MicroRNAs direct rapid deadenylation of mRNA
Ligang Wu*†, Jihua Fan*, and Joel G. Belasco*†‡
*Skirball Institute of Biomolecular Medicine and†Department of Microbiology, New York University School of Medicine, New York, NY 10016
Edited by Stanley N. Cohen, Stanford University School of Medicine, Stanford, CA, and approved January 19, 2006 (received for review December 18, 2005)
MicroRNAs (miRNAs) are ubiquitous regulators of eukaryotic gene
expression. In addition to repressing translation, miRNAs can
down-regulate the concentration of mRNAs that contain elements
to which they are imperfectly complementary. Using miR-125b and
let-7 as representative miRNAs, we show that in mammalian cells
this reduction in message abundance is a consequence of acceler-
ated deadenylation, which leads to rapid mRNA decay. The ability
of miRNAs to expedite poly(A) removal does not result from
require a poly(A) tail, a 3? histone stem-loop being an effec-
tive substitute. These findings suggest that miRNAs use two
distinct posttranscriptional mechanisms to down-regulate gene
let-7 ? miR-125b ? poly(A) ? translation
ulating protein synthesis. In animal cells, these small untrans-
lated RNAs repress gene expression by annealing to mRNAs to
which they are partially complementary. Unlike perfectly com-
plementary siRNAs, which guide mRNA cleavage at the sites to
the ability of their mRNA targets to function as templates for
protein synthesis, apparently by inhibiting translation initiation
by means of a mechanism that is poorly understood (2–5).
Despite this important difference, the regulatory influence of
both miRNAs and siRNAs is thought to be mediated by similar
protein complexes that deliver them to their mRNA targets
Although initial reports suggested that down-regulation by
partially complementary miRNAs was due entirely to decreased
also reduce the cellular concentration of the mRNAs that they
regulate, both in vitro and in vivo (12–16). For example, the
interaction of miR-125b or its paralog miR-125a with two
imperfectly complementary elements (miRE1 and miRE2) in
the 3? UTR of the mammalian lin-28 message leads to significant
reductions in both translation and mRNA abundance (15). This
decline in mRNA concentration has been shown to occur by a
posttranscriptional mechanism. In Caenorhabditis elegans, where
lin-28 plays an important role in larval development, a devel-
opmentally regulated miRNA homologous to miR-125b has a
similar effect on lin-28 message levels (14). These and other
findings have led to suggestions that miRNAs may be able to
destabilize mRNAs to which they are imperfectly complemen-
tary. However, the mechanism by which they do so is not known.
Here we report that in mammalian cells two different miRNAs,
miR-125b and let-7, expedite poly(A) tail removal as an initial step
in the accelerated degradation of mRNAs containing elements to
which they are imperfectly complementary. This increased rate of
deadenylation does not result from the diminished frequency of
translation caused by miRNA binding. Conversely, although
poly(A) removal appears to be a key step in miRNA-mediated
mRNA decay, a poly(A) tail is not required for translational
repression by miRNAs.
burgeoning body of evidence indicates that microRNAs
(miRNAs) play an important and widespread role in reg-
Down-regulation of mRNA by miR-125b has been observed
both in P19 mouse embryonal carcinoma cells, where the
increased production of this miRNA upon differentiation into
neurons contributes to a marked decline in the concentration of
lin-28 mRNA, and in 293T human embryonic kidney cells, where
the synthesis of miR-125b from a transfected gene significantly
reduces the cellular abundance of luciferase reporter mRNAs
bearing multiple copies of either miRE1 or miRE2 in the 3?
UTR (15). To determine whether miR-125b decreases the
concentration of mRNAs bearing these elements by expediting
mRNA degradation, we examined the effect of lin-28 miRE1 on
the decay of a ?-globin reporter mRNA (BG) expressed in 293T
cells under the control of a transiently inducible c-fos promoter.
This well established promoter-reporter system for studying
degradation of mRNA molecules that were similar in age (17).
