The Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Biochemistry, The Henry Wellcome Building of Cancer and Developmental Biology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK
* Author for correspondence (e-mail: eam29{at}cam.ac.uk)
SUMMARY
Five years into the `small RNA revolution' it is hard not to share in the excitement about the rapidly unravelling biology of microRNAs. Since the discovery of the first microRNA gene, lin-4, in the nematode Caenorhabditis elegans, many more of these short regulatory RNA genes have been identified in flowering plants, worms, flies, fish, frogs and mammals. Currently, about 2% of the known human genes encode microRNAs. MicroRNAs are essential for development and this review will summarise our current knowledge of animal microRNA function. We will also discuss the emerging links of microRNA biology to stem cell research and human disease, in particular cancer.
Introduction
MicroRNAs (miRNAs) are about 22-nucleotide, short, non-coding RNAs that are
thought to regulate gene expression through sequence-specific base pairing
with target mRNAs. Hundreds of microRNAs have been identified in worms, flies,
fish, frogs, mammals and flowering plants using molecular cloning and
bioinformatics prediction strategies
(Lagos-Quintana et al., 2001;
Lau et al., 2001
;
Lee and Ambros, 2001
;
Lim et al., 2003a
;
Llave et al., 2002
;
Reinhart et al., 2002
;
Watanabe et al., 2005
).
MicroRNAs are transcribed as long RNA precursors (pri-miRNAs) that contain a
stem-loop structure of about 80 bases. Pri-miRNAs are processed in the nucleus
by the RNase III enzyme Drosha and DGCR8/Pasha, which excises the stem-loop to
form the pre-miRNA (Denli et al.,
2004
; Gregory et al.,
2004
; Han et al.,
2004
; Landthaler et al.,
2004
; Lee et al.,
2003
). Pre-miRNAs are exported from the nucleus by Exportin-5
(Bohnsack et al., 2004
;
Lund et al., 2003
;
Yi et al., 2003
). In the
cytoplasm, another RNase III enzyme, Dicer, cuts the pre-miRNA to generate the
mature microRNA as part of a short RNA duplex. The RNA is subsequently unwound
by a helicase activity and incorporated into a RNA-induced silencing complex
(RISC). For more information on microRNA biogenesis and maturation, please see
the accompanying article by Du and Zamore
(Du and Zamore, 2005
).
Most microRNAs in animals are thought to function through the inhibition of
effective mRNA translation of target genes through imperfect base pairing with
the 3'-untranslated region (3'UTR) of target mRNAs
(Bartel, 2004). The underlying
mechanism is still poorly understood, but it appears to involve the inhibition
of translational initiation (Pillai et
al., 2005
). MicroRNA targets are largely unknown, but estimates
range from one to hundreds of target genes for a given microRNA, based on
target predictions using a variety of bioinformatics approaches
(Brennecke et al., 2005
;
John et al., 2004
;
Kiriakidou et al., 2004
;
Lewis et al., 2005
;
Lewis et al., 2003
;
Rajewsky and Socci, 2004
;
Stark et al., 2003
;
Xie et al., 2005
). In
addition, at least one microRNA, miR-196, can cleave a target mRNA, HOXB8, in
the same manner as a short interfering RNA (siRNA) does during the process of
RNA interference (RNAi) (Mansfield et al.,
2004
; Yekta et al.,
2004
). This mechanism is the preferred one for plant microRNAs
(Meins et al., 2005
).
MicroRNAs may also play a role in AU-rich element-mediated mRNA degradation
(Jing et al., 2005
). See Du
and Zamore for a further discussion of this
(Du and Zamore, 2005
).
Finally, the involvement of microRNAs in transcriptional gene silencing (TGS),
which has been observed in plants, remains an intriguing possibility
(Baulcombe, 2004
).
This review focuses on the function of animal microRNAs only. For a recent
review of our current understanding of the roles of microRNAs in plants,
please see (Kidner and Martienssen,
2005), and for recent accounts of the history of this field,
please see recent articles by the pioneers of the field themselves
(Lee et al., 2004a
;
Ruvkun et al., 2004
). Here, we
describe how microRNAs contribute to different aspects of animal development
and what we know of their involvement in human disease (see
Table 1).
|
Interest in the genes controlling developmental timing in C.
elegans (Ambros and Horvitz,
1984; Chalfie et al.,
1981
; Horvitz and Sulston,
1980
) led to the cloning of the first microRNA, lin-4
miRNA (Lee et al., 1993
), and
the identification of the first microRNA target, lin-14 mRNA
(Wightman et al., 1993
). The
developmental-timing, or heterochronic, pathway regulates stage-specific
processes during C. elegans larval development. For a recent,
detailed review of this pathway, please see Rougvie
(Rougvie, 2005
). One focus of
the study of the heterochronic pathway in C. elegans has been the
developmental fate of several stem cells in the lateral hypodermis,
collectively known as the seam cells. The seam cells undergo a cell division
pattern that is synchronised with the four larval molts of the animal
(Fig. 1A). Only at the adult
stage will the seam cells exit mitosis and terminally differentiate. In
lin-4 mutant animals, the seam cells repeat the cell division pattern
that characterises the first larval stage (L1) and fail to differentiate. This
mutant phenotype has been interpreted as a heterochronic change with the
developmental clock being stuck at the L1 stage, resulting in developmental
`retardation'. Gain-of-function mutations in the lin-4 miRNA target
lin-14 lead to an identical phenotype, whereas loss-of-function
mutations in lin-14 result in an opposite, `precocious' phenotype,
where the seam cells skip the cell division of the first larval stage. The
lin-4 and lin-14 gene products therefore act as a
developmental switch that controls the L1 to L2 transition
(Fig. 1A,B).
Three microRNAs of the let-7 family, mir-48, mir-84 and
mir-241 act redundantly to control the next developmental transition,
from the L2 to the L3 stage (Fig.
1B) (Abbott et al.,
2005; Lau et al.,
2001
; Lin et al.,
2005
; Reinhart et al.,
2000
). Loss-of-function mutations in these three microRNAs lead to
the repetition of the cell division pattern of the second larval stage,
whereas a gain-of-function mutation in mir-48 results in a precocious
phenotype. A likely target of mir-48, mir-84 and mir-241
during this transition is the C. elegans hunchback orthologue
hbl-1. The microRNA let-7, the second microRNA to be
identified (Reinhart et al.,
2000
), controls the transition from the fourth larval stage to the
adult stage, and two of its targets in the heterochronic pathway are the
lin-41 and hbl-1 genes, both of which are also heterochronic
genes (Abrahante et al., 2003
;
Lin et al., 2003
;
Slack et al., 2000
). More
recently, two additional let-7 target genes, the transcription factor
genes daf-12 and pha-4, were identified using a combination
of bioinformatics-based prediction and RNAi analyses
(Grosshans et al., 2005
).
daf-12 is also a regulator of the heterochronic pathway controlling
seam cell fate (Antebi et al.,
1998
). Finally, the two let-7 family microRNAs
mir-48 and mir-84 also control cessation of the larval
molting cycle at the adult stage, with mir-48; mir-84 double
mutant animals undergoing a supernumerary molt at the adult stage
(Fig. 1A,B)
(Abbott et al., 2005
).
