1775
Commentary
MicroRNAs and their roles in mammalian stem cells
Rui Yi1,* and Elaine Fuchs2,*
1
Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, CO 80309, USA
Laboratory of Mammalian Cell Biology and Development, Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10065, USA
2
*Authors for correspondence ([email protected]; [email protected])
Journal of Cell Science
Journal of Cell Science 124, 1775-1783
© 2011. Published by The Company of Biologists Ltd
doi:10.1242/jcs.069104
Summary
Discovered in Caenorhabditis elegans in 1993, microRNAs (miRNAs) make up a novel class of tiny, ~21–24 nucleotide, non-coding
RNA species. Since its identification as a key component of a broadly conserved mechanism that regulates gene expression posttranscriptionally, the miRNA pathway has emerged as one of the most extensively investigated pathways of the past decade. Because
of their potential to regulate a large number of protein-encoding genes, miRNAs have been implicated in numerous biological
processes, including development, stem cell regulation and human diseases. In this Commentary, we focus on miRNAs and their roles
in mammalian stem cells. Following an introduction to the miRNA biogenesis pathway with an emphasis on its regulatory features,
we then discuss what is currently known about the roles that miRNAs have in the differentiation and maintenance of embryonic and
somatic stem cells of diverse origins. In particular, their roles in stem cell differentiation have been well documented. Insights from
these studies provide a paradigm for the function of miRNAs in facilitating cellular transitions during differentiation. By contrast, the
roles that miRNAs have in the maintenance of stem cells are less well understood. However, with recent advances, their role as a
rheostat that fine-tunes stem cell self-renewal has begun to emerge. Finally, we discuss future studies that will hopefully lead to a
comprehensive understanding of the miRNA pathway in stem cells.
Key words: Differentiation, MicroRNA, Self-renewal, Stem cells
Introduction
A hallmark of stem cells is their ability to self-renew in the long
term to maintain their own population and to differentiate in order
to generate daughter cells with specific physiological functions
(Siminovitch et al., 1963). During embryonic development,
pluripotent embryonic stem cells (ESCs) residing in the inner cell
mass give rise to all tissues and organs of the body. Throughout
their lifetime, tissue-specific, multipotent somatic stem cells
maintain homeostasis of individual tissues and organs. Some of the
most fascinating questions in biology focus on understanding how
stem cells are specified, maintained and instructed to differentiate.
Whereas external cues provided by, for example, the stem cell
microenvironment or niche are required to regulate stem cell
behavior, internal cues, such as transcriptional regulation, also
govern the fate of stem cells. Ultimately, all regulatory networks
converge on the regulation of gene expression.
MicroRNAs (miRNAs) comprise a novel class of non-coding
regulatory RNAs that are widely expressed in both plants and
animals (reviewed in Ambros, 2004; Bartel, 2009). They regulate
mRNA stability and consequently protein production by recruiting
the RNA-induced silencing complex (RISC) to its cognate target
sites. They therefore have an important role in the posttranscriptional regulation of gene expression (reviewed in Bartel,
2009). The recognition of mRNA target sites, which are often
located in the 3⬘ untranslated region (3⬘-UTR), by miRNAs is
believed to be primarily mediated by base pairing between
nucleotides 2–8 of the miRNA, a region also referred to as the seed
sequence, and the cognate mRNA sequence (reviewed by Bartel,
2009). miRNA-mediated gene regulatory networks are rather
complex because a single miRNA can recognize hundreds of targets
and a single mRNA can be simultaneously regulated by multiple
miRNAs. The complexity of miRNA-mediated regulation, its
potential impact on the expression of a large number of proteins
and its control over the output from the transcriptome have drawn
increased attention to these tiny regulators. Over the past decade,
much progress has been made in deciphering miRNA expression
and function in animal development, including the differentiation
and maintenance of stem cells (reviewed by Ambros, 2004; Bartel,
2009).
The investigation of the hitherto unappreciated layer of gene
regulation mediated by miRNAs has also provided new insights
into our understanding of stem cell biology and pointed to new
directions for therapeutic applications of stem cells. In this
Commentary, we review the most recent developments in our
understanding of the biological role of miRNAs in the
differentiation and maintenance of mammalian stem cells, discuss
lessons learned from recent studies and offer our views on future
challenges. Because of the considerable interest in this topic, many
excellent reviews have been published in the past few years and
we also refer our readers to these articles for a comprehensive
overview of the field (Gangaraju and Lin, 2009; Ivey and
Srivastava, 2010; Martinez and Gregory, 2010).
miRNA biogenesis and its regulation
In the past decade, the miRNA biogenesis pathway has been
extensively investigated. We outline the biogenesis pathway in
Fig. 1, but would also like to refer our readers to several excellent
reviews for more detailed discussions (e.g. Kim et al., 2009; Krol
et al., 2010). Here, we primarily explore the regulatory features of
the miRNA biogenesis pathway and discuss the potential
implications for the role of miRNAs in stem cells.
miRNAs can be transcribed by both RNA polymerase II and III
(Borchert et al., 2006; Cai et al., 2004). It is widely recognized that
many highly expressed mammalian miRNAs are initially
transcribed by RNA polymerase II in the form of a long, primary
transcript (pri-miRNA) (Cai et al., 2004). Because transcription
1776
Journal of Cell Science 124 (11)
Transcriptional regulation
RNA pol II
transcription
RNA pol II or III
transcription
hnRNPA1
pri-miRNA
G
Lin28
Dgcr8
Mirtron
RNA pol III
transcription of
hairpin RNA
p53, SMAD
G
AAAAA
Exon
Exon
AAAAA
Spliceosome
Drosha
pre-miRNA
Splicing
pre-miRNA
Dicer
ing
Dic
Xpo5
Most
miR
Nucleus
NAs
Dicer
pre-miR-451
Cytoplasm
RISC
g
cin
Ago1–4
Ago2
N
NA
As
siR
iR
Ago2
Ago2
NA
M
iR
os
m
ta
w
ni
miR-451 RISC
fe
m
a
al
d
m
an
s
Journal of Cell Science
Sli
Translational inhibition
Deadenylation
mRNA cleavage
Ago2
Ago1–4
G
AAAAA
mRNA
G
AAAAA
mRNA
Fig. 1. miRNA biogenesis pathways and regulatory networks. miRNAs begin their journey as long, primary transcripts (pri-miRNAs) produced by either RNA
polymerase II or III (RNA pol II or RNA pol III) in the nucleus. Many highly expressed miRNAs are transcribed by RNA pol II and, as a result, are under diverse
transcriptional regulation and often have cell-specific expression patterns within a lineage. For most mammalian miRNAs, the pri-miRNA folds into a hairpin
structure characteristic of miRNA genes. The hairpin is recognized and, subsequently, cleaved by an RNase III processing complex formed by Drosha (an RNase III
enzyme) and Dgcr8 (a crucial cofactor of Drosha) to give rise to the pre-miRNA hairpin in the nucleus. This molecular event can be regulated by several proteins,
for example, hnRNPA1, p53 and SMAD as well as Lin28, in an miRNA-specific manner. Alternative pathways, for example, direct transcription of hairpin RNAs
and a splicing-mediated hairpin production mechanism, called the mirtron pathway, also exist. These miRNAs are generally expressed at a very low level and their
biological significance is currently not clear. Regardless of its origin, the pre-miRNA is recognized and exported to the cytoplasm by a nuclear RNA-export factor,
Xpo5. In the cytoplasm, the pre-miRNA hairpin is cleaved by a second RNase III enzyme, Dicer, to produce a short double-stranded RNA duplex. In mammals, all
but one miRNA (miR-451) is processed by Dicer. One strand of the duplex is embedded with one of four Argonaute proteins (Ago1–4) to form the RISC. When the
RISC is charged with a specific miRNA, it is guided to mRNA targets harboring miRNA-recognition sequences and, in most cases, it inhibits translation by
deadenylation and direct translational inhibition. However, in several rare cases, when the miRNA is perfectly matched to the target mRNA sequences, it leads to
the cleavage of the mRNA transcripts by the unique ‘slicer’ activity of Ago2.
mediated by RNA polymerase II is under extensive regulation by
transcription factors and diverse epigenetic mechanisms, a complex
network of transcriptional regulation can potentially control miRNA
expression and confer tissue- and cell-specific expression patterns
at the transcriptional level. Indeed, numerous miRNAs are
specifically regulated by transcription factors in stem cell lineages.
In ESCs, key transcriptional regulators such as Oct4, Sox2, Nanog
and Tcf3 specifically occupy the promoters of both active and
silent miRNA genes to activate and repress their expression,
respectively (Marson et al., 2008). In addition, specific histone
marks associated with actively transcribed regions (such as histone
H3 Lys4 trimethylation and histone H3 Lys36 trimethylation
marks), as well as those associated with silenced regions (such as
histone H3 Lys27 trimethylation marks), are also correlated with
active and silent miRNAs, respectively (Marson et al., 2008).
These observations provide strong evidence for an emerging
scheme, in which master transcriptional regulators control a network
of gene expression profiles, including the expression of both
miRNAs and mRNAs. Subsequently, miRNAs modulate the
expression level of their mRNA targets to further shape the output
generated from the transcriptome.
In addition to transcriptional regulation, various posttranscriptional regulatory features have a role in miRNA production.
During the formation of the primary transcript, the flanking
Journal of Cell Science
microRNA in mammalian stem cells
sequences of the miRNA fold into a hairpin structure, called premiRNA, that is characteristic of miRNA-encoding genes. For most
stereotypical miRNAs, the hairpin is sequentially processed, first
by the Drosha–DiGeorge syndrome critical region gene 8 (Dgcr8)
microprocessor complex in the nucleus and then by Dicer in the
cytoplasm. Following Dicer processing, the mature miRNA is
directly embedded with Argonaute proteins to form the RISC
(Fig. 1) (Kim et al., 2009; Krol et al., 2010).
For many miRNAs, a co-transcriptional processing model for
the pre-miRNA hairpin by the Drosha–Dgcr8 complex has been
proposed (Morlando et al., 2008). However, several studies have
shown that this step in the generation of the pre-miRNA can be
tightly regulated by several proteins, as illustrated by the following
examples (see also Fig. 1). Heterogeneous nuclear
ribonucleoprotein A1 (hnRNP A1), for instance, specifically
interacts with the stem-loop of the miR-18a pre-miRNA before
further processing by Drosha and thereby enhances miR-18a
maturation (Guil and Caceres, 2007). Furthermore, p53 and SMAD
proteins enhance Drosha–Dgcr8 processing of pri-miRNA by
interacting with the DEAD-box RNA helicase p68, a component
of the Drosha–Dgcr8 microprocessor complex, and by recruiting
the Drosha–Dgcr8 complex to a specific set of miRNAs (Davis et
al., 2008; Suzuki et al., 2009). By contrast, Lin28 specifically
inhibits the production of mature let-7 miRNA by interfering with
pre-let-7 cropping from the primary transcript in the nucleus and
destabilizing pre-let-7 in the cytoplasm (Heo et al., 2008;
Viswanathan et al., 2008). Notably, the inhibition of let-7 production
by Lin28 has an important role in the maintenance of ESCs (Melton
et al., 2010; Viswanathan et al., 2008). These mechanisms,
therefore, provide additional regulatory mechanisms by which cells
can control miRNA production and function after the transcription
of their primary transcripts.
After the miRNA duplex is released from the pre-miRNA hairpin
by Dicer, one strand of the duplex is selectively incorporated with
an Argonaute protein to form the RISC (Schwarz et al., 2003). In
mammals, there are four Argonaute proteins (Ago1–Ago4, also
known as eIF2c1–eIF2c4) that can associate with miRNAs to form
the RISC. However, it is not completely clear whether all four Ago
proteins associate with similar subset of miRNAs and have similar
functions in the miRNA pathway during development and in stem
cells. When the Ago proteins are knocked out individually in mice,
only the loss of Ago2 results in embryonic lethality, whereas the
loss of Ago1, Ago3 and Ago4 does not lead to discernible
phenotypes during mouse development.
Among the Ago proteins, Ago2 is the only one with so-called
‘slicer’ activity, which is required for the cleavage of target mRNA
by small interfering (si) RNA or perfectly matched miRNA (Liu et
al., 2004). In mammalian cells, however, the requirement for the
slicer activity of Ago2 was not immediately clear because very few
endogenous siRNAs are produced and these are mostly restricted
to the oocytes and ESCs (Tam et al., 2008; Watanabe et al., 2008).
Furthermore, there is only one example of an miRNA that cleaves
a single target, namely the cleavage of mRNA encoding HoxB8 by
miR-196 (Yekta et al., 2004). Apart from this example, there is
little evidence of mammalian miRNA-mediated cleavage of target
mRNAs. However, in a hunt to decipher the slicer-dependent Ago2
function, miR-451, an miRNA highly expressed in terminally
differentiated blood cells, was identified as a unique miRNA whose
biogenesis unusually requires the ‘slicer’ activity of Ago2, but not
the dicing activity of Dicer at the step of pre-miRNA processing
(Fig. 1) (Cheloufi et al., 2010). Slicer-deficient Ago2 mutant mice
1777
are anemic, a phenotype that resembles the defect in erythropoiesis
that was observed in miR-451-null mice (Cheloufi et al., 2010;
Patrick et al., 2010; Rasmussen et al., 2010; Yu et al., 2010).
However, the slicer-deficient Ago2 mutant pups died shortly after
birth, showing a more severe phenotype than miR-451-null animals.
These differences could be a sign that Ago2 is a unique Argonaute
protein that has the ability to cleave RNAs in an miRNA-dependent
or -independent manner (Karginov et al., 2010). Interestingly, a
recent study showed that Ago2 itself is regulated by the mouse
homolog of lin41 (mlin41, which in turn is a target of the let-7
miRNA) in several stem cells. Furthermore, the repression of Ago2
by mlin41 antagonizes the silencing of target mRNAs by let-7 and
miR-124, which are both implicated in stem cell differentiation
(see below) (Rybak et al., 2009). Together, these results suggest
that the association of miRNAs with individual Ago proteins also
has a role in regulating stem cell fate.
Whereas future studies will be needed to fully understand the
extent to which the production of individual miRNAs and their
association with individual Ago proteins are regulated, these results
clearly indicate that miRNA biogenesis is under delicate control at
multiple steps during and after transcription. In turn, although
functional analyses of regulated miRNA biogenesis are still rare,
such regulation could have a substantial impact on miRNA activity
in stem cells and their lineages, as demonstrated by Lin28-mediated
inhibition of let-7 in ESCs.
miRNA function in stem cell differentiation
When stem cells give rise to their progenies during differentiation,
the transition at the cellular level resembles closely the
developmental progression at the organismal level, that is, the
progression from the inner cell mass (which comprises pluripotent
ESCs) to the three germ layers (endoderm, mesoderm and
ectoderm) and eventually to fully functional tissues and organs.
Numerous pathways that have essential roles in stem cells, for
example the Wnt and Notch signaling pathways, were initially
characterized as a result of their roles during animal development.
Similarly, the founding members of the miRNA family, namely
lin-4 and let-7, were originally identified as heterochronic genes
that govern developmental transitions in the nematode
Caenorhabditis elegans (Lee et al., 1993; Pasquinelli et al., 2000;
Reinhart et al., 2000).
Lin-4 and let-7 negatively regulate the expression of master
regulators of differentiation, such as lin-14, lin-28 and lin-41.
These master regulators are essential for the maintenance of an
early developmental lineage, but must also be downregulated as
the animal transitions to a later lineage. For example, lin-4
downregulates lin-14 through the first larval stage (L1) and let-7
downregulates lin-28 and lin-41 when the animal develops from
the fourth larval stage (L4) to the adult stage (Lee et al., 1993;
Reinhart et al., 2000).
Studies on lin-4 and let-7 miRNAs provided the first evidence
that miRNAs have essential roles in development. The negative
feedback regulatory network between lin-4 and let-7 and their
mRNA targets provided the basis for a model in which miRNAs
help to deplete protein production from mRNAs inherited from an
earlier developmental stage. In doing so, miRNAs facilitate a
precise and robust transition through the developmental program.
Following this paradigm, the roles for miRNAs in stem cell
differentiation have been extensively documented in recent years.
Fueled by a rapid development in miRNA profiling techniques, it
quickly became evident that numerous miRNAs are differentially
1778
Journal of Cell Science 124 (11)
Journal of Cell Science
expressed as stem cells embark on differentiation (Houbaviy et al.,
2003; Landgraf et al., 2007; Sempere et al., 2004; Suh et al., 2004).
These observations provided the first clue and a molecular basis to
explore the crucial roles that miRNAs have in stem cell
differentiation. In the following sections, we will discuss current
evidence for the important roles of miRNAs in embryonic and
somatic stem cell differentiation.
miRNA in embryonic stem cell differentiation
Because of their ease to be cultivated as pluripotent stem cells and
the ability to differentiate them along different lineages in vitro,
ESCs have been extensively studied, yielding comprehensive
knowledge on their underlying gene regulatory network. Therefore,
it comes as no surprise that mouse ESCs have served as a fertile
ground for the first observations of the functional significance of
miRNAs in mammalian stem cells. The importance of miRNAs
was highlighted when, following knockout of Dicer and subsequent
loss of all miRNAs, ESCs failed to silence their self-renewal
program and displayed severe defects in their ability to differentiate
(Kanellopoulou et al., 2005; Murchison et al., 2005).
Because of the potential roles of Dicer in the biogenesis and
function of other small RNA species, it was not clear at the time
whether the loss of miRNAs in the Dicer-null ESCs was solely
responsible for the phenotype observed. However, when the second
major miRNA-processing gene, Dgcr8, was ablated in ESCs,
similar defects in differentiation were observed (Wang et al., 2007).
Together, these results unequivocally establish essential roles for
miRNAs in controlling the balance between ‘stemness’ and
differentiation in cultured ESCs.
A
Sox2, Oct4, KLF4
miR145
Lin28, Myc
let-7
To ensure robust differentiation of ESCs towards a specific cell
lineage, at least two distinct processes must be accurately controlled.
First, the self-renewal program has to be properly silenced. Second,
a robust program dictating the specific cell fate has to be
established. Recent studies have begun to reveal roles for individual
miRNAs in both events. miR-145 is expressed at low levels in selfrenewing human ESCs, but it is substantially upregulated during
differentiation (Xu et al., 2009). Acting as a key antagonist of ESC
maintenance, miR-145 directly targets and suppresses the mRNAs
encoding the transcription factors Oct4, Sox2 and Klf4, which are
required to maintain pluripotency (Fig. 2A) (Xu et al., 2009).
Several other miRNAs follow a similar pattern during ESC
differentiation. Among the most fascinating of these is the let-7
family, which is highly conserved from C. elegans to humans. In
mice and humans, the let-7 family comprises eleven members
dispersed over eight genomic loci. Let-7 antagonizes Myc and
Lin28 protein production and, as a result, destabilizes the selfrenewing capacity and promotes the differentiation of ESCs (Fig.
2A) (Melton et al., 2010). The potency of let-7 in promoting
differentiation is further demonstrated by the observation that the
inhibition of this miRNA family substantially enhances the
reprogramming of somatic cells into induced pluripotent stem cells
(Melton et al., 2010).
The ability to adjust culture conditions to coax ESCs along
particular lineages has also provided new insights into how
miRNAs function during the process of specifying lineage. Both
miR-1 and miR-133 are muscle-specific miRNAs that are induced
by serum response factor (SRF), a transcription factor that is
activated when ESCs differentiate to form cardiomyocytes (Ivey
D
Silencing the self-renewal program
p63
miR203
Exit of cell cycle
Inhibition of self-renewal
Epidermal stem cells
ESCs
Self-renewal
Self-renewal
B
Dll-1 (non-muscle fate)
E
Sox9
miR1
miR133
ESCs
Differentiation
miR9
STMN1(control proliferation and migration)
Self-renewal
ESCs
Self-renewal
hNPCs
Differentiation
Exit of cell cycle
Inhibition of self-renewal
Differentiation
F
Pax7
Maintenance of proliferation
Suppression of migration
miR124
Neuronal stem cells
Cardiomyocytes
Self-renewal
C
Differentiation
Differentiation
miR1
miR206
Exit of quiescence
Induction of differentiation
Muscle stem cells
(satellite cells)
Quiescence
Proliferation
Differentiation
Fig. 2. Roles of miRNAs in the differentiation of embryonic and somatic stem cells. The roles of miRNAs in the differentiation of ESCs can be summarized
into three distinct mechanisms: (A) promoting the silencing of the self-renewal program; (B) suppressing the gene expression of other differentiated lineages; and
(C) maintaining the state of the specific lineage. The roles of miRNAs in the differentiation of somatic stem cells are shown in (D–F). In skin, brain and muscle,
miR-203, miR-124, and miR-1 and miR-206, respectively, promote differentiation by inhibiting key transcriptional factors that are crucial for the maintenance of
each stem cell population.
Journal of Cell Science
microRNA in mammalian stem cells
et al., 2008; Zhao et al., 2005). When miR-1 and miR-133 are
ectopically activated in murine ESCs, they promote mesoderm
differentiation by repressing non-muscle gene expression through
direct downregulation of Dll-1, a key ligand in the Notch signaling
pathway (Fig. 2B) (Ivey et al., 2008). Interestingly, miR-1 and, to
a lesser extent, miR-133 can rescue the abnormal differentiation
phenotype observed in SRF-null embryoid body cells, arguing for
a major role for miR-1 and miR-133 in the SRF-mediated
differentiation program (Ivey et al., 2008). When taken together,
these observations support the hypothesis that tissue-specific
miRNAs preferentially target mRNAs that are expressed in other
lineages, narrowing the gene expression profile towards the defined
cell lineage and thereby maintaining tissue identity (Fig. 2B) (Farh
et al., 2005; Stark et al., 2005).
Whereas restricting other lineages is one method by which
miRNAs can ensure tissue selectivity of differentiating somatic
stem cells, miRNAs can also achieve this by regulating the
maintenance of somatic cells upon ESC differentiation. In this
regard, the recent findings of Delaloy et al. offer new insights
(Delaloy et al., 2010). This group showed that, when human ESCs
are induced to form proliferative aggregates (neurospheres) of
multipotent neuronal progenitor cells (hNPCs), they switch on the
expression of a brain-specific miRNA, miR-9. Moreover, when
antisense oligonucleotides are used to downregulate miR-9 activity
in hNPCs, their proliferation is restricted and their migration is
enhanced. These findings underline roles for miR-9 in specifically
promoting the differentiation of ESCs into cells with a neural fate
(Fig. 2C) (Delaloy et al., 2010).
Taken together, these observations show that miRNAs function
in three distinct ways to promote ESC differentiation: (1) silencing
the self-renewal program by targeting core pluripotency factors;
(2) restricting the expression of genes in other lineages and, hence,
dictating the differentiating ESCs to a defined lineage; (3)
maintaining the state of the specified lineage (Fig. 2A–C).
miRNA in somatic stem cell differentiation
In parallel to work carried out in ESCs, numerous studies focusing
on the differentiation of somatic stem cells have yielded
considerable insights into the requirement for miRNAs in regulating
homeostasis of somatic tissues (Fig. 2D–F).
In mammalian skin, miR-203 is the most abundantly expressed
miRNA (Yi et al., 2008). When the spatiotemporal expression of
miR-203 was visualized by in situ hybridization, it was shown that
this miRNA is rapidly switched on when epidermal stem cells
begin to differentiate (Yi et al., 2008). Illustrating the functional
significance of this expression pattern, premature activation of
miR-203 in epidermal stem cells depletes the stem cell pool by
restricting their proliferative potential and promoting their exit
from the cell cycle. Conversely, transient inhibition of endogenous
miR-203 by a chemically modified antisense oligonucleotide (an
antagomir) resulted in normally differentiating suprabasal cells
that proliferated and showed upregulation of the key miR-203
target Np63a (Fig. 2D) (Yi et al., 2008). Np63a is a transcription
factor essential for the maintenance of epidermal stem cells (Mills
et al., 1999; Senoo et al., 2007; Yang et al., 1999). Although it is
not yet clear whether Np63a is the only target that mediates the
function of miR-203, this study provides an important example of
miRNAs regulating the developmental transition by targeting a
key stem cell regulator at the point at which differentiation is
induced (Yi et al., 2008). Additionally, it is intriguing that, whereas
miR-203 potently represses epidermal stem cell proliferation,
1779
terminal differentiation markers such as Krt1, Krt10, loricrin and
involucrin were not activated when miR-203 was precociously
induced in the stem cells (Yi et al., 2008). This latter finding
suggests that, at least for the epidermis, the exit from the cell cycle
and the induction of terminal differentiation in somatic stem cells
can be regulated by distinct mechanisms.
In the adult brain, miR-9 has effects on neuronal stem cells that
differ from those that miR-203 has on epidermal stem cells. In the
brain, these neural stem cells reside at the subventricular zone
(SVZ). miR-9 functions in neural progenitor maintenance, in part
by directly targeting the nuclear receptor NR2E1 (nuclear receptor
subfamily 2, group E, member 1, previously known as TLX),
which is an essential governor of self-renewal of neural stem cells
(Zhao et al., 2009). Interestingly, NR2E1 also antagonizes the
expression of miR-9 by directly reducing the transcription of
pri-miR-9 (Zhao et al., 2009). Thus, NR2E1 and miR-9 form a
negative feedback regulatory network to balance both proliferation
and differentiation of neural stem cells.
By contrast, miR-124, which is one of the most specific and
abundant miRNAs in the brain, more closely mirrors the effects
shown by miR-203. Like miR-203, miR-124 is expressed at low
levels in the stem cell compartment in the SVZ, but is sharply
upregulated in mature granule and periglomerular neurons (Cheng
et al., 2009). Similarly, gain of function of miR-124 induces cell
cycle exit, whereas inhibition of miR-124 by antagomir in vivo
results in an increase in the population of precursor cells in the
SVZ. Moreover, Sox9, a key transcription factor whose
downregulation is required for neural differentiation, has been
identified as a direct target of miR-124 (Fig. 2E) (Cheng et al.,
2009).
A recent study of skeletal muscle satellite cells provided another
interesting parallel to the skin stem cell lineage. Analogous to
Np63a in epidermal stem cells, Pax7 is a crucial transcription
factor that is highly expressed in quiescent muscle stem cells
(satellite cells) and is required to maintain the stem cell population.
Upon injury, satellite stem cells are activated to repair the wound.
In such an event, Pax7 is rapidly downregulated to allow these
cells to enter differentiation and contribute to muscle regeneration.
Both miR-1 and miR-206 are markedly upregulated concomitant
with downregulation of Pax7 and satellite cell differentiation; both
of these miRNAs target mRNA encoding Pax7 (Fig. 2F) (Chen et
al., 2010). Conversely, specific inhibition of miR-1 and miR-206
by antagomirs delays Pax7 downregulation and interferes with the
differentiation program (Chen et al., 2010). Together, these findings
suggest that some somatic miRNAs are primarily required for
transitioning stem cells along a differentiation lineage.
The hematopoietic system, with its well-defined cell lineages,
offers an ideal system to map the lineage-specific expression
patterns of miRNAs and to decipher their roles in differentiation.
In one of the earliest studies of mammalian miRNAs, miR-181,
miR-223 and miR-142 were all found to be upregulated when bone
marrow progenitor cells differentiate towards B lymphocytes (Chen
et al., 2004). Moreover, forced expression of miR-181 in those
progenitors enhanced B-lymphocyte development at the expense
of T-lymphocyte differentiation. In another important study, it was
discovered that miR-155 is expressed in mature B and T
lymphocytes, and that it is also substantially upregulated in a
variety of lymphomas (Eis et al., 2005). Loss-of-function studies
in mice revealed that miR-155 is a crucial regulator of specific
differentiation processes in the immune response in vivo (Rodriguez
et al., 2007; Thai et al., 2007). In an effort to identify the targets
Journal of Cell Science
1780
Journal of Cell Science 124 (11)
of miR-155, the mRNAs upregulated in miR-155-null
T lymphocytes were analyzed for the presence of seed matches to
miR-155 (Rodriguez et al., 2007). Even though ~65% of the
upregulated genes contained miR-155 seed matches in their 3⬘UTRs, the key miR-155 targets seemed to be mRNAs encoding
cytokines, providing an explanation for the remarkable impact and
specificity of this miRNA on immune cell lineages.
In another elegant study of hematopoietic-specific miRNAs,
Xiao et al. focused on miR-150 and provided an example of a
single mRNA–miRNA regulatory pair that has crucial functions in
lymphocyte development (Xiao et al., 2007). Like miR-155, miR150 is primarily expressed in mature lymphocytes rather than in
the progenitor cells. In loss- and gain-of-function studies, miR-150
was shown to regulate lymphocyte terminal differentiation (Xiao
et al., 2007). Importantly, the expression of c-Myb, a key
transcription factor and the top predicted target of miR-150, was
inversely correlated with that of miR-150, suggesting that miR150 might be both necessary and sufficient to control c-Myb
expression. Interestingly, the c-Myb heterozygous knockout mice
showed phenotypes similar to those observed in miR-150 transgenic
mice, thereby lending support to the argument that miR-150 carries
out its function through the accurate control of c-Myb expression
(Xiao et al., 2007).
Studies of the erythroid lineage have also revealed an inverse
relationship between miRNA expression and stem cell progenitor
maintenance. When erythroid progenitors begin to differentiate
into red blood cells, the miR-144–miR-451 locus is directly
activated by a key erythroid transcription factor, Gata-1 (Dore et
al., 2008). Loss-of-function studies in both zebrafish and mouse
revealed that this conserved miRNA locus is required for
erythroblast maturation (Dore et al., 2008; Rasmussen et al., 2010).
Interestingly, the mRNA of 14-3-3, a key regulator of cytokine
signaling, was identified as a direct target of miR-451. More
importantly, knocking down 14-3-3 in miR-451-null hematopoietic
stem cells rescued these defects during erythroid differentiation in
vitro, providing another example whereby a single target might be
primarily responsible for mediating the effects of an miRNA in a
defined cellular context (Patrick et al., 2010; Yu et al., 2010).
Overall, the emerging literature on miRNAs in somatic tissues
provides fertile ground for additional studies into the mechanisms
by which miRNAs control the balance between somatic stem cells
and their differentiating lineages (Fig. 2D–F).
A ESCs
Rbl2
Role of miRNAs in stem cell self-renewal
Compared with their function in differentiation, the roles of
miRNAs in stem cell maintenance are poorly understood.
Nonetheless, studies on ESCs have shown that loss of either Dgcr8
or Dicer results in proliferation defects. Because Dgcr8 acts as a
cofactor for Drosha in the nucleus during miRNA biogenesis,
direct transfection of an miRNA duplex into the cell can bypass the
requirement for Drosha processing and thereby provide a means to
examine the role of an individual miRNA or an miRNA family in
the Dgcr8-null background. With this approach, it was shown that
members of the miR-290 family, an ESC-specific miRNA family,
are able to rescue the proliferation defects caused by loss of Dgcr8
function by directly controlling the expression of key regulators of
the cell cycle pathway, including Cdkn1a, Rbl2 and Lats2, which
are negative regulators of the cell cycle in ESCs (Wang et al.,
2008) (Fig. 3A).
Interestingly, the regulation of Rbl2 by the miR-290 family is
also a key link to the DNA methylation defects observed in Dicernull ESCs (Fig. 3A) (Benetti et al., 2008; Sinkkonen et al., 2008).
Derepression of Rbl2 in Dicer-null ESCs results in transcriptional
repression of DNA methyltransferases and, in turn, leads to a
decrease in de novo DNA methylation (Benetti et al., 2008;
Sinkkonen et al., 2008).
The miR-290 family also has an essential role in antagonizing
the effects of differentiation-related miRNAs, such as the let-7
family (Melton et al., 2010). The ectopic expression of let-7 family
members in Dgcr8- null but not in wild-type ESCs was able to
silence the self-renewal program. This observation suggests that
other miRNAs that are highly expressed in ESCs are capable of
blocking the effect of let-7 on self-renewal. Indeed, co-expression
of the miR-290 and let-7 family members largely abolished the
inhibition of let-7 miRNAs on self-renewal (Melton et al., 2010).
Although the direct targets of the miR-290 family that mediate the
antagonizing effects on the let-7 miRNAs remain elusive, it has
become increasingly clear that an underlying network centered on
the miR-290 family has a pivotal role in governing self-renewal
and cell cycle progression in ESCs (Fig. 3A). The systematic
identification of the targets of this family and functional dissection
of their contribution should provide substantial new insights into
our understanding of the self-renewal program.
In somatic stem cells, evidence for the involvement of miRNAs
in the maintenance of self-renewal has just begun to surface.
Maintain de novo DNA methylation
miR290~295
Self-renewal
?
let-7 miRNA
B Mammary gland stem cells
miR205
PTEN
Self-renewal
C Hair follicle stem cells
Blimp1
miR125b
VDR
?
Balance stem cell input to the sebaceous gland lineage
Balance stem cell input to the hair follicle lineage
Balance self-renewal and lineage commitment of skin stem cells
Fig. 3. Roles of miRNAs in the maintenance of stem cells.
(A)In ESCs, the miR-290~295 cluster miRNAs help to
maintain ‘stemness’ by two distinct mechanisms. The
miRNA cluster inhibits Rbl2 to facilitate cell cycle
progression and maintain de novo DNA methylation. It also
inhibits currently unknown targets to antagonize the
inhibitory effect of let-7 miRNA on the self-renewal
program. (B)In mammary gland stem cells, miR-205 targets
an important tumor suppressor, PTEN, to indirectly promote
self-renewal. (C)In hair follicle stem cells, miR-125b
directly targets Blimp1, a key transcription factor of the
sebaceous gland lineage, and VDR (vitamin D receptor), a
key regulator required for hair follicle differentiation, and
thereby provides a rheostat mechanism to fine-tune the input
of stem cells to the differentiated lineages. Unknown proteins
or factors are indicated by a question mark (?).
Journal of Cell Science
microRNA in mammalian stem cells
Notably, miR-205 was identified as the most abundantly expressed
miRNA in mammary gland progenitor cells (Ibarra et al., 2007).
When miR-205 was overexpressed in a mammary epithelial cell
line with a heterogeneous progenitor population, it expanded the
progenitor population, enhancing proliferation and leading to
increased colony formation (Greene et al., 2010). Intriguingly,
PTEN, a well-characterized tumor suppressor gene, was identified
as an miR-205 target (Greene et al., 2010). Because the function
of PTEN has an unusually intimate correlation with its expression
level (Alimonti et al., 2010), it is of particular interest to speculate
that the modulation of PTEN expression by miR-205 might have
a crucial role in balancing self-renewal of the stem cells of the
mammary gland and other tissues in which this miRNA is expressed
(Fig. 3B).
Most recently, miR-125b was identified as an miRNA
specifically expressed in skin stem cells (Zhang et al., 2011). When
the stem cells differentiate into outer root sheath (ORS) progenitor
cells, miR-125b is rapidly downregulated. When miR-125b is
elevated in both stem cells and ORS progenitor cells through an
inducible, transgenic strategy in mice, the balance between
maintenance of stem cells and differentiation into hair follicle
lineages is shifted towards the stem cell fate. More stem-cell-like
cells are generated and their differentiation is impaired. Intriguingly,
the maintenance of stem cells was intact and, upon withdrawal of
doxycycline to decrease the levels of miR-125b, the differentiation
block was lifted even after up to 4 months of continuous miR-125b
induction. These observations suggest an interesting new model
for the roles of miRNA in stem cells as a rheostat mechanism
precisely governing the input of stem cells to the differentiation
program (Fig. 3C).
New roles for miRNA regulation in injury and
aging
In addition to the roles of miRNAs in controlling stem cell
differentiation, which have become increasingly well established,
several new twists have emerged in the miRNA world. Among
them is the notion that miRNAs function in injury or stress
situations, as alluded to earlier when discussing the roles of Pax7.
Furthermore, knockout mouse models have yielded some intriguing
surprises. In a remarkable series of studies on miRNAs in cardiac
development, individual ablation of the genes encoding miR-133,
miR-143, miR-145, miR-206, miR-208 and miR-499 did not
manifest itself in discernible phenotypes during normal
development and tissue homeostasis (reviewed by Liu and Olson,
2010). However, in response to various stress conditions, all of
these mutant mice displayed strong phenotypes (Liu and Olson,
2010). Such observations reveal that, even though compensatory
networks might be able to partially rescue the loss of individual
miRNAs in a well-controlled environment in vivo, when exposed
to more stringent conditions, these backup circuits no longer suffice
to maintain normal tissue function. They also raise the interesting
possibility that the miRNA pathway is particularly important for
the proper maintenance and differentiation of stem cells when they
are under stress.
Culture conditions can present both a stressful situation to cells
and a platform for manipulating the microenvironment. As such,
systems making use of cultured stem cells often generate new
insights into miRNA functions. Examples of such a case are the
bi-cistronic miR-143 and miR-145 loci, which are substantially
upregulated in multipotent cardiac progenitors in vivo (Cordes et
al., 2009). When specifically examined during the reprogramming
1781
of fibroblasts into vascular smooth muscle cells (VSMCs) in vitro,
miR-145 but not the clustered miR-143, which shares no sequence
similarity with miR-145, was able to potentiate the reprogramming
effects of myocardin (Myocd), an essential component of the
molecular switch that activates genes encoding contractile muscle
proteins (Cordes et al., 2009). Furthermore, miR-145 was sufficient
to guide ~75% cells into VSMCs, when the reprogramming was
performed with neural crest stem cells (Cordes et al., 2009).
Whereas both assays demonstrated an essential role for miR-145
in controlling VSMC fate, the most remarkable finding was that,
instead of inhibiting Myocd, whose mRNA contains recognition
sites for miR-145, miR-145 activated the expression of a luciferase
reporter containing the 3⬘-UTR of Myocd by more than 100-fold
(Cordes et al., 2009). miRNAs have been shown previously to be
capable of promoting protein production of their target mRNA in
cultured cells when these are under cell cycle arrest (Vasudevan et
al., 2007). However, a second luciferase reporter containing the 3⬘UTR of another miR-145 target, Klf4, was specifically repressed
by miR-145 under the same experimental conditions. These findings
indicate that miR-145 can either activate or repress its targets, and
that the effect is probably determined by specific interactions
between miR-145 and individual mRNA targets (Cordes et al.,
2009).
Exactly how miR-145 exerts its effects at the mechanistic level
is not yet clear. One possibility is that miR-145 could prevent the
binding of a context-dependent repressive RNA-binding protein
(Cordes et al., 2009). Irrespective of the underlying mechanism,
another question to be addressed in the future is the extent to which
this presently unconventional regulatory mechanism emerges to
take a place in the multifaceted toolbox of miRNAs.
Finally, it is worth mentioning that miRNAs have also been
implicated in the regulation of stem cell aging. In this regard,
Hmga2, a key transcription factor for the self-renewal of neural
stem cells, is highly expressed in fetal neural stem cells in mice,
but its levels decline by more than 99% during their lifetime
(Nishino et al., 2008). The decreased level of Hmga2 reduces the
self-renewal capacity of neural stem cells and seems to be, in part,
caused by an approximately 30-fold age-induced increase in the
expression of let-7b, which is known to target and inhibit Hmga2
(Mayr et al., 2007; Nishino et al., 2008). The specific disruption of
Hmga2 regulation using let-7b with a truncated 3⬘-UTR showed
substantial rescue of the self-renewal capacity in an in vitro culture
assay, supporting the causative role of let-7b in the downregulation
of Hmga2 (Nishino et al., 2008).
Closing remarks and future challenges
Functional characterization of miRNAs in mammalian stem cells
is still in its infancy. So far, many studies used either in vitro cell
culture models or transgenic mouse models, as well as in vivo
delivery of antagomirs, to address the roles of miRNAs in stem
cells. To rigorously investigate the roles that miRNAs have in
governing stem cell fate in vivo and to examine their long-term
functions, knockout mouse models will be indispensable for future
studies. The development of accurate stem cell identification and
isolation, highly sensitive miRNA profiling techniques at a singlecell level, and rapidly generated conditional knockout mouse
models for individual miRNAs will hopefully allow us to address
several fundamental questions concerning miRNAs and stem cells
in the near future.
Among the myriad key issues that remain unresolved is the
question of how a single miRNA or miRNA family regulates stem
Journal of Cell Science
1782
Journal of Cell Science 124 (11)
cells in response to various endogenous and exogenous stimuli.
When C. elegans miRNAs are knocked out individually or in
combination as a whole family that shares the same seed sequence,
only a few exhibit developmental defects (Alvarez-Saavedra and
Horvitz, 2010; Miska et al., 2007). Similarly, with more than 30
miRNAs having been knocked out individually without effect in
mice, it is evident that the loss of a single miRNA or even a whole
family might not cause the severe developmental defects in cell
fate specification that would be expected from ablation of a gene
encoding a master regulator. However, when these mutant mice are
subject to physiological stresses such as injury or DNA damage,
substantially disrupted phenotypes appear (reviewed by Leung and
Sharp, 2010; Liu and Olson, 2010). These observations also suggest
that miRNA-mediated regulation has an important role in managing
the robustness of the biological system (reviewed by Herranz and
Cohen, 2010).
These observations begin to link the miRNA pathway, one of
the most ancient pathways (Christodoulou et al., 2010), to stress
responses (reviewed by Leung and Sharp, 2010). It is worth noting
that, because of the central role of stem cells during the development
and homeostasis of adult tissues, a defective stress response in
stem cells could be magnified and manifested as defects in their
differentiated daughters. This illustrates the crucial need to
investigate how stress signals regulate miRNA expression and how
miRNA-mediated regulation in turn balances the output of gene
expression and protects stem cells from the various and diverse
stresses that they encounter throughout their life.
A second key issue to address will be how a single miRNA can
execute its physiological function by regulating its targets. Directly
related to this is the question whether a single miRNA can perform
different functions when placed in distinct cellular contexts. As
miRNA functions in mammalian stem cells begin to unfold, some
of the most challenging questions to be answered are which mRNAs
are targeted by a specific miRNA and which of its many targets are
the most important for a specific miRNA in a given cellular context.
To answer these questions, the complexities of miRNA-mediated
regulation must be considered. First, a single miRNA can target
more than 100 genes in a defined cellular context. Second, a single
gene can be targeted by multiple, potentially unrelated miRNAs.
Third, the effect of an miRNA on its targets is somewhat subtle,
for example, <2-fold downregulation, as demonstrated by recent
studies using genome-wide approaches to quantify the impact of
miRNA on protein output (Baek et al., 2008; Selbach et al., 2008).
However, in both studies, the number of proteins analyzed was
limited to ~3000 of the most abundantly expressed proteins. It is
possible that miRNA targets, which are expressed at a relatively
low level, can be more potently regulated, as demonstrated by
numerous individual studies. Recently, experimental identification
approaches have been applied to identify RISC-associated mRNA
species as miRNA target candidates (Chi et al., 2009; Hafner et al.,
2010). With future development in these promising techniques,
especially given the reduced requirement for input materials, these
approaches should provide exciting insights into the spectrum of
miRNA targets and the extent to which they are regulated by
miRNAs in mammalian stem cells.
A third key area to be addressed in the future is the question of
how miRNA-mediated regulation integrates with other regulatory
mechanisms, for example, transcriptional regulation, to modulate
stem cell fate. Although biologists now appreciate the importance
of the regulatory layer provided by miRNAs, the interactions
between the miRNA pathway and other regulatory mechanisms
must be elucidated to fully understand how miRNAs work.
Furthermore, it will be important to unveil the characteristics of
the miRNA pathway that are distinct from other mechanisms such
as cellular-context-dependent function. The answers to these
fascinating questions are certainly going to not only provide
substantial new insights into the functions of miRNAs, but also
point to new directions in how to use and target miRNAs in the
manipulation of stem cells for regenerative medicine.
This publication was made possible by grants R00AR054704 and
R01AR059697 (R.Y.), and R01AR031737 (E.F.) from the NIH. E.F. is
a Howard Hughes Medical Institute Investigator. Deposited in PMC
for release after 12 months.
References
Alimonti, A., Carracedo, A., Clohessy, J. G., Trotman, L. C., Nardella, C., Egia, A.,
Salmena, L., Sampieri, K., Haveman, W. J., Brogi, E. et al. (2010). Subtle variations
in Pten dose determine cancer susceptibility. Nat. Genet. 42, 454-458.
Alvarez-Saavedra, E. and Horvitz, H. R. (2010). Many families of C. elegans microRNAs
are not essential for development or viability. Curr. Biol. 20, 367-373.
Ambros, V. (2004). The functions of animal microRNAs. Nature 431, 350-355.
Baek, D., Villen, J., Shin, C., Camargo, F. D., Gygi, S. P. and Bartel, D. P. (2008). The
impact of microRNAs on protein output. Nature 455, 64-71.
Bartel, D. P. (2009). MicroRNAs: target recognition and regulatory functions. Cell 136,
215-233.
Benetti, R., Gonzalo, S., Jaco, I., Munoz, P., Gonzalez, S., Schoeftner, S., Murchison,
E., Andl, T., Chen, T., Klatt, P. et al. (2008). A mammalian microRNA cluster controls
DNA methylation and telomere recombination via Rbl2-dependent regulation of DNA
methyltransferases. Nat. Struct. Mol. Biol. 15, 268-279.
Borchert, G. M., Lanier, W. and Davidson, B. L. (2006). RNA polymerase III transcribes
human microRNAs. Nat. Struct. Mol. Biol. 13, 1097-1101.
Cai, X., Hagedorn, C. H. and Cullen, B. R. (2004). Human microRNAs are processed
from capped, polyadenylated transcripts that can also function as mRNAs. RNA 10,
1957-1966.
Cheloufi, S., Dos Santos, C. O., Chong, M. M. and Hannon, G. J. (2010). A dicerindependent miRNA biogenesis pathway that requires Ago catalysis. Nature 465, 584589.
Chen, C. Z., Li, L., Lodish, H. F. and Bartel, D. P. (2004). MicroRNAs modulate
hematopoietic lineage differentiation. Science 303, 83-86.
Chen, J. F., Tao, Y., Li, J., Deng, Z., Yan, Z., Xiao, X. and Wang, D. Z. (2010).
microRNA-1 and microRNA-206 regulate skeletal muscle satellite cell proliferation
and differentiation by repressing Pax7. J. Cell Biol. 190, 867-879.
Cheng, L. C., Pastrana, E., Tavazoie, M. and Doetsch, F. (2009). miR-124 regulates
adult neurogenesis in the subventricular zone stem cell niche. Nat. Neurosci. 12, 399408.
Chi, S. W., Zang, J. B., Mele, A. and Darnell, R. B. (2009). Argonaute HITS-CLIP
decodes microRNA-mRNA interaction maps. Nature 460, 479-486.
Christodoulou, F., Raible, F., Tomer, R., Simakov, O., Trachana, K., Klaus, S.,
Snyman, H., Hannon, G. J., Bork, P. and Arendt, D. (2010). Ancient animal
microRNAs and the evolution of tissue identity. Nature 463, 1084-1088.
Cordes, K. R., Sheehy, N. T., White, M. P., Berry, E. C., Morton, S. U., Muth, A. N.,
Lee, T. H., Miano, J. M., Ivey, K. N. and Srivastava, D. (2009). miR-145 and miR143 regulate smooth muscle cell fate and plasticity. Nature 460, 705-710.
Davis, B. N., Hilyard, A. C., Lagna, G. and Hata, A. (2008). SMAD proteins control
DROSHA-mediated microRNA maturation. Nature 454, 56-61.
Delaloy, C., Liu, L., Lee, J. A., Su, H., Shen, F., Yang, G. Y., Young, W. L., Ivey, K. N.
and Gao, F. B. (2010). MicroRNA-9 coordinates proliferation and migration of human
embryonic stem cell-derived neural progenitors. Cell Stem Cell 6, 323-335.
Dore, L. C., Amigo, J. D., Dos Santos, C. O., Zhang, Z., Gai, X., Tobias, J. W., Yu, D.,
Klein, A. M., Dorman, C., Wu, W. et al. (2008). A GATA-1-regulated microRNA
locus essential for erythropoiesis. Proc. Natl. Acad. Sci. USA 105, 3333-3338.
Eis, P. S., Tam, W., Sun, L., Chadburn, A., Li, Z., Gomez, M. F., Lund, E. and
Dahlberg, J. E. (2005). Accumulation of miR-155 and BIC RNA in human B cell
lymphomas. Proc. Natl. Acad. Sci. USA 102, 3627-3632.
Farh, K. K., Grimson, A., Jan, C., Lewis, B. P., Johnston, W. K., Lim, L. P., Burge,
C. B. and Bartel, D. P. (2005). The widespread impact of mammalian MicroRNAs on
mRNA repression and evolution. Science 310, 1817-1821.
Gangaraju, V. K. and Lin, H. (2009). MicroRNAs: key regulators of stem cells. Nat. Rev.
Mol. Cell Biol. 10, 116-125.
Greene, S. B., Gunaratne, P. H., Hammond, S. M. and Rosen, J. M. (2010). A putative
role for microRNA-205 in mammary epithelial cell progenitors. J. Cell Sci. 123, 606618.
Guil, S. and Caceres, J. F. (2007). The multifunctional RNA-binding protein hnRNP A1
is required for processing of miR-18a. Nat. Struct. Mol. Biol. 14, 591-596.
Hafner, M., Landthaler, M., Burger, L., Khorshid, M., Hausser, J., Berninger, P.,
Rothballer, A., Ascano, M., Jr, Jungkamp, A. C., Munschauer, M. et al. (2010).
Transcriptome-wide identification of RNA-binding protein and microRNA target sites
by PAR-CLIP. Cell 141, 129-141.
Journal of Cell Science
microRNA in mammalian stem cells
Heo, I., Joo, C., Cho, J., Ha, M., Han, J. and Kim, V. N. (2008). Lin28 mediates the
terminal uridylation of let-7 precursor MicroRNA. Mol. Cell 32, 276-284.
Herranz, H. and Cohen, S. M. (2010). MicroRNAs and gene regulatory networks:
managing the impact of noise in biological systems. Genes Dev. 24, 1339-1344.
Houbaviy, H. B., Murray, M. F. and Sharp, P. A. (2003). Embryonic stem cell-specific
microRNAs. Dev. Cell 5, 351-358.
Ibarra, I., Erlich, Y., Muthuswamy, S. K., Sachidanandam, R. and Hannon, G. J.
(2007). A role for microRNAs in maintenance of mouse mammary epithelial progenitor
cells. Genes Dev. 21, 3238-3243.
Ivey, K. N. and Srivastava, D. (2010). MicroRNAs as regulators of differentiation and
cell fate decisions. Cell Stem Cell 7, 36-41.
Ivey, K. N., Muth, A., Arnold, J., King, F. W., Yeh, R. F., Fish, J. E., Hsiao, E. C.,
Schwartz, R. J., Conklin, B. R., Bernstein, H. S. et al. (2008). MicroRNA regulation
of cell lineages in mouse and human embryonic stem cells. Cell Stem Cell 2, 219-229.
Kanellopoulou, C., Muljo, S. A., Kung, A. L., Ganesan, S., Drapkin, R., Jenuwein, T.,
Livingston, D. M. and Rajewsky, K. (2005). Dicer-deficient mouse embryonic stem
cells are defective in differentiation and centromeric silencing. Genes Dev. 19, 489-501.
Karginov, F. V., Cheloufi, S., Chong, M. M., Stark, A., Smith, A. D. and Hannon, G.
J. (2010). Diverse endonucleolytic cleavage sites in the mammalian transcriptome
depend upon microRNAs, Drosha, and additional nucleases. Mol. Cell 38, 781-788.
Kim, V. N., Han, J. and Siomi, M. C. (2009). Biogenesis of small RNAs in animals. Nat.
Rev. Mol. Cell Biol. 10, 126-139.
Krol, J., Loedige, I. and Filipowicz, W. (2010). The widespread regulation of microRNA
biogenesis, function and decay. Nat. Rev. Genet. 11, 597-610.
Landgraf, P., Rusu, M., Sheridan, R., Sewer, A., Iovino, N., Aravin, A., Pfeffer, S.,
Rice, A., Kamphorst, A. O., Landthaler, M. et al. (2007). A mammalian microRNA
expression atlas based on small RNA library sequencing. Cell 129, 1401-1414.
Lee, R. C., Feinbaum, R. L. and Ambros, V. (1993). The C. elegans heterochronic gene
lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75, 843-854.
Leung, A. K. and Sharp, P. A. (2010). MicroRNA functions in stress responses. Mol. Cell
40, 205-215.
Liu, J., Carmell, M. A., Rivas, F. V., Marsden, C. G., Thomson, J. M., Song, J. J.,
Hammond, S. M., Joshua-Tor, L. and Hannon, G. J. (2004). Argonaute2 is the
catalytic engine of mammalian RNAi. Science 305, 1437-1441.
Liu, N. and Olson, E. N. (2010). MicroRNA regulatory networks in cardiovascular
development. Dev. Cell 18, 510-525.
Marson, A., Levine, S. S., Cole, M. F., Frampton, G. M., Brambrink, T., Johnstone,
S., Guenther, M. G., Johnston, W. K., Wernig, M., Newman, J. et al. (2008).
Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic
stem cells. Cell 134, 521-533.
Martinez, N. J. and Gregory, R. I. (2010). MicroRNA gene regulatory pathways in the
establishment and maintenance of ESC identity. Cell Stem Cell 7, 31-35.
Mayr, C., Hemann, M. T. and Bartel, D. P. (2007). Disrupting the pairing between let7 and Hmga2 enhances oncogenic transformation. Science 315, 1576-1579.
Melton, C., Judson, R. L. and Blelloch, R. (2010). Opposing microRNA families regulate
self-renewal in mouse embryonic stem cells. Nature 463, 621-626.
Mills, A. A., Zheng, B., Wang, X. J., Vogel, H., Roop, D. R. and Bradley, A. (1999).
p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 398,
708-713.
Miska, E. A., Alvarez-Saavedra, E., Abbott, A. L., Lau, N. C., Hellman, A. B.,
McGonagle, S. M., Bartel, D. P., Ambros, V. R. and Horvitz, H. R. (2007). Most
Caenorhabditis elegans microRNAs are individually not essential for development or
viability. PLoS Genet. 3, e215.
Morlando, M., Ballarino, M., Gromak, N., Pagano, F., Bozzoni, I. and Proudfoot, N.
J. (2008). Primary microRNA transcripts are processed co-transcriptionally. Nat. Struct.
Mol. Biol. 15, 902-909.
Murchison, E. P., Partridge, J. F., Tam, O. H., Cheloufi, S. and Hannon, G. J. (2005).
Characterization of Dicer-deficient murine embryonic stem cells. Proc. Natl. Acad. Sci.
USA 102, 12135-12140.
Nishino, J., Kim, I., Chada, K. and Morrison, S. J. (2008). Hmga2 promotes neural
stem cell self-renewal in young but not old mice by reducing p16Ink4a and p19Arf
Expression. Cell 135, 227-239.
Pasquinelli, A. E., Reinhart, B. J., Slack, F., Martindale, M. Q., Kuroda, M. I., Maller,
B., Hayward, D. C., Ball, E. E., Degnan, B., Muller, P. et al. (2000). Conservation of
the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature
408, 86-89.
Patrick, D. M., Zhang, C. C., Tao, Y., Yao, H., Qi, X., Schwartz, R. J., Jun-Shen
Huang, L. and Olson, E. N. (2010). Defective erythroid differentiation in miR-451
mutant mice mediated by 14-3-3zeta. Genes Dev. 24, 1614-1619.
Rasmussen, K. D., Simmini, S., Abreu-Goodger, C., Bartonicek, N., Di Giacomo, M.,
Bilbao-Cortes, D., Horos, R., Von Lindern, M., Enright, A. J. and O’Carroll, D.
(2010). The miR-144/451 locus is required for erythroid homeostasis. J. Exp. Med. 207,
1351-1358.
Reinhart, B. J., Slack, F. J., Basson, M., Pasquinelli, A. E., Bettinger, J. C., Rougvie,
A. E., Horvitz, H. R. and Ruvkun, G. (2000). The 21-nucleotide let-7 RNA regulates
developmental timing in Caenorhabditis elegans. Nature 403, 901-906.
1783
Rodriguez, A., Vigorito, E., Clare, S., Warren, M. V., Couttet, P., Soond, D. R., van
Dongen, S., Grocock, R. J., Das, P. P., Miska, E. A. et al. (2007). Requirement of
bic/microRNA-155 for normal immune function. Science 316, 608-611.
Rybak, A., Fuchs, H., Hadian, K., Smirnova, L., Wulczyn, E. A., Michel, G., Nitsch,
R., Krappmann, D. and Wulczyn, F. G. (2009). The let-7 target gene mouse lin-41 is
a stem cell specific E3 ubiquitin ligase for the miRNA pathway protein Ago2. Nat. Cell
Biol. 11, 1411-1420.
Schwarz, D. S., Hutvagner, G., Du, T., Xu, Z., Aronin, N. and Zamore, P. D. (2003).
Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199-208.
Selbach, M., Schwanhausser, B., Thierfelder, N., Fang, Z., Khanin, R. and Rajewsky,
N. (2008). Widespread changes in protein synthesis induced by microRNAs. Nature
455, 58-63.
Sempere, L. F., Freemantle, S., Pitha-Rowe, I., Moss, E., Dmitrovsky, E. and Ambros,
V. (2004). Expression profiling of mammalian microRNAs uncovers a subset of brainexpressed microRNAs with possible roles in murine and human neuronal differentiation.
Genome Biol. 5, R13.
Senoo, M., Pinto, F., Crum, C. P. and McKeon, F. (2007). p63 is essential for the
proliferative potential of stem cells in stratified epithelia. Cell 129, 523-536.
Siminovitch, L., McCulloch, E. A. and Till, J. E. (1963). The distribution of colonyforming cells among spleen colonies. J. Cell. Physiol. 62, 327-336.
Sinkkonen, L., Hugenschmidt, T., Berninger, P., Gaidatzis, D., Mohn, F., Artus-Revel,
C. G., Zavolan, M., Svoboda, P. and Filipowicz, W. (2008). MicroRNAs control de
novo DNA methylation through regulation of transcriptional repressors in mouse
embryonic stem cells. Nat. Struct. Mol. Biol. 15, 259-267.
Stark, A., Brennecke, J., Bushati, N., Russell, R. B. and Cohen, S. M. (2005). Animal
MicroRNAs confer robustness to gene expression and have a significant impact on
3⬘UTR evolution. Cell 123, 1133-1146.
Suh, M. R., Lee, Y., Kim, J. Y., Kim, S. K., Moon, S. H., Lee, J. Y., Cha, K. Y., Chung,
H. M., Yoon, H. S., Moon, S. Y. et al. (2004). Human embryonic stem cells express a
unique set of microRNAs. Dev. Biol. 270, 488-498.
Suzuki, H. I., Yamagata, K., Sugimoto, K., Iwamoto, T., Kato, S. and Miyazono, K.
(2009). Modulation of microRNA processing by p53. Nature 460, 529-533.
Tam, O. H., Aravin, A. A., Stein, P., Girard, A., Murchison, E. P., Cheloufi, S., Hodges,
E., Anger, M., Sachidanandam, R., Schultz, R. M. et al. (2008). Pseudogene-derived
small interfering RNAs regulate gene expression in mouse oocytes. Nature 453, 534538.
Thai, T. H., Calado, D. P., Casola, S., Ansel, K. M., Xiao, C., Xue, Y., Murphy, A.,
Frendewey, D., Valenzuela, D., Kutok, J. L. et al. (2007). Regulation of the germinal
center response by microRNA-155. Science 316, 604-608.
Vasudevan, S., Tong, Y. and Steitz, J. A. (2007). Switching from repression to activation:
microRNAs can up-regulate translation. Science 318, 1931-1934.
Viswanathan, S. R., Daley, G. Q. and Gregory, R. I. (2008). Selective blockade of
microRNA processing by Lin28. Science 320, 97-100.
Wang, Y., Medvid, R., Melton, C., Jaenisch, R. and Blelloch, R. (2007). DGCR8 is
essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal.
Nat. Genet. 39, 380-385.
Wang, Y., Baskerville, S., Shenoy, A., Babiarz, J. E., Baehner, L. and Blelloch, R.
(2008). Embryonic stem cell-specific microRNAs regulate the G1-S transition and
promote rapid proliferation. Nat. Genet. 40, 1478-1483.
Watanabe, T., Totoki, Y., Toyoda, A., Kaneda, M., Kuramochi-Miyagawa, S., Obata,
Y., Chiba, H., Kohara, Y., Kono, T., Nakano, T. et al. (2008). Endogenous siRNAs
from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature 453, 539543.
Xiao, C., Calado, D. P., Galler, G., Thai, T. H., Patterson, H. C., Wang, J., Rajewsky,
N., Bender, T. P. and Rajewsky, K. (2007). MiR-150 controls B cell differentiation by
targeting the transcription factor c-Myb. Cell 131, 146-159.
Xu, N., Papagiannakopoulos, T., Pan, G., Thomson, J. A. and Kosik, K. S. (2009).
MicroRNA-145 regulates OCT4, SOX2, and KLF4 and represses pluripotency in
human embryonic stem cells. Cell 137, 647-658.
Yang, A., Schweitzer, R., Sun, D., Kaghad, M., Walker, N., Bronson, R. T., Tabin, C.,
Sharpe, A., Caput, D., Crum, C. et al. (1999). p63 is essential for regenerative
proliferation in limb, craniofacial and epithelial development. Nature 398, 714-718.
Yekta, S., Shih, I. H. and Bartel, D. P. (2004). MicroRNA-directed cleavage of HOXB8
mRNA. Science 304, 594-596.
Yi, R., Poy, M. N., Stoffel, M. and Fuchs, E. (2008). A skin microRNA promotes
differentiation by repressing ‘stemness’. Nature 452, 225-229.
Yu, D., dos Santos, C. O., Zhao, G., Jiang, J., Amigo, J. D., Khandros, E., Dore, L.
C., Yao, Y., D’Souza, J., Zhang, Z. et al. (2010). miR-451 protects against erythroid
oxidant stress by repressing 14-3-3zeta. Genes Dev. 24, 1620-1633.
Zhang, L., Stokes, N., Polak, L. and Fuchs, E. (2011). Specific microRNAs are
preferentially expressed by skin stem cells to balance self-renewal and early lineage
commitment. Cell Stem Cell 8, 294-308.
Zhao, C., Sun, G., Li, S. and Shi, Y. (2009). A feedback regulatory loop involving
microRNA-9 and nuclear receptor TLX in neural stem cell fate determination. Nat.
Struct. Mol. Biol. 16, 365-371.
Zhao, Y., Samal, E. and Srivastava, D. (2005). Serum response factor regulates a
muscle-specific microRNA that targets Hand2 during cardiogenesis. Nature 436, 214220.