The presence of two copies of miRE1 in the 3? UTR (BG?2E1)
markedly accelerated the decay of the reporter mRNA in cells
that had been engineered to produce miR-125b at a concentra-
tion comparable with that in differentiating P19 cells (Fig. 1 A
and B; see also Fig. 6, which is published as supporting infor-
mation on the PNAS web site). Inserting additional copies of
miRE1 resulted in even faster decay (data not shown). No such
effect was observed in cells that lacked miR-125b (Fig. 1B; see
also Fig. 6).
decay did not result from endonucleolytic cleavage within the
lin-28 element. Luciferase reporter mRNAs that bore either
miRE1 or a synthetic element (element P) perfectly comple-
mentary to miR-125b were extracted from 293T cells containing
miR-125b and analyzed by ligation to a synthetic oligoribonu-
cleotide and RT-PCR with primers related to the oligoribonu-
cleotide or complementary to a 3? UTR segment downstream of
the regulatory element. This procedure made it possible to
detect any 3?-terminal degradation intermediates that might
result from mRNA cleavage within the target element, as the 5?
end of such intermediates could be joined to the oligoribonu-
cleotide by T4 RNA ligase (18). Although miR-125b directed
cleavage within the RNA element to which it was perfectly
complementary, no such cleavage could be detected in or near
the imperfectly complementary miRE1 element, which none-
theless mediated significant reductions in luciferase mRNA and
protein levels (Fig. 1C and other data not shown; see also Fig.
7, which is published as supporting information on the PNAS
web site). We conclude that miR-125b accelerates the decay of
mRNA containing imperfectly complementary elements by a
process characteristic of RNA silencing mediated by perfectly
complementary siRNAs (1).
Closer examination of the decay of BG?2E1 mRNA revealed
that, in the presence of miR-125b, it underwent noticeable
shortening within 3 h after its transient synthesis (Fig. 1B).
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviation: miRNA, microRNA.
See Commentary on page 3951.
‡To whom correspondence should be addressed at: Skirball Institute of Biomolecular
© 2006 by The National Academy of Sciences of the USA
March 14, 2006 ?
vol. 103 ?
Site-specific cleavage in vitro by RNase H before gel electro-
phoresis showed that this decrease in length was due to trimming
of the 3? poly(A) tail (Fig. 1D), most likely by an exonuclease. By
measuring poly(A) length as a function of time after inducing
transient transcription in cells containing miR-125b, we found
that the presence of miRE1 caused extensive deadenylation and
that decay of the transcribed portion of BG?2E1 mRNA did not
begin until the shortest poly(A) tails had reached a length of
20–30 nt or less. No such effect of miR-125b was observed for
the poly(A) tail of an otherwise identical reporter mRNA (BG)
lacking miRE1; nor did miRE1 expedite deadenylation in the
absence of miR-125b. That BG?2E1 mRNA undergoing miR-
125b-mediated deadenylation still retained its 5? cap was evident
from its resistance to 5?-exonucleolytic digestion in vitro, which
was lost upon treatment with tobacco acid pyrophosphatase to
remove the cap (Fig. 1E). These findings indicate that miR-125b
can destabilize mRNA by interacting with imperfectly comple-
mentary 3? UTR elements and hastening deadenylation as an
initial step in accelerated decay.
To identify other transcripts down-regulated by miR-125b,
we examined its effect on mRNA levels in P19 cells, which
produce this miRNA when induced to differentiate into neu-
rons (15, 19). Cytoplasmic RNA extracted from undifferenti-
ated P19 cells that had or had not been transfected with
chemically synthesized miR-125b (Fig. 6) was used to probe a
mouse genome microarray. With a high degree of confidence
(P ? 0.003), 22 mRNAs were found to decrease in abundance
by at least a factor of 1.4 within 24 h after exposure to
miR-125b. As expected, these included lin-28 mRNA, whose
concentration fell by a factor of 1.7, a change comparable with
the 2-fold increase in Lin-28 protein levels previously observed
when the function of miR-125b was inhibited in differentiating
P19 cells (15). Most of the affected mRNAs contained one or
more 3? UTR elements with the potential to interact produc-
tively with miR-125b, whereas such elements were compara-
tively rare in P19 mRNAs judged not to be affected (Fig. 2A;
see also Table 1, which is published as supporting information
on the PNAS web site).