It is striking that at least two microRNA families and at least four
microRNAs are involved in the control of developmental timing in C.
elegans. As the lin-4 and let-7 microRNA families are
conserved, they might play similar roles in other organisms. This notion is
supported by the temporal regulation of let-7 expression in several
species (Pasquinelli et al.,
2000). However, at least one potential role for let-7
family microRNAs outside of the heterochronic pathway has been reported, as
discussed below (Johnson et al.,
2005
).
|
In the absence of loss-of-function mutants for most microRNA genes,
organisms with defective microRNA biogenesis are a useful tool for
investigating the biological roles of microRNAs, particularly during
embryogenesis and early development. This is because such organisms allow one
to study the roles of the first set of microRNAs required during development.
Dicer knockout mutants have been particularly useful in this regard, although
RNAi, and other biological processes as well as microRNA function, might be
de-regulated in these mutants. Dicer was first analyzed genetically in C.
elegans, where it is called DCR-1, and it was found to be essential for
germline development (Knight and Bass,
2001). A similar phenotype was observed for C. elegans
mutants of the other RNase III enzyme required for microRNA biogenesis, Drosha
(DRSH-1) (Denli et al., 2004
).
As dcr-1 mutants are sterile, all homozygous animals had to be
derived from heterozygous mothers. It is likely that maternal contribution of
DCR-1 masks earlier abnormal phenotypes. This is supported by the occurrence
of additional abnormal phenotypes when dcr-1 mRNA is inactivated by
RNAi (Grishok et al., 2001
).
RNAi of dcr-1 mRNA results in a mixed phenotype that includes
embryonic lethality and developmental timing defects that are reminiscent of
the lin-4 and let-7 mutants
(Grishok et al., 2001
). These
observations suggest that microRNAs have essential roles in C.
elegans embryogenesis. This hypothesis was further supported by RNAi
knockdown of the mRNA transcripts for the two C. elegans argonaute
proteins required for miRNA biogenesis, ALG-1 and ALG-2
(Grishok et al., 2001
).
RNAi-treated worms showed a mixed phenotype that included embryonic and larval
lethality and heterochronic defects.
The fruitfly Drosophila melanogaster has two Dicer genes,
Dicer-1 and Dicer-2, and genetic analysis suggests that
Dicer-1 is the major Dicer gene required for microRNA biogenesis
(Lee et al., 2004b). Although
the phenotype of Dicer-1 mutant D. melanogaster has not been
fully reported, it appears that Dicer-1 is required for wild-type development
of both somatic tissues and the germline
(Hatfield et al., 2005
;
Lee et al., 2004b
) (for
details see below).
In the zebrafish Danio rerio, a likely null allele of Dicer leads
to a developmental arrest at 7 to 10 days post-fertilization
(Wienholds et al., 2003).
This late terminal phenotype is again likely to be due to maternal provision
of Dicer and/or of microRNAs. Indeed, removal of the maternal Dicer
contribution through the generation of germline clones leads to a more severe
defect (Giraldez et al.,
2005
). In maternal-zygotic Dicer mutants, axis formation and early
differentiation are normal, but many embryos have morphogenesis defects
affecting gastrulation, brain formation, somitogenesis and heart
development.
In the mouse, Mus musculus, Dicer1 mutants die around 7.5 days of
gestation (Bernstein et al.,
2003). A maternal contribution of Dicer is likely to have a much
smaller effect in M. musculus due to the much smaller size of the
egg. Homozygous Dicer1 null mutants from heterozygous mothers die
around 7.5 days of gestation (Bernstein et
al., 2003
). Mutant embryos have defects in axis formation and
gastrulation, and are depleted of Oct4-positive stem cells
(Bernstein et al., 2003
). In
all cases where only Dicer1 mutants have been analysed, one cannot
easily distinguish between defects that are due to a loss of microRNA
processing and those that are due to a loss of endogenous RNAi or other
pathways regulated by Dicer. For example, Dicer appears to have important
roles in heterochromatin formation and chromosome segregation in the fission
yeast Schizosaccharomyces pombe, in the ciliated protozoan
Tetrahymena and in vertebrate cells
(Fukagawa et al., 2004
;
Mochizuki and Gorovsky, 2005
;
Provost et al., 2002
). As
S. pombe does not encode any known microRNAs, these defects are
unlikely to be caused by their loss.
A more direct approach to investigating the role of microRNAs during
embryogenesis has been taken in D. melanogaster, where 2'
O-Methyl antisense oligoribonucleotides were used in microRNA depletion
studies (see Box 1)
(Leaman et al., 2005).
A single injection of 2' O-Methyl antisense oligoribonucleotides complementary to the 46 microRNAs known to be expressed in the D. melanogaster embryo resulted in a total of twenty-five different, abnormal phenotypes. These phenotypes included defects in blastoderm cellularization and patterning, morphogenesis and cell survival. Increased programmed cell death was observed in embryos injected with 2' O-Methyl antisense oligoribonucleotides that targeted the D. melanogaster miR-2 family, and miR-6, miR-11 and miR-308.
Clearly, our current understanding of microRNA function during
embryogenesis is only rudimentary. However, it is noteworthy that the only
evidence for a role of miRNAs in tissue patterning during embryogenesis to
date comes from depletion studies of miR-31 in D. melanogaster
(Leaman et al., 2005). Could
pattern formation be largely independent of regulation by microRNAs? With this
question in mind, it will be exciting to see functional studies of mammalian
miR-196, a microRNA that is located in a HOX cluster and can cleave HOXB8 mRNA
(Mansfield et al., 2004
;
Yekta et al., 2004
).
Differentiation and organogenesis
The heterochronic phenotypes of the lin-4 microRNA and the
let-7 family of microRNAs in C. elegans are clear examples
of cell differentiation defects. However, an example of a microRNA regulating
differentiation that is uncoupled from cell division was first uncovered
through the study of left-right asymmetry in C. elegans
(Johnston and Hobert, 2003).
In the worm, two bilateral taste receptor neurons, ASE left (ASEL) and ASE
right (ASER), display a left/right asymmetrical expression pattern of
gcy-5, gcy-6 and gcy-7, three putative chemoreceptor genes
(Chang et al., 2003
;
Hobert et al., 2002
)
(Fig. 2). In a genetic screen
for mutants in which the normally ASEL-specific expression of gcy-7
is disrupted, the microRNA gene lsy-6 was isolated
(Chang et al., 2003
). In
lsy-6 mutants, ASEL neurons do not express gcy-7, but
instead express the ASER-specific gcy-5 gene. Genetic interaction and
GFP reporter studies showed that lsy-6 is a negative regulator of the
NKX-type homeobox gene cog-1, which was identified in the same
genetic screen. Interestingly, a second microRNA, miR-273, might act upstream
in the same pathway as a regulator of die-1, which encodes a C2H2
zinc finger transcription factor (Chang et
al., 2004
). The transcription factor die-1 shows
ASEL-specific expression and acts upstream of lsy-6.
