REPORTS

17. L. Wu, J. Fan, J. G. Belasco, Proc. Natl. Acad. Sci. U.S.A.
103, 4034 (2006).
18. E. Huntzinger, E. Izaurralde, Nat. Rev. Genet. 12, 99
(2011).
19. T. Fukaya, Y. Tomari, EMBO J. 30, 4998 (2011).
20. Y. Mishima et al., Proc. Natl. Acad. Sci. U.S.A. 109,
1104 (2012).
21. M. R. Fabian et al., Mol. Cell 35, 868 (2009).
22. M. R. Fabian, Y. V. Svitkin, N. Sonenberg, Methods Mol.
Biol. 725, 207 (2011).
23. A. Zdanowicz et al., Mol. Cell 35, 881 (2009).
24. A. Eulalio et al., RNA 15, 21 (2009).
25. T. H. Beilharz et al., PLoS ONE 4, e6783 (2009).
26. M. Chekulaeva et al., Nat. Struct. Mol. Biol. 18, 1218
(2011).
27. M. R. Fabian et al., Nat. Struct. Mol. Biol. 18, 1211
(2011).
28. J. E. Braun, E. Huntzinger, M. Fauser, E. Izaurralde, data analysis. A.A.B., M.T.L., and A.J.G. interpreted the data
Mol. Cell 44, 120 (2011). and wrote the manuscript. Sequencing data are deposited
29. A. Cooke, A. Prigge, M. Wickens, J. Biol. Chem. 285, in the Gene Expression Omnibus (GEO) database with
28506 (2010). accession number GSE34743.
30. E. Wu et al., Mol. Cell 40, 558 (2010).
Supporting Online Material
Acknowledgments: We thank S. Wolin and the Wolin www.sciencemag.org/cgi/content/full/science.1215704/DC1
laboratory for vital intellectual and technical support and Materials and Methods
access to equipment and reagents; N. Ignolia, J. Weissman, SOM Text
D. Schoenberg, D. Patil, A. Staton, C. Takacs, and Y. Mishima Figs. S1 to S11
for reagents and discussions; and S. Baserga, D. Cazalla, Tables S1 and S2
D. Cifuentes, and S. Moxon for discussions. Supported by the Reference (31)
Pew Fellows Program in Biomedical Sciences (A.A.B.), the Database S1
Pew Scholars Program in the Biomedical Sciences, the Yale
Scholar Program, and NIH grants R01GM081602-05 (A.J.G). 24 October 2011; accepted 5 March 2012
Contributions: A.A.B. and A.J.G. designed the project. A.A.B. Published online 15 March 2012;
performed the experiments. A.A.B. and M.T.L. performed 10.1126/science.1215704

zation and translational repression (11, 12). Be-

miRNA-Mediated Gene Silencing cause only slightly more translational repression

Downloaded from http://science.sciencemag.org/ on July 22, 2016

is observed than mRNA destabilization, it is pos-
by Translational Repression Followed sible that most of the loss in protein synthesis
could directly result from effects on mRNA sta-
by mRNA Deadenylation and Decay bility. Most of these studies have not, however,
evaluated the kinetics of the miRNA-related cel-
lular processes (5, 10, 13, 14). Exceptions include
Sergej Djuranovic, Ali Nahvi, Rachel Green* several analyses of in vitro systems that con-
cluded that the effects of miRNAs on transla-
microRNAs (miRNAs) regulate gene expression through translational repression and/or messenger tional repression precede effects on mRNA target
RNA (mRNA) deadenylation and decay. Because translation, deadenylation, and decay are deadenylation or decay (1517), but concerns
closely linked processes, it is important to establish their ordering and thus to define the molecular remain that the in vitro reactions may not fully
mechanism of silencing. We have investigated the kinetics of these events in miRNA-mediated recapitulate the in vivo situation.
gene silencing by using a Drosophila S2 cell-based controllable expression system and show that We used an in vivo luciferase-based reporter
mRNAs with both natural and engineered 3 untranslated regions with miRNA target sites are system in Drosophila melanogaster S2 cells un-
first subject to translational inhibition, followed by effects on deadenylation and decay. We next der the control of an inducible metallothionein
used a natural translational elongation stall to show that miRNA-mediated silencing inhibits promoter (Mtn) (18). The reporter constructs con-
translation at an early step, potentially translation initiation. sist of one of the luciferase reporter genes [Firefly
(F-Luc) or Renilla (R-Luc)] fused at its 5 end to

m
expression
icroRNAs (miRNAs) are short en-
dogenous RNAs that regulate protein
1 from targeted genes by pairing to
studies showed that miRNAs principally affect
protein expression of miRNA-targeted genes with-
out obvious effects on mRNA abundance (710).
the Mtn promoter and at its 3 end to synthetic or