mRNAs bearing miRE1 or a synthetic element (element P) perfectly comple-
mentary to miR-125b were extracted from 293T cells that did or did not
produce miR-125b, ligated to an RNA oligonucleotide, and amplified by
RT-PCR, using primers corresponding to sites within the ligated oligonucleo-
tide or 0.08 kb downstream of the inserted element. The RT-PCR products
were analyzed by gel electrophoresis beside DNA size markers (M). DNA
sequencing confirmed that band C represented a degradation intermediate
resulting from miR-125b-directed mRNA cleavage in the middle of the per-
fectly complementary element P. Band X, which resulted from miR-125b-
independent mRNA cleavage upstream of the insertion site of miRE1 or
is due to the diminished concentration of this reporter mRNA in the presence
of miR-125b and competition with band C for PCR amplification. Calibration
is in base pairs. (D) Deadenylation mediated by miR-125b. Equal amounts of
the total cytoplasmic RNA samples examined in B were subjected to site-
specific cleavage by RNase H in the presence of an oligodeoxynucleotide
mRNA. The 5? and 3? RNA fragments thereby produced were analyzed by
electrophoresis and blotting, using markers (M) that corresponded in size to
reporter mRNA 3? fragments bearing no poly(A) or a 160-nt poly(A) tail. (E)
Retention of the 5? cap on mRNA undergoing deadenylation. The 1.5 and 5 h
BG?2E1 mRNA samples examined in B were treated or not treated with a
5?-phosphate-dependent exonuclease and analyzed by electrophoresis and
blotting. Prior treatment of the 1.5 h sample with tobacco acid pyrophos-
phatase (TAP) released the 5? cap and rendered the mRNA susceptible to 5?
exonuclease digestion. 28S rRNA, which lacks a 5? cap, served as an additional
positive control for exonuclease activity. AG-GAPDH mRNA was used as a
normalization standard. The percentage of BG?2E1 mRNA that was capped
was calculated from the ratio of band intensities in the presence versus the
absence of exonuclease treatment.
(A) Duplex expected for lin-28 miRE1 (top) base-paired with miR-125b (bot-
tom). (B) Decay of BG?2E1 and BG mRNA in the presence or absence of
miR-125b. Total cytoplasmic RNA was extracted from transfected 293T cells at
reporter gene from its c-fos promoter. Equal amounts of each RNA sample
were analyzed by electrophoresis and blotting. The relative quantity of re-
porter mRNA remaining at each time was calculated after normalization to
AG-GAPDH mRNA (a cotransfected internal standard). (C) Inability of miR-
125b to direct endonucleolytic cleavage within miRE1. Luciferase reporter
Wu et al.
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Besides lin-28, the P19 messages down-regulated by miR-
125b included Ajuba mRNA, whose 3? UTR contains a likely
miR-125b-responsive element in both mice and humans (A1;
Fig. 2B). Ajuba is a LIM-domain protein that plays an
important role in controlling the entry of vertebrate cells into
mitosis and meiosis (20, 21). In P19 cells induced to differen-
tiate, the concentration of this protein initially rises and then
declines (22), suggesting a delayed-onset repression mecha-
nism to which miR-125b may contribute as it accumulates over
time. Like lin-28 miRE1, the Ajuba A1 element was found to
direct rapid deadenylation and subsequent decay of a reporter
mRNA in transfected 293T cells that produced miR-125b (Fig.
8, which is published as supporting information on the PNAS
web site). mRNA destabilization involving fast poly(A) re-
moval was likewise observed for an element derived from
MKK7 (MAPK kinase 7) mRNA, another message down-
regulated by miR-125b in P19 cells (Fig. 2B; see also Fig. 9,
which is published as supporting information on the PNAS web
site). These findings indicate that miR-125b can expedite the
deadenylation and decay of many different mRNAs bearing a
variety of elements to which it is imperfectly complementary.
A second miRNA, let-7, also helps to down-regulate lin-28 gene
expression in differentiating P19 cells by interacting with a distinct
3? UTR element (L7; Fig. 3A) (15). In combination, L7 and let-7
proved to be as effective as miRE1 and miR-125b at repressing
protein synthesis (15), as judged from the 89% decrease in expres-
sion of an L7-containing luciferase reporter (Luc?6L7) in the
presence of let-7a. Furthermore, down-regulation of protein syn-
thesis was accompanied by a smaller yet substantial decline in
mRNA concentration (Fig. 3B). This reduction in mRNA was a
consequence of accelerated deadenylation and decay, as evidenced
of probable miR-125b-responsive elements. Mouse Genome 430A 2.0 arrays
(Affymetrix) were used to compare the relative concentrations of mRNAs in
undifferentiated P19 cells 24 h after mock transfection or transfection with a
chemically synthesized miR-125b duplex (see Fig. 6). Two groups of mRNAs
were identified whose concentration could be said with a high degree of
certainty to have decreased significantly (by at least a factor of 1.40, with
?95% confidence that the change was by at least a factor of 1.30) (see Table
1) or to have remained unchanged (?95% confidence of a concentration
change no greater than ?5%) in the presence of miR-125b. These mRNAs (22
in the first group, and 669 in the second) were then examined for 3? UTR
elements with the potential to interact productively with miR-125b [comple-
A1 element or the MKK7 M7 element with miR-125b.
miR-125b-responsive elements in down-regulated mRNAs of P19
lin-28 L7 element. (A) Duplex expected for the lin-28 L7 element (top) base-
paired with let-7a (bottom). (B) Influence of let-7a on the concentration of
blotting of total cytoplasmic RNA from 293T cells that had been transiently
cotransfected with a luciferase reporter gene containing 0 or 6 copies of the
let-7a, and a ?-galactosidase gene (internal standard). (C) Influence of let-7a
on the degradation of BG?L7 mRNA in 293T cells. Analyses of mRNA dead-
enylation and decay similar to those in Fig. 1 were performed with RNA
reporter gene, a gene encoding (?) or not encoding (?) human let-7a, and a
gene encoding AG-GAPDH mRNA (internal standard). (D) Deadenylation and
decay of BG?L7 and BG mRNA in HeLa cells. Analyses of mRNA degradation
that had been transiently cotransfected with a reporter gene containing
Effect of let-7 on the deadenylation and decay of mRNA bearing the
www.pnas.org?cgi?doi?10.1073?pnas.0510928103Wu et al.
by the ability of one copy of L7 to cause rapid poly(A) shortening
and subsequent degradation of BG?L7 mRNA in 293T cells
transfected with a gene encoding let-7a (Fig. 3C; see also Fig. 6).
That these effects were not cell-type-specific and could be caused
by an endogenous miRNA was demonstrated by showing a similar
influence of L7 on mRNA deadenylation and decay in HeLa cells
(Fig. 3D), which produce let-7 naturally (23).
To demonstrate that the rapid deadenylation and decay
mediated by miRNAs is not a consequence of the reduced
frequency with which ribosomes transit the coding region,
(40-nt) stem-loop structure within its 5? UTR to create
BG?2E1?hp. This stem-loop had previously been shown to
prevent assembly of 80S ribosomes when present at the same
location within another mRNA containing an identical 5? UTR
and coding region (24), and its inhibitory effect on translation of
BG?2E1?hp mRNA was confirmed by in-frame fusion to a
luciferase coding region, which showed it to reduce protein
synthesis by ?99.5%. Blocking translation initiation in this
manner caused only a slight increase in the slow rate of poly(A)
shortening previously observed for BG?2E1 in the absence of
miR-125b, and this change was dwarfed by the much larger
increase in the rate of deadenylation and decay caused by
miR-125b (Fig. 4). Thus, impaired translation per se is not
sufficient to accelerate deadenylation markedly, nor is transla-
tion required for miR-125b to increase the rate of poly(A)
Conversely, a poly(A) tail is not required for miR-125b to
repress translation. This was shown by comparing the effect of
miR-125b on luciferase reporter mRNAs bearing miRE1 and
either a 3? poly(A) tail (Luc?6E1) or a 3?-terminal stem-loop
derived from a histone mRNA (Luc?6E1.HSL). As expected,
miR-125b diminished the abundance of only the polyadenylated
reporter mRNA (Fig. 5 A and B). Interestingly, assays of
luciferase protein synthesis showed that, whereas the overall
the inhibitory effect of miR-125b on translation efficiency
(protein synthesis per mRNA molecule) was the same for both
mRNAs (Fig. 5C). This finding suggested that the regulatory
effects of translational repression and accelerated decay were
additive, such that decreases in translation efficiency and mRNA
concentration contributed equally to down-regulation of the
polyadenylated reporter, whereas repression of the reporter
bearing a histone stem-loop was achieved entirely at the level of
translation. We conclude that, whereas the mRNA decay mech-
anism triggered by the interaction of miR-125b with miRE1
Analyses of mRNA degradation similar to those in Fig. 1 were performed with
RNA samples from 293T cells that had been transiently cotransfected with a
modified BG?2E1 gene bearing a 40-bp inverted repeat in the 5? UTR
encoding AG-GAPDH mRNA (internal standard).