Evidence for a role of microRNAs in organogenesis has come from studies of
vulval development in C. elegans. The C. elegans vulva is a
ring-like structure that forms the connection between the hermaphrodite
gonadal arms and the exterior, and is essential for egg-laying and sperm
entry. It derives from a group of cells in the ventral hypodermis that are
induced to undergo a series of cell divisions and differentiation by a signal
from the gonadal anchor cell (Sulston and
Horvitz, 1977). Vulval induction requires RAS/LET-60 signalling
(Beitel et al., 1990
).
let-7 loss-of-function mutants die by bursting at the vulva
(Reinhart et al., 2000
;
Slack et al., 2000
), and this
bursting is suppressed by RNAi against the C. elegans RAS orthologue
LET-60 (Johnson et al., 2005
).
Furthermore, overexpression of the let-7 family microRNA miR-84
suppresses the let-60 gain-of-function phenotype. 3'UTR
reporter experiments suggest that RAS/LET-60 expression levels are regulated
post-transcriptionally and may be directly regulated by the let-7
family of microRNAs (Johnson et al.,
2005
).
In D. melanogaster, the discovery of an important role for
post-transcriptional control of the Notch signalling pathway predates the
discovery of the first microRNA in D. melanogaster by over a decade.
The Notch signalling pathway is an evolutionary conserved signal transduction
cascade that is required for patterning and normal development
(Lai, 2004). Two clusters of
Notch signalling target genes exist in D. melanogaster: the Enhancer
of split-Complex and the Bearded-Complex, which encode transcription factors
of the basic helix-loop-helix repressor and the Bearded families, respectively
(Knust et al., 1992
;
Lai et al., 2000a
;
Lai et al., 2000b
;
Wurmbach et al., 1999
).
Gain-of-function alleles in members of these gene families were found to be
due to short deletions in conserved regions of their 3'UTRs
(Knust, 1997
;
Leviten et al., 1997
). These
6- to 7-nucleotide motifs were named the GY-box, the Brd-box and the K-box.
Some of these motifs have been shown to post-transcriptionally control the
Enhancer of split-Complex and the Bearded-Complex genes
(Lai et al., 1998
;
Lai and Posakony, 1997
). More
recently, it has been noted that GY-, Brd- and K-box sequences are
complimentary to the newly identified D. melanogaster microRNAs
(Lagos-Quintana et al., 2001
;
Lai, 2002
). Candidate
microRNAs regulating these motifs include three microRNA families and the
following microRNAs: miR-2, miR-4, miR-5, miR-6, miR-7, miR-11 and miR-79
(Brennecke et al., 2005
;
Lai, 2002
;
Lai et al., 2005
;
Stark et al., 2003
). Although
no loss-of-function analyses of any of these microRNAs have been carried out,
overexpression of some of them causes defects reminiscent of Notch
loss-of-function mutants. These include notched wings and wing vein
abnormalities, an increased number of micro- and macrochaetes (small and large
bristles) in the adult notum, tufted sternopleural bristles and an increase in
sensory organ precursor cells in imaginal discs
(Lai et al., 2005
).
Interestingly, the list of microRNAs that might regulate Notch overlaps
considerably with the microRNAs that were found to play important roles during
embryogenesis in the 2' O-Methyl antisense oligoribonucleotides microRNA
depletion studies previously discussed
(Leaman et al., 2005
).
|
Similar to D. rerio, and with the same caveats, M. musculus
Dicer1 mutant animals indicate that microRNAs have wide-ranging roles in
differentiation and development during mouse embryogenesis
(Bernstein et al., 2003) (see
above). In the mouse, additional insights can be gained from studying
conditional-knockout strains, which allow one to the study the requirement for
Dicer and microRNAs in different tissues at different developmental time
points. In vitro, Dicer mutant embryonic stem (ES) cells, derived from
conditional gene targeting, have severe differentiation defects
(Kanellopoulou et al., 2005
).
One study that analyzed the effects of genetically inactivating
Dicer1 early during T-cell development found evidence for the
functioning of microRNAs in
ß cell, but not in CD4/CD8, lineage
commitment (Cobb et al.,
2005
). Another study found that knocking out Dicer1
during T-cell development blocked peripheral CD8+ T-cell
development, whereas CD4+ T cells, although reduced in numbers,
were viable; however, upon stimulation, these CD4+ T cells
proliferated poorly and underwent increased programmed cell death
(Muljo et al., 2005
).
The particular caveat with these conditional-knockout studies in mice is
that it is often unclear how efficiently Dicer and any existing microRNA pools
are depleted upon the somatic deletion of Dicer1. Indeed, microRNAs
seem to persist for some time (Cobb et
al., 2005). One specific microRNA that has been directly
implicated in B-cell development is miR-181
(Chen et al., 2004
). This
microRNA is highly expressed in B-lymphoid cells of mouse bone marrow. When
overexpressed in hematopoietic progenitor cells, it leads to an increase in
the fraction of B-lineage cells in in vitro differentiation experiments and in
vivo in adult mice. Conditionally inactivating Dicer1 in discrete
areas of the limb mesoderm in mice led to severe growth defects in the limbs
of mutant embryos, but no defect in basic limb patterning or in
tissue-specific differentiation was observed
(Harfe et al., 2005
). This is
a striking finding that is somewhat reminiscent of the Dicer1
knockout in D. rerio. However, it remains unknown whether residual
Dicer activity or microRNA pools could have disguised earlier roles of
microRNAs in limb development.
MicroRNA expression analysis has led to the discovery of a potential role
for the microRNA miR-1 in mammalian heart development. The microRNA miR-1,
which is the product of two genes, mir-1-1 and mir-1-2, is
highly expressed in mouse heart and muscle
(Lagos-Quintana et al., 2001;
Lee and Ambros, 2001
). An
analysis of the upstream-regulatory sequence of these two genes has led to the
identification of serum response factor (Srf), myocardin, Mef2 and Myod as
transcriptional regulators of miR-1 expression in vitro
(Zhao et al., 2005
). Of
these, Srf was found to be required for miR-1 expression in the developing
mouse heart, using a conditional Srf-knockout strain
(Zhao et al., 2005
).
Overexpression of miR-1 under the ß-myosin heavy chain promoter resulted
in developmental arrest at embryonic day 13.5, after heart failure. Transgenic
embryos developed thin ventricle walls and ventricular cardiomyocyte
proliferation defects. One candidate target for miR-1 in myocardial
development is the transcription factor Hand2, which was found to be reduced
in transgenic mice overexpressing miR-1 without an apparent change in
Hand2 mRNA levels (Zhao et al.,
2005
).
Growth control and programmed cell death
The D. melanogaster bantam gene was identified in a
gain-of-function screen for regulators of cell growth
(Hipfner et al., 2002).
