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We analyzed the intracellular localization of
the miRNP components. Previous work has indicated
that translationally repressed mRNAs
can be sequestered in specific cellular structures
such as germline (13) or stress (14) granules.
Immunofluorescence analysis indicated that
HA-tagged hAgo2-4 proteins, expressed in transfected
HeLa and human embryonic kidney
(HEK) 293 cells (fig. S7) or in stable HeLa cell
lines (figs. S8, A to C, and S9A), localize to
processing bodies (PBs; visualized as structures
enriched in the marker proteins Lsm1, Dcp1a,
and Xrn1), suborganelles identified as sites of
mRNA degradation (15). Association of Ago
proteins with PBs was further confirmed by
co-immunoprecipitation experiments (fig. S8,
D and E). Notably, the GFP-tagged Dcp1
was found to interact with HA-hAgo2 but not
HA-hAgo2 DPRP , a mutant of hAgo2 that does
not function as a repressor (8) (fig. S8E).
Next, we analyzed the cellular distribution
of reporter mRNAs by in situ hybridization in
HeLa cells. The RL-3xBulge mRNA but not its
mutant version, RL-3xBulgeMut, co-localized
with the PB marker Dcp1a, expressed as a fusion
with green fluorescent protein (GFP) (Fig.
3A and table S1). The co-localization of RL-
3xBulge mRNA with PBs was let-7–dependent
(table S2). Moreover, RL-3xBulgeMut RNA
A
RL (arbitrary units)
7
6
5
4
3
2
1
0
RL
Con
C
FL (normalized)
2.5
2
1.5
1
0.5
0
Perf
NHA-4E
Con
1xBulge
+
P olyA
-
PolyA
3xBulge
FL
NHA-4G
3xBulge
NHA-LacZ
B
FL or RL activities
(normalized)
2 BoxB
accumulated in PBs when co-transfected with
the siRNA-like duplex, let-7Mut, which carries
mutations restoring base pairing between let-7
and RL-3xBulgeMut RNAs (Fig. 3A and fig.
S10). The RL-Perf mRNA could not be
visualized in PBs, arguing against a possibility
that RL-3xBulge or RL-3xBulgeMut signals in
PBs originate from an mRNA targeted for degradation
(fig. S11). Closer examination of the
images provided additional evidence that RL-
3xBulge RNA localization is not related to the
degradative function of PBs. RL-3xBulge RNA
was often found adjacent to Dcp1 foci and not
overlapping with them (Fig. 3A, fig. S11, and
table S3). Furthermore, we also observed that
some Dcp1 foci did not contain detectable
amounts of RL-3xBulge RNA and, conversely,
that a few mRNA foci were negative for Dcp1
(9). These data point to some heterogeneity
and/or compartmentalization of PBs.
We used HeLa cells stably expressing either
RL-3xBulge or RL-3xBulgeMut reporters
(fig. S9, B and C) to further document the association
of repressed mRNAs with cellular
structures. The cells were permeabilized with
digitonine, and the resulting S14 supernatant and
pellet fractions were analyzed for the presence
of mRNAs and PB and miRNP components.
The RL-3xBulgeMut RNA was enriched in a
RL (normalized)
1.5
1
0.5
0
1.5
1
0.5
0
Con
FL
Con
3xBulge
RL
NHA-4E
Con
EMCV-FL
3xBulge
Con
EMCV-RL
3xBulge
Anti-miR-122a
Anti-let-7
NHA-4G
Con or
3xBulge
3xBulge
NHA-LacZ
Fig. 2. Cap-dependent but not cap-independent translation is repressed by let-7. The values represent
means of three transfections T SD. (A) Translation of the in vitro–transcribed capped RL-RNA reporters,
either poly(A) þ (200 ng) or poly(A) – (400 ng), in transfected HeLa cells. (B) IRES-mediated translation is
immune to repression by let-7. HeLa cells were transfected with 200 ng of the indicated noncapped
EMCV-FL or -RL RNAs or capped FL RNA. Oligonucleotides were included as indicated. (C) Translation
driven by the tethered initiation factor eIF4E or eIF4GI is immune to let-7 repression. Cells were
transfected with 100 and 500 ng of plasmids expressing dicistronic reporters and indicated NHA fusions.
Activities in co-transfections performed with pFL-2BoxB-RL-Con and pNHA-eIF4E were set to 1.
soluble fraction, but the RL-3xBulge reporter
was found almost exclusively in the pellet fraction
containing cellular structures. Likewise,
endogenous N-Ras and K-Ras mRNAs, recently
established targets of let-7 RNA (16),
were enriched in the pellet as were all tested
PB and miRNP components (fig. S12). Importantly,
treatment of cells with anti-let-7 oligonucleotide
specifically released a substantial
fraction of the pellet-associated mRNAs to the
supernatant (fig. S12A).
Lastly, we investigated whether miRNAs
localize to specific cellular structures. The let-7
and miR-122a probes stained dot-like structures
in HeLa and human hepatoma Huh7 cells, respectively,
but not in control cells known not to
express these miRNAs (16, 17) (Fig. 3B). No
staining was observed with the mutant or premiRNA–specific
probes (fig. S13). Because the
in situ hybridization was not compatible with
the antibody labeling of PBs, we microinjected
the in vitro–transcribed, Cy3-labeled pre–let-7
RNA into nuclei of HeLa cells and analyzed its
distribution. Remarkably, exported let-7 RNA
accumulated in PB foci also labeled with
antibodies against Dcp1 (Fig. 3C). The presence
of let-7 in the cytoplasm was inhibited by
wheat germ agglutinin, indicating that export of
pre-miRNA from the nucleus occurred by a
physiological mechanism (fig. S14). As with
the RL-3xBulge RNA foci, let-7 foci were
frequently adjacent to, rather than exactly colocalizing
with, Dcp1 foci. Some let-7 foci not
labeled by Dcp1, and vice versa, were also
observed (Fig. 3C and tables S1 and S3).
Our data indicate that, in mammalian cells,
let-7 miRNP inhibits translation at the initiation
step. The observation that the cap-independent
translation is immune to the repression suggests
that miRNPs target an early step of initiation,
likely involving the m 7 G cap. Importantly, the
results of experiments involving an entirely independent
methodology—a direct tethering of
hAgo2 to mRNA reporters, an approach that
mimics the miRNP-mediated inhibition (8)—
are consistent with the above interpretation.
Inhibition of initiation by factors binding to the
mRNA 3-UTR is a common theme in translational
regulation. Such inhibition may involve
interference either with the recruitment
of eIF4E to the m 7 G cap or with the eIF4EeIF4G
interaction (18–20). Our findings that
the polysomal status of mRNAs repressed by
let-7 in mammalian cells differs from that of
mRNAs repressed by lin-4 in C. elegans (1, 2)
suggest that the inhibition of productive translation
by different miRNAs, or in different
organisms, can follow diverse routes.
After initial submission of this work, other
reports implicating PBs in miRNA-mediated
repression have appeared (21, 22). Our data extend
these findings by directly demonstrating
that miRNAs and repressed mRNAs localize to
PBs. We believe that relocalization of the repressed
mRNA to PBs is a consequence rather
www.sciencemag.org SCIENCE VOL 309 2 SEPTEMBER 2005 1575