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 ?
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.).
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