Overexpression of bantam causes the overgrowth of wing and eye
tissue, whereas bantam loss-of-function mutant animals are smaller
than wild-type animals and have reduced cell numbers. bantam was
found to interact with the growth regulatory gene expanded, but was
epistatic to the CycD/Cdk4 pathway. Subsequent cloning of
bantam identified it as a microRNA-encoding gene
(Brennecke et al., 2003
). The
bantam microRNA regulates tissue growth cell autonomously. Overgrowth
phenotypes due to overexpression of bantam do not result in increased
levels of programmed cell death, and bantam overexpression rescued
programmed cell death induced by overexpression of the transcription factor
E2A element-binding factor (E2F) and its dimerisation partner (DP).
bantam overexpression also blocked programmed cell death induced by
overexpression of the pro-apoptotic gene hid/Wrinked
(Fig. 3). Furthermore, Hid was
shown to be the likely direct target of bantam mRNA
(Brennecke et al., 2003
). It
is unclear whether the small body size of bantam mutant flies is due
to the increased activity of Hid or the de-regulation of other target genes. A
second D. melanogaster microRNA, miR-14, was also found to suppress
programmed cell death (Xu et al.,
2003
). Whether this is a direct effect of the de-regulation of
pro-apoptotic genes remains to be determined. Increased levels of programmed
cell death were also found in depletion experiments using 2' O-Methyl
antisense oligoribonucleotides targeting the D. melanogaster miR-2
family, miR-6, miR-11 and miR-308 (Leaman
et al., 2005
) (see previous discussion). Finally, the deletion of
Dicer-1 in Drosophila results in a growth defect in germline
stem cells (Hatfield et al.,
2005
) (see below). Together, these observations suggest an
important role for microRNAs in growth control during D. melanogaster
development.
Additional links between microRNAs, growth control and programmed cell
death have also come from other species. The phenotype of mir-48; mir-84;
mir-241 mutants in C. elegans is one of cellular overgrowth
(Abbott et al., 2005). In
D. rerio, the zygotic removal of Dicer results in a larval growth
arrest (Wienholds et al.,
2003
) (see above). And, finally, in M. musculus, removal
of Dicer in the limb mesoderm leads to a dramatic programmed cell death in the
developing limb (Harfe et al.,
2005
).
Stem cells and the germline
The first two microRNAs to be identified, the C. elegans microRNAs
lin-4 and let-7, control cell divisions in the hypodermal
blast lineage (Ambros and Horvitz,
1984; Chalfie et al.,
1981
; Horvitz et al.,
1983
). In the absence of either gene, this stem cell lineage fails
to differentiate and continues its proliferative cycle. More recently, other
let-7 family members have also been shown to be involved in the
differentiation of this stem cell lineage
(Abbott et al., 2005
;
Lin et al., 2005
). In the
mouse, Dicer is required for embryonic stem cell differentiation in vitro
(Kanellopoulou et al., 2005
)
(see above). And in early Dicer1 mutant mouse embryos, the pool of
pluripotent stem cells that is required for the proliferation of cells in the
inner cell mass of the blastocyst is diminished, as assayed by in situ
hybridization using probes against Oct4 mRNA
(Bernstein et al., 2001
). It is
unclear whether the pool of Oct4 mRNA-positive cells fails to expand,
differentiates or undergoes programmed cell death. The identities of any
microRNAs that may be required for stem cell maintenance in the mouse are
currently unknown. However, expression studies in ES cell lines and in mouse
embryoid bodies have identified microRNAs (miR-302 family) that are
specifically expressed in ES cells but not in adult mouse tissues
(Houbaviy et al., 2003
;
Suh et al., 2004
). These
microRNAs may be candidates for stem cell renewal factors.
|
Human disease
Many of the functional roles of microRNAs discussed above hint at the
potential involvement of microRNAs in human disease. For example, the
lin-4 and let-7 mutant phenotypes observed in C.
elegans can be interpreted as growth defects
(Ambros and Horvitz, 1984;
Chalfie et al., 1981
;
Horvitz et al., 1983
). The
let-7 family of microRNAs may also be regulators of the
proto-oncogene RAS. In D. melanogaster, the bantam
microRNA and miR-14 are required for growth control; for example, through the
regulation of programmed cell death
(Brennecke et al., 2003
;
Xu et al., 2003
). If
microRNAs are major regulators of growth and proliferation, is there also
evidence for roles of microRNAs in human cancer? Many microRNAs are
de-regulated in primary human tumours
(Calin et al., 2002
;
Calin et al., 2004a
;
He et al., 2005
;
Lu et al., 2005
). Moreover,
many human microRNAs are located at genomic regions linked to cancer
(Calin et al., 2004b
;
McManus, 2003
). Of particular
interest is the mir-17 microRNA cluster, which is in a region on
human chromosome 13 that is frequently amplified in B-cell lymphomas
(He et al., 2005
).
Overexpression of the mir-17 cluster was found to co-operate with
Myc to accelerate tumour development in a mouse B-cell lymphoma
model. Further evidence for such a link between Myc and the
mir-17 cluster has come from microarray expression studies, which
showed that mir-17 cluster gene expression was induced by the
overexpression of Myc (O'Donnell et al.,
2005
). Predicted targets for the mir-17 cluster microRNAs
include members of the E2F and retinoblastoma families
(Lewis et al., 2003
);
mir-17 cluster microRNAs have been found to downregulate E2F1
expression (O'Donnell et al.,
2005
).
Another potential link between microRNAs and human disease comes from the
identification of an essential co-factor for the microRNA biogenesis enzyme
Drosha. This cofactor is encoded by DGCR8, which maps to chromosomal region
22q11.2, which is commonly deleted in DiGeorge syndrome
(Denli et al., 2004;
Gregory et al., 2004
;
Han et al., 2004
;
Landthaler et al., 2004
;
Lee et al., 2003
;
Lindsay, 2001
;
Shiohama et al., 2003
).
Haploinsufficiency of this region accounts for over 90% of individuals with
DiGeorge syndrome, a disorder that affects 1 in 3,000 live births and results
in heterogeneous defects including heart defects, immunodeficiency,
schizophrenia and obsessive-compulsive disorder, among others. If indeed
haploinsufficiency of DGCR8 contributes to DiGeorge syndrome, reduced levels
of specific miRNAs may be to blame.
Potential roles of microRNAs in the development of the immune system have
been discussed above; however, microRNAs might also be involved in immune
defence. A cellular microRNA, miR-32, can regulate primate foamy virus type 1
(PFV-1) proliferation in cell culture
(Lecellier et al., 2005). In
addition, large DNA viruses of the herpesvirus family, including EBV
(Pfeffer et al., 2005
;
Pfeffer et al., 2004
) and SV40
(Sullivan et al., 2005
),
encode viral microRNA genes. These viral microRNAs have no apparent homologues
in host genomes and their function is currently not understood.
Evolution
The labelling of C. elegans lin-4 and let-7 as
heterochronic genes (Ambros and Horvitz,
1984) was highly provocative, suggesting potential roles for these
genes during evolutionary change, but was well received by the evolutionary
biology community (Gould,
2000
). With the identification of lin-4 and
let-7 as microRNAs, their small size added only to the attractiveness
of an evolutionary role for these genes. But how quickly do microRNAs
themselves evolve? One of the early, exciting findings in the field was the
realisation that the let-7 microRNA is 100% conserved between C.
elegans and humans (Pasquinelli et
al., 2003
). Overall, about 40% of the C. elegans microRNA
families are conserved in humans (Lim et
al., 2003b
) (Fig.
4). By contrast, many primate-specific microRNAs that have no
counterparts in the mouse have been identified
(Berezikov et al., 2005
), and
several microRNA-encoding genes occur in highly repetitive and fast-evolving
regions of the genome, such as in LINE-2 transposable elements
(Smalheiser and Torvik,
2005
). For the mir-17 cluster and the microRNAs in the
HOX gene cluster, an evolutionary analysis has been reported
(Tanzer et al., 2005
;
Tanzer and Stadler, 2004
).
These initial studies suggest that microRNA families, rather than single
microRNAs, are evolutionary conserved. One of the next questions to answer
will be how the interactions between microRNAs and their targets evolve.
|
Research over the past five years has put microRNAs at centre stage. Early
cloning experiments (Lagos-Quintana et
al., 2001; Lau et al.,
2001
; Lee and Ambros,
2001
) combined with expression studies (e.g.
Wienholds et al., 2005
),
global approaches (such as Dicer knockouts) and selected functional studies
have generated a tremendous amount of excitement about microRNAs in many areas
of biology (see Table 1). In
parallel, many new tools for the study of microRNAs have been developed (see
Du and Zamore, 2005
). We
expect that soon we will appreciate that what we have learned about microRNAs
to date is just the tip of the iceberg.
ACKNOWLEDGMENTS
We would like to thank Victor Ambros, Ann Rougvie and Phil Zamore for sharing information prior to publication.
REFERENCES
Abbott, A. L., Alvarez-Saavedra, E., Miska, E. A., Lau, N. C., Bartel, D. P., Horvitz, H. R. and Ambros, V. (2005). The let-7 MicroRNA family members mir-48, mir-84 and mir-241 function together to regulate. developmental timing in Caenorhabditis elegans. Dev. Cell 9, 403-414.[CrossRef][Medline]
Abrahante, J. E., Daul, A. L., Li, M., Volk, M. L., Tennessen, J. M., Miller, E. A. and Rougvie, A. E. (2003). The Caenorhabditis elegans hunchback-like gene lin-57/hbl-1 controls developmental time and is regulated by microRNAs. Dev. Cell 4,625 -637.[CrossRef][Medline]
Ambros, V. and Horvitz, H. R. (1984). Heterochronic mutants of the nematode Caenorhabditis elegans.Science 226,409 -416.[Medline]
Antebi, A., Culotti, J. G. and Hedgecock, E. M.
(1998). daf-12 regulates developmental age and the dauer
alternative in Caenorhabditis elegans. Development
125,1191
-1205.
Bartel, D. P. (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116,281 -297.[CrossRef][Medline]
Baulcombe, D. (2004). RNA silencing in plants. Nature 431,356 -363.[CrossRef][Medline]
Beitel, G. J., Clark, S. G. and Horvitz, H. R. (1990). Caenorhabditis elegans ras gene let-60 acts as a switch in the pathway of vulval induction. Nature 348,503 -509.[CrossRef][Medline]
Berezikov, E., Guryev, V., van de Belt, J., Wienholds, E., Plasterk, R. H. and Cuppen, E. (2005). Phylogenetic shadowing and computational identification of human microRNA genes. Cell 120,21 -24.[CrossRef][Medline]
Bernstein, E., Caudy, A. A., Hammond, S. M. and Hannon, G. J. (2001). Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409,363 -366.[CrossRef][Medline]
Bernstein, E., Kim, S. Y., Carmell, M. A., Murchison, E. P., Alcorn, H., Li, M. Z., Mills, A. A., Elledge, S. J., Anderson, K. V. and Hannon, G. J. (2003). Dicer is essential for mouse development. Nat. Genet. 35,215 -217.[CrossRef][Medline]
Bohnsack, M. T., Czaplinski, K. and Gorlich, D.
(2004). Exportin 5 is a RanGTP-dependent dsRNA-binding protein
that mediates nuclear export of pre-miRNAs. RNA
10,185
-191.
Brennecke, J., Hipfner, D. R., Stark, A., Russell, R. B. and Cohen, S. M. (2003). bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila Cell 113,25 -36.[CrossRef][Medline]
Brennecke, J., Stark, A., Russell, R. B. and Cohen, S. M. (2005). Principles of microRNA-target recognition. PLoS Biol. 3,E85 .[CrossRef][Medline]
Calin, G. A., Dumitru, C. D., Shimizu, M., Bichi, R., Zupo, S.,
Noch, E., Aldler, H., Rattan, S., Keating, M., Rai, K. et al.
(2002). Frequent deletions and down-regulation of micro-RNA genes
miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc.
Natl. Acad. Sci. USA 99,15524
-15529.
Calin, G. A., Liu, C. G., Sevignani, C., Ferracin, M., Felli,
N., Dumitru, C. D., Shimizu, M., Cimmino, A., Zupo, S., Dono, M. et
al. (2004a). MicroRNA profiling reveals distinct signatures
in B cell chronic lymphocytic leukemias. Proc. Natl. Acad. Sci.
USA 101,11755
-11760.
Calin, G. A., Sevignani, C., Dumitru, C. D., Hyslop, T., Noch,
E., Yendamuri, S., Shimizu, M., Rattan, S., Bullrich, F., Negrini, M.
et al. (2004b). Human microRNA genes are frequently located
at fragile sites and genomic regions involved in cancers. Proc.
Natl. Acad. Sci. USA 101,2999
-3004.
Chalfie, M., Horvitz, H. R. and Sulston, J. E. (1981). Mutations that lead to reiterations in the cell lineages of C. elegans. Cell 24,59 -69.[CrossRef][Medline]
Chang, S., Johnston, R. J., Jr and Hobert, O.
(2003). A transcriptional regulatory cascade that controls
left/right asymmetry in chemosensory neurons of C. elegans. Genes
Dev. 17,2123
-2137.
Chang, S., Johnston, R. J., Jr, Frokjaer-Jensen, C., Lockery, S. and Hobert, O. (2004). MicroRNAs act sequentially and asymmetrically to control chemosensory laterality in the nematode. Nature 430,785 -789.[CrossRef][Medline]
Chen, C. Z., Li, L., Lodish, H. F. and Bartel, D. P.
(2004). MicroRNAs modulate hematopoietic lineage differentiation.
Science 303,83
-86.
Cobb, B. S., Nesterova, T. B., Thompson, E., Hertweck, A.,
O'Connor, E., Godwin, J., Wilson, C. B., Brockdorff, N., Fisher, A. G.,
Smale, S. T. et al. (2005). T cell lineage choice and
differentiation in the absence of the RNase III enzyme Dicer. J.
Exp. Med. 201,1367
-1373.
Denli, A. M., Tops, B. B., Plasterk, R. H., Ketting, R. F. and Hannon, G. J. (2004). Processing of primary microRNAs by the Microprocessor complex. Nature 432,231 -235.[CrossRef][Medline]
Du, T. and Zamore, P. D. (2005). microPrimer: An introduction to microRNA. Development (in press).
Fukagawa, T., Nogami, M., Yoshikawa, M., Ikeno, M., Okazaki, T., Takami, Y., Nakayama, T. and Oshimura, M. (2004). Dicer is essential for formation of the heterochromatin structure in vertebrate cells. Nat. Cell. Biol. 6, 784-791.[CrossRef][Medline]
Giraldez, A. J., Cinalli, R. M., Glasner, M. E., Enright, A. J.,
Thomson, J. M., Baskerville, S., Hammond, S. M., Bartel, D. P. and
Schier, A. F. (2005). MicroRNAs regulate brain morphogenesis
in zebrafish. Science
308,833
-838.
Gould, S. J. (2000). Of coiled oysters and big brains: how to rescue the terminology of heterochrony, now gone astray. Evol. Dev. 2,241 -248.[CrossRef][Medline]
Gregory, R. I., Yan, K. P., Amuthan, G., Chendrimada, T., Doratotaj, B., Cooch, N. and Shiekhattar, R. (2004). The Microprocessor complex mediates the genesis of microRNAs. Nature 432,235 -240.[CrossRef][Medline]
Griffiths-Jones, S. (2004). The microRNA
Registry. Nucleic Acids Res.
32,D109
-D111.
Grishok, A., Pasquinelli, A. E., Conte, D., Li, N., Parrish, S., Ha, I., Baillie, D. L., Fire, A., Ruvkun, G. and Mello, C. C. (2001). Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106, 23-34.[CrossRef][Medline]
Grosshans, H., Johnson, T., Reinert, K. L., Gerstein, M. and Slack, F. J. (2005). The temporal patterning microRNA let-7 regulates several transcription factors at the larval to adult transition in C. elegans. Dev. Cell. 8, 321-330.[CrossRef][Medline]
Han, J., Lee, Y., Yeom, K. H., Kim, Y. K., Jin, H. and Kim, V.
N. (2004). The Drosha-DGCR8 complex in primary microRNA
processing. Genes Dev.
18,3016
-3027.
Harfe, B. D., McManus, M. T., Mansfield, J. H., Hornstein, E.
and Tabin, C. J. (2005). The RNaseIII enzyme Dicer is
required for morphogenesis but not patterning of the vertebrate limb.
Proc. Natl. Acad. Sci. USA
102,10898
-10903.
Hatfield, S. D., Shcherbata, H. R., Fischer, K. A., Nakahara, K., Carthew, R. W. and Ruohola-Baker, H. (2005). Stem cell division is regulated by the microRNA pathway. Nature 435,974 -978.[CrossRef][Medline]
He, L., Thomson, J. M., Hemann, M. T., Hernando-Monge, E., Mu, D., Goodson, S., Powers, S., Cordon-Cardo, C., Lowe, S. W., Hannon, G. J. et al. (2005). A microRNA polycistron as a potential human oncogene. Nature 435,828 -833.[CrossRef][Medline]
Hipfner, D. R., Weigmann, K. and Cohen, S. M.
(2002). The bantam gene regulates Drosophila growth.
Genetics 161,1527
-1537.
Hobert, O., Johnston, R. J., Jr and Chang, S. (2002). Left-right asymmetry in the nervous system: the Caenorhabditis elegans model. Nat. Rev. Neurosci. 3, 629-640.[CrossRef][Medline]
Horvitz, H. R. and Sulston, J. E. (1980).
Isolation and genetic characterization of cell-lineage mutants of the nematode
Caenorhabditis elegans. Genetics
96,435
-454.
Horvitz, H. R., Sternberg, P. W., Greenwald, I. S., Fixsen, W. and Ellis, H. M. (1983). Mutations that affect neural cell lineages and cell fates during the development of the nematode Caenorhabditis elegans. Cold Spring Harb. Symp. Quant. Biol. 48,453 -463.[Medline]
Houbaviy, H. B., Murray, M. F. and Sharp, P. A. (2003). Embryonic stem cell-specific MicroRNAs. Dev. Cell 5,351 -358.[CrossRef][Medline]
Hutvagner, G., Simard, M. J., Mello, C. C. and Zamore, P. D. (2004). Sequence-specific inhibition of small RNA function. PLoS Biol. 2,E98 .[Medline]
Jing, Q., Huang, S., Guth, S., Zarubin, T., Motoyama, A., Chen, J., Di Padova, F., Lin, S. C., Gram, H. and Han, J. (2005). Involvement of microRNA in AU-rich element-mediated mRNA instability. Cell 120,623 -634.[CrossRef][Medline]
John, B., Enright, A. J., Aravin, A., Tuschl, T., Sander, C. and Marks, D. S. (2004). Human MicroRNA targets. PLoS Biol. 2,E363 .[CrossRef][Medline]
Johnson, S. M., Grosshans, H., Shingara, J., Byrom, M., Jarvis, R., Cheng, A., Labourier, E., Reinert, K. L., Brown, D. and Slack, F. J. (2005). RAS is regulated by the let-7 microRNA family. Cell 120,635 -647.[CrossRef][Medline]
Johnston, R. J. and Hobert, O. (2003). A microRNA controlling left/right neuronal asymmetry in Caenorhabditis elegans. Nature 426,845 -849.[CrossRef][Medline]
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.
Kidner, C. A. and Martienssen, R. A. (2005). The developmental role of microRNA in plants. Curr. Opin. Plant Biol. 8,38 -44.[CrossRef][Medline]
Kiriakidou, M., Nelson, P. T., Kouranov, A., Fitziev, P.,
Bouyioukos, C., Mourelatos, Z. and Hatzigeorgiou, A.
(2004). A combined computational-experimental approach predicts
human microRNA targets. Genes Dev.
18,1165
-1178.
Knight, S. W. and Bass, B. L. (2001). A role
for the RNase III enzyme DCR-1 in RNA interference and germ line development
in Caenorhabditis elegans. Science
293,2269
-2271.
Knight, S. W. and Bass, B. L. (2002). The role of RNA editing by ADARs in RNAi. Mol. Cell 10,809 -817.[CrossRef][Medline]
Knust, E. (1997). Drosophila morphogenesis: movements behind the edge. Curr. Biol. 7,R558 -R561.[CrossRef][Medline]
Knust, E., Schrons, H., Grawe, F. and Campos-Ortega, J. A.
(1992). Seven genes of the Enhancer of split complex of
Drosophila melanogaster encode helix-loop-helix proteins.
Genetics 132,505
-518.
Lagos-Quintana, M., Rauhut, R., Lendeckel, W. and Tuschl, T.
(2001). Identification of novel genes coding for small expressed
RNAs. Science 294,853
-858.
Lai, E. C. (2002). Micro RNAs are complementary to 3' UTR sequence motifs that mediate negative post-transcriptional regulation. Nat. Genet. 30,363 -364.[CrossRef][Medline]
Lai, E. C. (2004). Notch signaling: control of
cell communication and cell fate. Development
131,965
-973.
Lai, E. C. and Posakony, J. W. (1997). The
Bearded box, a novel 3' UTR sequence motif, mediates negative
post-transcriptional regulation of Bearded and Enhancer of split Complex gene
expression. Development
124,4847
-4856.
Lai, E. C., Burks, C. and Posakony, J. W.
(1998). The K box, a conserved 3' UTR sequence motif,
negatively regulates accumulation of enhancer of split complex transcripts.
Development 125,4077
-4088.
Lai, E. C., Bodner, R., Kavaler, J., Freschi, G. and Posakony,
J. W. (2000a). Antagonism of notch signaling activity by
members of a novel protein family encoded by the bearded and enhancer of split
gene complexes. Development
127,291
-306.
Lai, E. C., Bodner, R. and Posakony, J. W.
(2000b). The enhancer of split complex of Drosophila includes
four Notch-regulated members of the bearded gene family.
Development 127,3441
-3455.
Lai, E. C., Tam, B. and Rubin, G. M. (2005).
Pervasive regulation of Drosophila Notch target genes by GY-box-, Brd-box-,
and K-box-class microRNAs. Genes Dev.
19,1067
-1080.
Landthaler, M., Yalcin, A. and Tuschl, T. (2004). The human DiGeorge syndrome critical region gene 8 and Its D. melanogaster homolog are required for miRNA biogenesis. Curr. Biol. 14,2162 -2167.[CrossRef][Medline]
Lau, N. C., Lim, L. P., Weinstein, E. G. and Bartel, D. P.
(2001). An abundant class of tiny RNAs with probable regulatory
roles in Caenorhabditis elegans. Science
294,858
-862.
Leaman, D., Chen, P. Y., Fak, J., Yalcin, A., Pearce, M., Unnerstall, U., Marks, D. S., Sander, C., Tuschl, T. and Gaul, U. (2005). Antisense-mediated depletion reveals essential and specific functions of microRNAs in Drosophila development. Cell 121,1097 -1108.[CrossRef][Medline]
Lecellier, C. H., Dunoyer, P., Arar, K., Lehmann-Che, J.,
Eyquem, S., Himber, C., Saib, A. and Voinnet, O.
(2005). A cellular microRNA mediates antiviral defense in human
cells. Science 308,557
-560.
Lee, R. C. and Ambros, V. (2001). An extensive
class of small RNAs in Caenorhabditis elegans. Science
294,862
-864.
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.[CrossRef][Medline]
Lee, R., Feinbaum, R. and Ambros, V. (2004a). A short history of a short RNA. Cell 116,S89 -S96.[Medline]
Lee, Y., Ahn, C., Han, J., Choi, H., Kim, J., Yim, J., Lee, J., Provost, P., Radmark, O., Kim, S. et al. (2003). The nuclear RNase III Drosha initiates microRNA processing. Nature 425,415 -419.[CrossRef][Medline]
Lee, Y. S., Nakahara, K., Pham, J. W., Kim, K., He, Z., Sontheimer, E. J. and Carthew, R. W. (2004b). Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell 117,69 -81.[CrossRef][Medline]
Leviten, M. W., Lai, E. C. and Posakony, J. W.
(1997). The Drosophila gene Bearded encodes a novel small protein
and shares 3' UTR sequence motifs with multiple Enhancer of split
complex genes. Development
124,4039
-4051.
Lewis, B. P., Shih, I. H., Jones-Rhoades, M. W., Bartel, D. P. and Burge, C. B. (2003). Prediction of mammalian microRNA targets. Cell 115,787 -798.[CrossRef][Medline]
Lewis, B. P., Burge, C. B. and Bartel, D. P. (2005). Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120,15 -20.[CrossRef][Medline]
Lim, L. P., Glasner, M. E., Yekta, S., Burge, C. B. and Bartel,
D. P. (2003a). Vertebrate microRNA genes.
Science 299,1540
.
Lim, L. P., Lau, N. C., Weinstein, E. G., Abdelhakim, A., Yekta,
S., Rhoades, M. W., Burge, C. B. and Bartel, D. P.
(2003b). The microRNAs of Caenorhabditis elegans.
Genes Dev. 17,991
-1008.
Lin, M., Jones-Rhoades, M. W., Lau, N. C., Bartel, D. P. and Rougvie, A. E. (2005). Regulatory mutations upstream of mir-48, a C. elegans let-7 family microRNA cause developmental timing defects. Dev. Cell 9, 415-422.[CrossRef][Medline]
Lin, S. Y., Johnson, S. M., Abraham, M., Vella, M. C., Pasquinelli, A., Gamberi, C., Gottlieb, E. and Slack, F. J. (2003). The C elegans hunchback homolog, hbl-1, controls temporal patterning and is a probable microRNA target. Dev. Cell 4,639 -650.[CrossRef][Medline]
Lindsay, E. A. (2001). Chromosomal microdeletions: dissecting del22q11 syndrome. Nat. Rev. Genet. 2,858 -868.[CrossRef][Medline]
Llave, C., Kasschau, K. D., Rector, M. A. and Carrington, J.
C. (2002). Endogenous and silencing-associated small RNAs in
plants. Plant Cell 14,1605
-1619.
Lu, J., Getz, G., Miska, E. A., Alvarez-Saavedra, E., Lamb, J., Peck, D., Sweet-Cordero, A., Ebert, B. L., Mak, R. H., Ferrando, A. A. et al. (2005). MicroRNA expression profiles classify human cancers. Nature 435,834 .[CrossRef][Medline]
Lund, E., Guttinger, S., Calado, A., Dahlberg, J. E. and Kutay, U. (2003). Nuclear export of microRNA precursors. Science 303,95 -98.[Medline]
Mansfield, J. H., Harfe, B. D., Nissen, R., Obenauer, J., Srineel, J., Chaudhuri, A., Farzan-Kashani, R., Zuker, M., Pasquinelli, A. E., Ruvkun, G. et al. (2004). MicroRNA-responsive `sensor' transgenes uncover Hox-like and other developmentally regulated patterns of vertebrate microRNA expression. Nat. Genet. 36,1079 -1083.[CrossRef][Medline]
McManus, M. T. (2003). MicroRNAs and cancer. Semin. Cancer Biol. 13,253 -258.[CrossRef][Medline]
Meins, F., Jr, Si-Ammour, A. and Blevins, T. (2005). RNA silencing systems and their relevance to plant development. Annu. Rev. Cell Dev. Biol. (in press).
Meister, G., Landthaler, M., Dorsett, Y. and Tuschl, T.
(2004). Sequence-specific inhibition of microRNA- and
siRNA-induced RNA silencing. Rna
10,544
-550.
Mochizuki, K. and Gorovsky, M. A. (2005). A
Dicer-like protein in Tetrahymena has distinct functions in genome
rearrangement, chromosome segregation, and meiotic prophase. Genes
Dev. 19,77
-89.
Muljo, S. A., Ansel, K. M., Kanellopoulou, C., Livingston, D.
M., Rao, A. and Rajewsky, K. (2005). Aberrant T cell
differentiation in the absence of Dicer. J. Exp. Med.
202,261
-269.
O'Donnell, K. A., Wentzel, E. A., Zeller, K. I., Dang, C. V. and Mendell, J. T. (2005). c-Myc-regulated microRNAs modulate E2F1 expression. Nature 435,839 -843.[CrossRef][Medline]
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.[CrossRef][Medline]
Pasquinelli, A. E., McCoy, A., Jimenez, E., Salo, E., Ruvkun, G., Martindale, M. Q. and Baguna, J. (2003). Expression of the 22 nucleotide let-7 heterochronic RNA throughout the Metazoa: a role in life history evolution? Evol. Dev. 5, 372-378.[CrossRef][Medline]
Petersen, M. and Wengel, J. (2003). LNA: a versatile tool for therapeutics and genomics. Trends Biotechnol. 21,74 -81.[CrossRef][Medline]
Pfeffer, S., Zavolan, M., Grasser, F. A., Chien, M., Russo, J.
J., Ju, J., John, B., Enright, A. J., Marks, D., Sander, C. et al.
(2004). Identification of virus-encoded microRNAs.
Science 304,734
-736.
Pfeffer, S., Sewer, A., Lagos-Quintana, M., Sheridan, R., Sander, C., Grasser, F. A., van Dyk, L. F., Ho, C. K., Shuman, S., Chien, M. et al. (2005). Identification of microRNAs of the herpesvirus family. Nat. Methods 2, 269-276.[CrossRef][Medline]
Pillai, R. S., Bhattacharyya, S. N., Artus, C. G., Zoller, T., Cougot, N., Basyuk, E., Bertrand, E. and Filipowicz, W. (2005). Inhibition of translational initiation by let-7 microRNA in human cells. Science (in press).
Poy, M. N., Eliasson, L., Krutzfeldt, J., Kuwajima, S., Ma, X., Macdonald, P. E., Pfeffer, S., Tuschl, T., Rajewsky, N., Rorsman, P. et al. (2004). A pancreatic islet-specific microRNA regulates insulin secretion. Nature 432,226 -230.[CrossRef][Medline]
Provost, P., Silverstein, R. A., Dishart, D., Walfridsson, J.,
Djupedal, I., Kniola, B., Wright, A., Samuelsson, B., Radmark, O. and
Ekwall, K. (2002). Dicer is required for chromosome
segregation and gene silencing in fission yeast cells. Proc. Natl.
Acad. Sci. USA 99,16648
-16653.
Rajewsky, N. and Socci, N. D. (2004). Computational identification of microRNA targets. Dev. Biol. 267,529 -535.[CrossRef][Medline]
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.[CrossRef][Medline]
Reinhart, B. J., Weinstein, E. G., Rhoades, M. W., Bartel, B.
and Bartel, D. P. (2002). MicroRNAs in plants.
Genes Dev. 16,1616
-1626.
Rougvie, A. E. (2005). Keeping time with microRNAs. Development (in press).
Ruvkun, G., Wightman, B. and Ha, I. (2004). The 20 years it took to recognize the importance of tiny RNAs. Cell 116,S93 -S96.[Medline]
Shiohama, A., Sasaki, T., Noda, S., Minoshima, S. and Shimizu, N. (2003). Molecular cloning and expression analysis of a novel gene DGCR8 located in the DiGeorge syndrome chromosomal region. Biochem. Biophys. Res. Commun. 304,184 -190.[CrossRef][Medline]
Slack, F. J., Basson, M., Liu, Z., Ambros, V., Horvitz, H. R. and Ruvkun, G. (2000). The lin-41 RBCC gene acts in the C. elegans heterochronic pathway between the let-7 regulatory RNA and the LIN-29 transcription factor. Mol. Cell 5,659 -669.[CrossRef][Medline]
Smalheiser, N. R. and Torvik, V. I. (2005). Mammalian microRNAs derived from genomic repeats. Trends Genet. 21,322 -326.[CrossRef][Medline]
Stark, A., Brennecke, J., Russell, R. B. and Cohen, S. M. (2003). Identification of Drosophila microRNA targets. PLoS Biol. 1,E60 .[Medline]
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.[CrossRef][Medline]
Sullivan, C. S., Grundhoff, A. T., Tevethia, S., Pipas, J. M. and Ganem, D. (2005). SV40-encoded microRNAs regulate viral gene expression and reduce susceptibility to cytotoxic T cells. Nature 435,682 -686.[CrossRef][Medline]
Sulston, J. E. and Horvitz, H. R. (1977). Post-embryonic cell lineages of the nematode, Caenorhabditis elegans. Dev. Biol. 56,110 -156.[CrossRef][Medline]
Tanzer, A. and Stadler, P. F. (2004). Molecular evolution of a microRNA cluster. J. Mol. Biol. 339,327 -335.[CrossRef][Medline]
Tanzer, A., Amemiya, C. T., Kim, C. B. and Stadler, P. F. (2005). Evolution of microRNAs located within Hox gene clusters. J. Exp. Zoolog. B Mol. Dev. Evol. 304, 75-85.[Medline]
Watanabe, T., Takeda, A., Mise, K., Okuno, T., Suzuki, T., Minami, N. and Imai, H. (2005). Stage-specific expression of microRNAs during Xenopus development. FEBS Lett. 579,318 -324.[CrossRef][Medline]
Wienholds, E., Koudijs, M. J., van Eeden, F. J., Cuppen, E. and Plasterk, R. H. (2003). The microRNA-producing enzyme Dicer1 is essential for zebrafish development. Nat. Genet. 35,217 -218.[CrossRef][Medline]
Wienholds, E., Kloosterman, W. P., Miska, E., Alvarez-Saavedra,
E., Berezikov, E., de Bruijn, E., Horvitz, R. H., Kauppinen, S. and
Plasterk, R. H. (2005). MicroRNA Expression in Zebrafish
Embryonic Development. Science
309,310
-311.
Wightman, B., Ha, I. and Ruvkun, G. (1993). Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans.Cell 75,855 -862.[CrossRef][Medline]
Wurmbach, E., Wech, I. and Preiss, A. (1999). The Enhancer of split complex of Drosophila melanogaster harbors three classes of Notch responsive genes. Mech. Dev. 80,171 -180.[CrossRef][Medline]
Xie, X., Lu, J., Kulbokas, E. J., