35S] in vitro cell labeling mix (Amersham Biosciences) at 30°C for 10
min. Reaction products were separated on 12% SDS/PAGE and transferred onto
PVDF (BioRad) or subjected to dual luciferase assay.
Dual Luciferase Assay. Dual luciferase assays were performed according to the
manufacturer’s protocols (Promega). Firefly luciferase activity was measured by
adding 2 ␮l of each reaction with LAR I (20 ␮l) into one well of a 96-well plate and
read in Victor3 V (PerkinElmer) for 5 sec. Renilla luciferase activity was measured
by adding Stop & Glo (20 ␮l, Promega) to each well and reread for 5 sec.
Ribosome-Binding Assay. Ribosome-binding assays were performed as described
in ref. 18. In vitro translation repression reactions supplemented with cyclohexPNAS 兩 April 8, 2008 兩 vol. 105 兩 no. 14 兩 5347
BIOCHEMISTRY
60S
ribosome recruitment to translationally repressed mRNAs in
worms, humans (16), and flies (15). In worm and human cells, the
60S antiassociation factor eIF6 (23–26) associates with RNAinduced silencing complexes but not necessarily with miRNAtargeted mRNAs. Like the data presented here, in fly extracts,
pseudopolysomes, nonpolysomal complexes of a molecular mass
⬎80S, form on miRNA-targeted mRNAs in the presence of both
cycloheximide and GMP-PNP, indicating the absence of 60S subunits (15). Contrary to the cap dependency of the high molecular
mass complex presented here, pseudopolysomes form on mRNAs
lacking a 7-methyl guanosine cap. These observations suggest
important similarities between miRNA-mediated translation repression across species but also imply distinguishing details in the
mechanisms of miRNA-mediated repression in these organisms.
The formation of a high molecular mass complex on miRNAtargeted mRNAs containing 40S but lacking 60S ribosome components in ribosome-binding assays described here provides one
possible explanation for the rapid sedimentation of miRNAtargeted mRNAs in polysome profiling assays observed in worms
(2, 3) and humans (4). Further analyses in cell-based and -free
systems will more precisely define the mechanism(s) of miRNA
function in mammals and their similarities and differences across
species.
Ago2 (co-eIF2A) was originally defined as a ribosome-associated
protein that eluted in high salt (27) and that stabilized 40Scontaining complexes in the presence of mRNAs (28). The high
molecular mass complex formed on translationally repressed mRNAs possesses 40S ribosome subunits but lacks 60S ribosome
subunits. Consistent with a role in stabilizing 40S ribosomes associated with mRNAs, Ago2 is recruited to unrepressed mRNAs
[fraction 8, Ago2 (⫺), Fig. 3B Upper]. Ago2 is also recruited to
translationally repressed mRNA (fraction 14, Fig. 3B), possibly
because these mRNAs possess increased amounts of 40S subunits
without joined 60S subunits. It has been shown that Ago2 interacts
with the antiassociation factor eIF6 and thus 60S through TRBP
and prevents 60S subunit joining to translationally repressed mRNAs (16). Our data present a complimentary mechanism that Ago2
interacts with translationally repressed mRNAs and prevents 60S
subunit joining. Ago2 has also been shown to directly interact with
caps of translationally repressed mRNAs (22). Together, these data
suggest that Ago2 may function in more than one way to repress
translation.
imide (600 ␮M), GMP-PNP (1 mM), or hippuristanol (50 ␮M) were loaded onto
10 –30% glycerol gradient containing 1⫻ HSB (500 mM NaCl; 20 mM Hepes-KOH,
pH 7.5; 30 mM MgOAc; and 2 mM DTT). Glycerol gradients were ultracentrifuged
by using an SW41 rotor (Beckman) at 39,000 rpm for 3.5 h, sequentially fractionated (500 ␮l) from the top, and subjected to Cerenkov scintillation counting.
Precipitation of Biotinylated mRNAs. Streptavidin agarose (SAA) beads (Invitrogen) were used to precipitate biotinylated mRNA reporters. SAA beads (100 ␮l)
were washed in 1⫻ HSB buffer three times and incubated with glycerol gradient
fractions (200 ␮l) or whole lysate reactions at 4°C for 60 min. Reactions were
centrifuged, and supernatants were removed. Precipitates were washed twice in
1⫻ HSB and subjected to RNA extraction and precipitation or to Western blot
analysis.
Northern Blot Analysis. The Northern blot analysis was performed by PAGE as
described in ref. 6. RNAs extracted from SAA precipitates were separated on
8% PAGE containing urea (7 M) and transferred to Hybond n ⫹ membranes
(Amersham Biosciences) for 2.5 h at 350 mA. After UV cross-linking, membranes were hybridized with 5⬘ end-labeled primers for 5S rRNA, 5⬘TTAGCTTCCGAGATCA-3⬘; 5.8S rRNA, 5⬘-GCTAGCGCTGCGTTCTTCATCGACGC3⬘; 28S rRNA5⬘-AACGATCAGAGTAGTGGTATTTCACC-3⬘; 18S rRNA, 5⬘CGGAACTACGACGGTATCTG-3⬘; and tRNAi-Met, 5⬘-GGTAGCAGAGGATGGTTTCGATCC-3⬘. Membranes were washed, visualized, and analyzed by
PhosphorImager (Molecular Dynamics).
anti-Ago2 antibody (Upstate) recognizes residues 7– 48 of human Ago2, which
are conserved amino acids between human, mouse, cattle, dog, and frog. AntieIF2␣, -eIF4A, and -eIF4E were kind gifts from Jerry Pelletier. The anti-eIF3g
antibody was a kind gift from Hiroaki Imataka, Riken Genomic Sciences Center,
Wako, Japan. Anti-RPS7 and -RPL18 antibodies were used according to the
manufacturer’s protocol (Abnova). Membranes were washed three times with
PBST, incubated with horseradish peroxidase-conjugated secondary antibodies
(Jackson ImmunoResearch) at 1:5,000 in 1% nonfat milk powder–PBST, and
developed by ECL (Pierce).
To strip Western blots of antibody complexes, membranes were incubated in
stripping buffer (100 mM 2-mercaptoethanol; 2% SDS; and 62.5 mM Tris䡠HCl, pH
6.7) at 50°C for 30 min. These membranes were washed with PBST for 2 ⫻ 10 min,
blocked in 5% milk–PBST, and reprobed with appropriate antibodies.
Toeprinting Assay. Translation repression reactions containing mRNA (0.1 pmol)
and CXCR4 siRNA (0.6 pmol), RRL (7 ␮l), and MgOAc (2 mM) with or without
GMP-PNP (1 mM) or Hisppuristanol (50 ␮M) proceeded for 5 min at 30°C. Then,
reverse transcription (RT) mix containing dNTPs (5 mM), 1⫻ reconstitution buffer
(20 mM Tris䡠HCl, pH 7.5; 100 mM KCl; and 1 mM DTT), 5⬘ end-labeled primer (0.2
pmol, 5⬘-TTATGCAGTTGCTCTCCAGCG-3⬘), and M-MLV RT (1 ␮l, Invitrogen) was
added to translation repression reactions. These mixtures were incubated for 15
min at 30°C and subjected to deproteinization and ethanol precipitation. RNAs
were resolved on a 10% sequencing gel (National Diagnotics) and visualized by
PhosphorImager analysis (Molecular Dynamics).
See SI Materials and Methods for additional details.
Western Blot Analysis. The SAA precipitates were resuspended in 1⫻ SDS loading
buffer, boiled at 95°C for 5 min, and centrifuged. Supernatants were resolved on
SDS–10% PAGE and transferred onto PVDF membranes (BioRad). Membranes
were blocked in 5% nonfat milk powder in PBST (10 mM phosphate buffer, pH
7.2; 150 mM NaCl; and 0.1% Tween 20) for 60 min, washed twice with PBST, and
incubated with antibodies in 1% nonfat milk powder–PBST at 4°C overnight. The
ACKNOWLEDGMENTS. We thank Jerry Pelletier, John Doench, Steffen Schubert,
and Tara Love for critically reading this manuscript. We thank Helen Cargill and
Etienne Gagnon for help with Fig. 4. A.Y. was supported by National Institutes of
Health Training Grant GM07266. This work was supported by a grant from the
Claudia Adams Barr Program in Cancer Research (C.D.N.).
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Wang et al.