Howard Hughes Medical Institute (HHMI) and Department of

sites in the 3 untranslated region (3UTR) (1). By simultaneously measuring translational effi- Molecular Biology and Genetics, Johns Hopkins University
Although some studies showed a strong corre- ciencies (thus indirectly levels of protein synthe- School of Medicine, Baltimore, MD 21205, USA.
lation between the diminution of protein and sis) and mRNA abundance, global analyses have *To whom correspondence should be addressed. E-mail:
mRNA levels of miRNA-targeted genes (26), other shown evidence of significant mRNA destabili- ragreen@jhmi.edu

Fig. 1. Steady state evaluation of miRNA-mediated gene silencing using a copper-inducible in vivo reporter system. (A)
Measured protein amounts (luminescence) from transfected nontargeted (NT), targeted (T), and control (con) constructs
24 hours after induction. Additional expression of bantam miRNA, not Argonaute 1, results in increased repression for
synthetic bantam targeted constructs (fig. S1). (B to E) Ratios of steady-state protein amounts for synthetic and natural miRNA-targeted constructs 48 hours
after induction. In each case, mean values T SD from three independent triplicate experiments are shown as a normalized ratio of protein amounts (NT/T).

www.sciencemag.org SCIENCE VOL 336 13 APRIL 2012 237

REPORTS
natural 3UTRs that contain miRNA binding sites
responsive to either endogenously expressed
(bantam and miR-279) or ectopically introduced
miRNAs (miR-9b) (fig. S1); control constructs
not subject to miRNA-mediated gene regulation
are detailed in fig. S1. We first asked whether
miRNA-mediated responsiveness is limited by en-
dogenous components of the microRNA-induced
silencing complex (miRISC) (Argonaute protein
or miRNAs). By using a previously character-
ized bantam-responsive synthetic construct and
ectopically expressed additional miRISC compo-
nents (bantam and Ago1), we followed repres-
sion levels of the target mRNAs 24 hours after
induction (Fig. 1A) (19). The results indicate that
Ago1 is not limiting for repression in Drosophila
S2 cells, whereas bantam is limiting because greater
repression was observed when it was overex-
pressed (Fig. 1A). In the remaining experiments,
We next used a set of synthetic and natural
3UTRs with miRNA binding sites responding to
either bantam or miR-9b and miR-279 and their
corresponding controls (fig. S1 and tables S1 and
S2). We chose natural (endogenous) 3UTRs
from hid and Vha68-1 and a pair of synthetic
3UTR targets that contain six natural tandem
sites for either bantam or miR-9b and miR-279
miRNAs (19, 20). After transfection and consti-
tutive induction, luciferase expression levels were
measured after 48 hours and normalized to assess
the end-point effects of miRNA-mediated repres-
sion (Fig. 1, B to E). The synthetic bantam
3UTR exhibited repression levels up to 80-fold
when compared with the control (Fig. 1B); the
reporter containing the 3UTR of the hid gene
with as many as eight miRNA binding sites ex-
hibited ~ninefold repression (Fig. 1D) (19). Ad-
ditionally, both synthetic and natural (20) 3UTRs
antagomirs to the cell cultures induced levels of
derepression for the reporter constructs similar to
those observed when comparing reporter protein
expression from constructs with intact and altered
miRNA binding sites (fig. S2).
For regulated induction, transcriptional shut-
off of the Mtn promoter was accomplished by
using a specific copper chelator, bathocuproine
disulphonate (BCS). Although BCS chelates any
residual copper in the S2 cell medium, it does not
penetrate the cell membrane and thus does not
affect normal cellular homeostasis (21). A 90-min
pulse induction by copper (II) sulfate induced ex-
pression of the various reporter constructs to lev-
els that could be monitored over the subsequent
48 hours. By using this optimized pulse-induction
protocol (fig. S3), we determined how our reporter
pairs with synthetic and natural 3UTRs respond to
miRNAs during an extended period of time. Nor-

Downloaded from http://science.sciencemag.org/ on July 22, 2016

we expressed additional amounts of endogenous- responding to ectopically expressed miR-9b and malized levels of luciferase luminescence for both
ly present miRNAs (bantam and miR-279) from miR-279 exhibited strong repression 48 hours miRNA-targeted and nontargeted constructs were
plasmids under the constitutive actin promoter. after induction (Fig. 1, C and E). The addition of used to assess miRNA-mediated gene silencing at

Fig. 2. Time-resolved progression of miRNA-mediated gene silencing es- reporter genes from oligo(dT)25 resin precipitation or from total RNA
tablishes that repression of protein synthesis precedes mRNA deadenylation presented as ratios of poly(A) and total mRNA, respectively. Each data point
and decay. (A to D) Normalized levels of protein amounts for both miRNA- represents the mean value T SD calculated from three independent
targeted (T) and nontargeted (NT) constructs; normalized mRNA levels for experiments.

238 13 APRIL 2012 VOL 336 SCIENCE www.sciencemag.org


the protein level. mRNA levels for reporter and
control genes were determined by using quantita-
tive reverse transcription polymerase chain reaction
(qRT-PCR) from RNA samples isolated with oligo
(dT)25 resin, as well as from total RNA. Both val-
ues were normalized with respect to the parallel
transfected control genes and were used to calculate
repression ratios between miRNA-targeted and
nontargeted constructs (Fig. 2). We emphasize that
there is substantial mRNA degradation (for all re-
porters) during the experiment (fig. S4), but here
we are interested in the relative amount of decay of
the targeted and nontargeted mRNAs. The results
of the pulse-induction experiments were consistent
and show that the repressive effects of miRNAs on
synthesis of all four proteins precede any effects on
mRNA deadenylation or decay (Fig. 2).
These four examples include effects on pro-
tein and mRNA levels that are both well cor-
related and poorly correlated (5, 1012, 19, 20).
For reporters with natural 3 UTRs, the repression
effects on protein and mRNA levels are corre-
lated at later (but not early) time points (Fig. 2, C
and D), consistent with global studies on miRNA-
mediated silencing (11, 12). For reporters with
synthetic 3UTRs, the repression effects on protein
and mRNA levels are uncorrelated at both early
and late time points (Fig. 2, A and B) (5, 10, 19, 20).
The differences in overall stability of the mRNAs
likely reflect the complexity of RNA degrada-
tion as specified by various 3UTR-located se-
quence motifs. In all experiments here, repression
of protein synthesis is consistently seen just 2
hours after induction, whereas mRNA destabili-
zation comes later. Additionally, in experiments
where less bantam miRNA was present (by not
supplying an exogenous source), the observed
timing of protein repression and mRNA dead-
REPORTS
enylation and decay were unaffected (fig. S5,
A and B).
We performed a similar set of experiments
with use of actinomycin D to more rapidly shut
off transcription (fig. S6, A to D). At a single
time point after induction, all constructs showed
a substantial reduction in protein production
but no reduction in mRNA abundance (Fig. 2).
Consistent with BCS shut-off data, translation
rates for targeted constructs are substantially re-
duced with respect to nontargeted ones (fig. S6,
A to D).
mRNA polyadenylate [poly(A)] tails are in-
volved in translation initiation and thus in de-
termining translational efficiency (22, 23). Potential
subtle changes in poly(A) tail length not detect-
able using an oligo(dT)25 isolation procedure were
analyzed for the complete set of RNA samples
by using a poly(A) tail-length assay that allows

Downloaded from http://science.sciencemag.org/ on July 22, 2016

Fig. 3. mRNA deadenylation is not required for miRNA-mediated trans-
lational repression. (A to D) Length of poly(A) tail was determined by using
G/I tailing PCR-based amplification (materials and methods). Positions
of overamplified products with or without (C) poly(A) tail are indicated.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA poly(A) tail
length is shown as a control. M lane represents 100base pair markers. (E)
Time-resolved progression of miRNA-mediated gene silencing for histone H3
constructs. Normalized levels of protein and mRNA amounts for miRNA-
targeted (T) and nontargeted (NT) constructs are shown. Each data point
represents an average value T SD from three independent experiments.

www.sciencemag.org SCIENCE VOL 336 13 APRIL 2012 239

REPORTS
Fig. 4. Translational elongation stalling assay in-
dicates that miRNA-mediated gene silencing targets
early steps in translation. (A to E) Time-resolved pro-
gression of miRNA-mediated effects on the stability
of various mRNA constructs containing the lysine-
induced elongation stall. Normalized mRNA amounts
for the miRNA-targeted (T) and nontargeted (NT) con-
structs are shown as the ratio of NT/T; note that
values are less than one and decreasing. Each data
point represents an average T SD from three inde-
pendent experiments.
for specific amplification of poly(A)-containing
mRNAs. Subtle changes in poly(A) tail length
could be documented with this assay when cells
were treated with puromycin (fig. S7) (24). How-
ever, we did not observe any shortening of poly(A)
tails in reporter constructs responding to bantam
or mir-9b and mir-279 (Fig. 3, A to D). Because
no intermediates are observed, these data suggest
that deadenylation in vivo is processive and close-
ly coupled to mRNA decay. These data argue that
translational inhibition is not triggered by dead-
enylation, either partial or complete, in the system
that we have established [in contrast with earlier
studies (6, 12, 25)].
We also evaluated the timing and extent of
silencing of reporter gene pairs carrying histone
H3 terminal stem loops [and no poly(A) tail] (26)
(fig. S8). This reporter pair is translationally
repressed rapidly (Fig. 3E) and at levels compa-
rable to those observed with equivalent constructs
carrying a poly(A) tail (Fig. 2A versus Fig. 3E).
These data provide further support for the idea
that deadenylation is not required for translational
repression by miRNAs (5). However, deadenyl-
ases may play a direct role in translational repres-
sion independent of their deadenylation activity
(2729), and deadenylation may consolidate the
observed translational repression.
To define where in the translation cycle the
miRNA-mediated stalling occurs, we developed
an approach that uses as a read-out the specific
cleavage of mRNAs when ribosomes stall during
translation elongation (30) (fig. S9). We inserted
12 lysine codons at position 6 in both the natural
and synthetic reporters; as a control, we inserted
12 arginines and 12 more neutral glutamine res-
idues (fig. S10). By using qRT primers that en-
compassed the predicted cleavage site as well as
the stall sequence, we determined the extent of
cleavage of both targeted and nontargeted report-
240 13 APRIL 2012
er mRNAs over time (Fig. 4, A to D, and fig.
S11, A to C). Ratios of the targeted and nontar-
geted proteins and mRNAs were normalized to
the amount of a parallel control reporter. In all
cases, mRNAs of nontargeted reporters were
rapidly cleaved and degraded, whereas mRNAs
of the targeted reporters were relatively stabilized.
Similar results were obtained in a stalling exper-
iment with histone H3 constructs, confirming
that the poly(A) tail is not essential for miRNA-
mediated translational repression (Fig. 4E). These
data establish that miRNA-mediated translational
silencing happens in the Drosophila system dur-
ing the initiation or early elongation phase of pro-
tein synthesis.
We find that miRNA-mediated gene silencing
in Drosophila S2 cells is first manifested through
effects on translation, and in particular the early
events thereof, and is subsequently consolidated
by mRNA deadenylation and decay. Although it
is possible that the order of events is different in
other systems or in a fashion that is mRNA-
specific, our data in Drosophila are consistent.
Moreover, these observations are consistent with
earlier studies on miRNA-mediated silencing
in vitro (15, 17) and with previous studies of trans-
lation as affected by iron levels (24). With these
insights into the relative timing of the events in-
volved in miRNA-mediated gene silencing, we
can now focus subsequent molecular mechanistic
analysis on these earliest triggering steps.
References and Notes
1. D. P. Bartel, Cell 136, 215 (2009).
2. S. Bagga et al., Cell 122, 553 (2005).
3. L. P. Lim et al., Nature 433, 769 (2005).
4. A. J. Giraldez et al., Science 312, 75 (2006); 10.1126/
science.1122689.
5. A. Eulalio et al., RNA 15, 21 (2009).
6. T. H. Beilharz et al., PLoS ONE 4, e6783 (2009).
7. P. H. Olsen, V. Ambros, Dev. Biol. 216, 671 (1999).
VOL 336 SCIENCE www.sciencemag.org
Downloaded from http://science.sciencemag.org/ on July 22, 2016
8. R. S. Pillai et al., Science 309, 1573 (2005); 10.1126/
science.1115079.
9. C. P. Petersen, M. E. Bordeleau, J. Pelletier, P. A. Sharp,
Mol. Cell 21, 533 (2006).
10. X. C. Ding, H. Grosshans, EMBO J. 28, 213 (2009).
11. D. G. Hendrickson et al., PLoS Biol. 7, e1000238
(2009).
12. H. Guo, N. T. Ingolia, J. S. Weissman, D. P. Bartel,
Nature 466, 835 (2010).
13. L. Wu, J. Fan, J. G. Belasco, Proc. Natl. Acad. Sci. U.S.A.
103, 4034 (2006).
14. M. Wakiyama, K. Takimoto, O. Ohara, S. Yokoyama,
Genes Dev. 21, 1857 (2007).
15. G. Mathonnet et al., Science 317, 1764 (2007).
16. A. Zdanowicz et al., Mol. Cell 35, 881 (2009).
17. M. R. Fabian et al., Mol. Cell 35, 868 (2009).
18. H. Johansen et al., Genes Dev. 3, 882 (1989).
19. A. Nahvi, C. J. Shoemaker, R. Green, RNA 15, 814
(2009).
20. I. Behm-Ansmant et al., Genes Dev. 20, 1885 (2006).
21. A. Selvaraj et al., Genes Dev. 19, 891 (2005).
22. D. R. Gallie, Genes Dev. 5, 2108 (1991).
23. T. H. Beilharz, T. Preiss, RNA 13, 982 (2007).
24. M. Muckenthaler, N. Gunkel, R. Stripecke, M. W. Hentze,
RNA 3, 983 (1997).
25. S. Iwasaki, T. Kawamata, Y. Tomari, Mol. Cell 34, 58
(2009).
26. W. F. Marzluff, E. J. Wagner, R. J. Duronio, Nat. Rev.
Genet. 9, 843 (2008).
27. J. E. Braun, E. Huntzinger, M. Fauser, E. Izaurralde,
Mol. Cell 44, 120 (2011).
28. M. Chekulaeva et al., Nat. Struct. Mol. Biol. 18, 1218
(2011).
29. M. R. Fabian et al., Nat. Struct. Mol. Biol. 18, 1211
(2011).
30. K. Kuroha et al., EMBO Rep. 11, 956 (2010).
Acknowledgments: We thank K. Wehner, J. Doudna,
M. Jinek, N. Guydosh, and J. Coller for helpful comments.
Funding is from HHMI.
Supplementary Materials
www.sciencemag.org/cgi/content/full/336/6078/237/DC1
Materials and Methods
Figs. S1 to S11
Tables S1 to S4
References
24 October 2011; accepted 13 March 2012
10.1126/science.1215691
 miRNA-Mediated Gene Silencing by Translational Repression
Followed by mRNA Deadenylation and Decay
Sergej Djuranovic, Ali Nahvi and Rachel Green (April 12, 2012)
Science 336 (6078), 237-240. [doi: 10.1126/science.1215691]

Editor's Summary

Translation Block
MicroRNAs (miRNAs) are small, noncoding RNA genes that are found in the genomes of most
eukaryotes, where they play an important role in the regulation of gene expression. Although whether
gene activity is repressed by blocking translation of messenger RNA (mRNA) targets, or by promoting
their deadenylation and then degradation, has been open to debate. Bazzini et al. (p. 233, published
online 15 March) and Djuranovic et al. (p. 237) looked at early points in the repression reaction in the
zebrafish embryo or in Drosophila tissue culture cells, respectively, and found that translation was

Downloaded from http://science.sciencemag.org/ on July 22, 2016

blocked before target mRNAs were significantly deadenylated and degraded. Thus, miRNAs appear to
interfere with the initiation step of translation.

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