Accelerated deadenylation and decay in the absence of translation.
ential effect of a 3? histone stem-loop or a poly(A) tail on the reduction in
by electrophoresis and blotting of total cytoplasmic RNA from 293T cells that
had been transiently cotransfected with a gene that encoded a luciferase
reporter mRNA bearing six copies of miRE1 in the 3? UTR and ending with
either a 3? poly(A) tail (Luc?6E1) or a 3?-terminal histone H1.3 stem-loop
(Luc?6E1.HSL), a gene that encoded (?) or did not encode (?) miR-125b, and
a ?-galactosidase gene (internal standard). (B) Confirmation of the dissimilar
nature of the 3? ends. The Luc?6E1 and Luc?6E1.HSL RNA samples from cells
lacking miR-125b were also analyzed by electrophoresis and blotting after
treatment, in the presence or absence of oligo(dT), with RNase H and an
oligodeoxynucleotide complementary to a segment 361–386 nt upstream of
the poly(A) addition site of Luc?6E1 or 376–395 nt upstream of the 3? end of
Luc?6E1.HSL (right). Calibration is in nucleotides. (C) Contributions of trans-
After normalizing the concentrations of Luc?6E1 and Luc?6E1.HSL mRNA to
?-galactosidase mRNA, the ratio of each in the absence or presence of miR-
125b was calculated and superposed on a bar graph showing the overall
degree of repression of the same reporters, as judged from relative luciferase
protein levels (normalized to ?-galactosidase). The effect of miR-125b on
translation efficiency (black bars) corresponds to the ratio of its overall effect
case of the polyadenylated reporter (Luc?6E1), which was repressed 10.7-
0.3 and mRNA abundance by a factor of 3.5 ? 0.2. In the case of the reporter
ending in a histone stem-loop (Luc?6E1.HSL), which was repressed 3.9-fold,
miR-125b reduced translation efficiency by a factor of 3.6 ? 0.3 and mRNA
abundance by a factor of 1.1 ? 0.1.
Translational repression in the absence of a poly(A) tail. (A) Differ-
Wu et al.
March 14, 2006 ?
vol. 103 ?
no. 11 ?
involves accelerated deadenylation, the mechanism of transla-
tional repression mediated by the same interaction does not
require a poly(A) tail.
The discovery that both miR-125b and let-7 can expedite
poly(A) removal as an initial step in the rapid degradation of
mRNAs bearing imperfectly complementary elements sug-
gests that accelerated deadenylation and decay are likely to
be frequent consequences of the interaction of mammalian
miRNAs with their targets. By analogy to known pathways for
mRNA degradation (25), poly(A) tail loss would be expected
to facilitate decay of the transcribed portion of targeted
mRNAs by exposing either their 5? ends to cap removal and
subsequent degradation by the 5? exonuclease Xrn1 or their 3?
ends to exonucleolytic degradation by the exosome. The
former pathway would help to explain the detection of 3?-
terminal decay intermediates resulting from let-7-mediated
mRNA degradation in C. elegans (14).
mRNA decay triggered by rapid deadenylation appears to be
an important aspect of the mechanism by which miR-125b and
let-7 down-regulate gene expression. Thus, replacing an
mRNA poly(A) tail with a 3? histone stem-loop not only
eliminates the effect of miR-125b on message abundance but
also diminishes its influence on protein synthesis by an amount
equal to its effect on the concentration of an otherwise
identical mRNA bearing a poly(A) tail. This finding suggests
that, by reducing both the translation efficiency and the
concentration of targeted mRNAs that are polyadenylated,
miRNAs can have a greater overall impact on gene expression
than if down-regulation were limited to translational repres-
sion. Moreover, unlike translation inhibition, the regulatory
consequences of mRNA degradation are irreversible. By ex-
trapolation, accelerated deadenylation may also contribute to
the off-target effects of siRNAs, which can likewise reduce the
abundance of mRNAs to which they are imperfectly comple-
Although it is possible that miRNA-mediated deadenylation
and translational repression may both result from a single
precipitating event, our findings imply that these two regulatory
mechanisms can operate independently. Translation is not re-
quired for miRNAs to hasten poly(A) shortening, nor is a
poly(A) tail needed for them to down-regulate translation
efficiency. The latter observation is of particular significance in
view of recent evidence that miRNAs inhibit translation initia-
tion (5), as poly(A)-binding protein (PABP) facilitates transla-
tion initiation by interacting with a protein component of the 5?
cap binding complex (27, 28). Thus, miRNA-mediated poly(A)
tail loss might have been expected to impair translation effi-
ciency were the deadenylated messages not rapidly degraded.
The ability of miR-125b to inhibit translation of a reporter that
ends in a histone stem-loop instead of a poly(A) tail suggests that
translational repression by miRNAs occurs by a mechanism
where PABP and the protein that binds histone stem-loops
function analogously (28, 29).
Materials and Methods
Plasmid Construction, Cell Culture, and Transfection. Methods for
plasmid construction, cell culture, and DNA transfection are
published as Supporting Materials and Methods on the PNAS web
Monitoring the Decay of ?-Globin Reporter mRNAs. DNA mixtures
for transient transfection contained a ?-globin reporter plasmid
(pBG, pBG?2E1, pBG?A1, pBG?M7, or pBG?L7; 0.8 ?g), a
plasmid encoding or not encoding a miRNA (pMIR125b or
pMIR125b?; pLET7a or pLET7a?; 1.0–3.0 ?g), and a plasmid
encoding an ?-globin-GAPDH mRNA chimera (AG-GAPDH)
under the control of a constitutive SV40 promoter (pSV?1-
GAPDH; 0.2 ?g), which served as an internal standard. After
serum-starving the transfected cells for 24 h (0.5% serum),
transcription from the c-fos promoter of the reporter gene was
transiently induced by increasing the serum concentration to
20%, and total cytoplasmic RNA was isolated at time intervals
and analyzed by electrophoresis (1.5% agarose) and blotting, as
described in ref. 15.
To monitor the degradation of 5?- and 3?-terminal segments
of reporter mRNAs, equal amounts of cytoplasmic RNA were
treated with RNase H in the presence of an oligodeoxynucle-
otide (oligo BBB243: GGTTGTCCAGGTGACTCAGAC-
CCTC) complementary to codons 74–81 within the ?-globin
coding region, and the digested RNA samples were analyzed
by electrophoresis and blotting, using a 5% polyacrylamide?8
M urea gel. Size markers for fully deadenylated mRNA and for
mRNA bearing a 160-nt poly(A) tail were generated by
treating reporter mRNAs in cytoplasmic extracts with RNase
H, oligo(dT), and either oligo BBB243 or an oligodeoxynucle-
otide (oligo BBB81: CCTCACCACCAACTTCTTCCAC-
ATT) complementary to codons 20–27 within the ?-globin
coding region. Alternatively, RNA markers of the same size
and sequence were generated by in vitro transcription with T7
Similar procedures were used to examine let-7-mediated
deadenylation and decay in HeLa cells, except that the cells
were transfected with a ?-globin reporter plasmid (pBG
or pBG?L7; 0.8 ?g), pSV?1-GAPDH (0.2 ?g), and pUC19
To test for the presence of a 5? cap, cytoplasmic RNA samples
(10 ?g) were treated with Terminator 5?-phosphate-dependent
exonuclease (1 unit; EPICENTRE Biotechnologies) for 2 h at
30°C, according to the manufacturer’s protocol. As a positive
control, samples were pretreated with tobacco acid pyrophos-
phatase (5 units; EPICENTRE Biotechnologies) for 2 h at 37°C
to release the 5? cap.
Luciferase mRNA and Protein Assays. Relative steady-state levels
of luciferase and ?-galactosidase mRNA or protein were
assayed in extracts of transiently transfected 293T cells by
RNA electrophoresis and blotting or by measuring enzyme
activity, as described in ref. 15. To confirm the differential
nature of the 3? ends of Luc?6E1 and Luc?6E1.HSL mRNA,
cytoplasmic RNA from 293T cells transfected with pCL-6E1 or
pCL-6E1.HSL was treated, in the presence or absence of
oligo(dT), with RNase H and an oligodeoxynucleotide com-
plementary either to a segment 361–386 nt upstream of the
poly(A) addition site of Luc?6E1 (oligo HIVRH: CCGTTC-
ACTAATCGAATGGATCTGTC) or to a segment 376–395 nt
upstream of the 3? end of Luc?6E1.HSL (oligo LUC32:
TTCCGCCCTTCTTGGCCTTT). The resulting RNA sam-
ples were analyzed by electrophoresis on a 5% polyacryl-
amide?8 M urea gel beside a set of radiolabeled RNA size
markers, followed by blotting and probing.
A description of the use of oligoribonucleotide ligation and
RT-PCR to determine whether miR-125b can direct cleavage of
a luciferase reporter mRNA within miRE1 can be found in
Supporting Materials and Methods.
Microarray Analysis of mRNA in P19 Cells. Triplicate cultures of
undifferentiated P19 cells growing in ?-MEM (GIBCO) sup-
plemented with 10% FBS were mock-transfected or transfected
with a chemically synthesized miR-125b duplex (5?-UCCCU-
GAGACCCUAACUUGUGA-3? and 5?-ACAAGUUAGGG-
UCUCAGGGAUU-3?, 40 nM; Dharmacon) in the presence of
Lipofectamine 2000 (Invitrogen; see manufacturer’s protocol).
After 12 h, the transfection medium was replaced with fresh
www.pnas.org?cgi?doi?10.1073?pnas.0510928103 Wu et al.
medium, and the cells were grown for an additional 12 h before
total cytoplasmic RNA was extracted (15). The resulting RNA
Mouse Genome 430A 2.0 arrays (Affymetrix). The microarrays
were scanned with an Affymetrix GeneChip Scanner 3000, and
the raw data were processed with Affymetrix GCOS software.
Calculations of relative mRNA concentration, including nor-
malization and model-based analysis, were performed by using
DCHIP software (30).
This work was supported by National Institutes of Health Grant
GM55624 (to J.G.B.).
1. Zamore, P. D., Tuschl, T., Sharp, P. A. & Bartel, D. P. (2000) Cell 101, 25–33.
2. Lee, R. C., Feinbaum, R. L. & Ambros, V. (1993) Cell 75, 843–854.
3. Wightman, B., Ha, I. & Ruvkun, G. (1993) Cell 75, 855–862.
4. Moss, E. G., Lee, R. C. & Ambros, V. (1997) Cell 88, 637–646.
5. Pillai, R. S., Bhattacharyya, S. N., Artus, C. G., Zoller, T., Cougot, N., Basyuk,
E., Bertrand, E. & Filipowicz, W. (2005) Science 309, 1573–1576.
6. Hutva ´gner, G. & Zamore, P. D. (2002) Science 297, 2056–2060.
7. Martinez, J., Patkaniowska, A., Urlaub, H., Luhrmann, R. & Tuschl, T. (2002)
Cell 110, 563–574.
8. Mourelatos, Z., Dostie, J., Paushkin, S., Sharma, A., Charroux, B., Abel, L.,
Rappsilber, J., Mann, M. & Dreyfuss, G. (2002) Genes Dev. 16, 720–728.
9. Doench, J. G., Petersen, C. P. & Sharp, P. A. (2003) Genes Dev. 17, 438–442.
10. Zeng, Y., Yi, R. & Cullen, B. R. (2003) Proc. Natl. Acad. Sci. USA 100,
11. Meister, G., Landthaler, M., Patkaniowska, A., Dorsett, Y., Teng, G. & Tuschl,
T. (2004) Mol. Cell 15, 185–197.
12. Lim, L. P., Lau, N. C., Garrett-Engele, P., Grimson, A., Schelter, J. M., Castle,
J., Bartel, D. P., Linsley, P. S. & Johnson, J. M. (2005) Nature 433, 769–773.
13. Jing, Q., Huang, S., Guth, S., Zarubin, T., Motoyama, A., Chen, J., Di Padova,
F., Lin, S. C., Gram, H. & Han, J. (2005) Cell 120, 623–634.
14. Bagga, S., Bracht, J., Hunter, S., Massirer, K., Holtz, J., Eachus, R. &
Pasquinelli, A. E. (2005) Cell 122, 553–563.
15. Wu, L. & Belasco, J. G. (2005) Mol. Cell. Biol. 25, 9198–9208.
16. Kru ¨tzfeldt, J., Rajewsky, N., Braich, R., Rajeev, K. G., Tuschl, T., Manoharan,
M. & Stoffel, M. (2005) Nature 438, 685–689.
17. Shyu, A. B., Greenberg, M. E. & Belasco, J. G. (1989) Genes Dev. 3, 60–72.
18. Yekta, S., Shih, I. H. & Bartel, D. P. (2004) Science 304, 594–596.
19. Sempere, L. F., Freemantle, S., Pitha-Rowe, I., Moss, E., Dmitrovsky, E. &
Ambros, V. (2004) Genome Biol. 5, R13.
20. Goyal, R. K., Lin, P., Kanungo, J., Payne, A. S., Muslin, A. J. & Longmore,
G. D. (1999) Mol. Cell. Biol. 19, 4379–4389.
21. Hirota, T., Kunitoku, N., Sasayama, T., Marumoto, T., Zhang, D., Nitta, M.,
Hatakeyama, K. & Saya, H. (2003) Cell 114, 585–598.
22. Kanungo, J., Pratt, S. J., Marie, H. & Longmore, G. D. (2000) Mol. Biol. Cell
23. Hutva ´gner, G., McLachlan, J., Pasquinelli, A. E., Ba ´lint, E., Tuschl, T. &
Zamore, P. D. (2001) Science 293, 834–838.
24. Chen, C. Y. A., Xu, N. & Shyu, A. B. (1995) Mol. Cell. Biol. 15, 5777–5788.
25. Parker, R. & Song, H. (2004) Nat. Struct. Mol. Biol. 11, 121–127.
26. Jackson, A. L., Bartz, S. R., Schelter, J., Kobayashi, S. V., Burchard, J., Mao,
M., Li, B., Cavet, G. & Linsley, P. S. (2003) Nat. Biotechnol. 21, 635–637.
27. Tarun, S. Z., Jr., & Sachs, A. B. (1996) EMBO J. 15, 7168–7177.
28. Kahvejian, A., Svitkin, Y. V., Sukarieh, R., M’Boutchou, M. N. & Sonenberg,
N. (2005) Genes Dev. 19, 104–113.
29. Ling, J., Morley, S. J., Pain, V. M., Marzluff, W. F. & Gallie, D. R. (2002) Mol.
Cell. Biol. 22, 7853–7867.
30. Li, C. & Wong, W. H. (2001) Proc. Natl. Acad. Sci. USA 98, 31–36.
31. Lewis, B. P., Shih, I. H., Jones-Rhoades, M. W., Bartel, D. P. & Burge, C. B.
(2003) Cell 115, 787–798.
Wu et al.
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vol. 103 ?
no. 11 ?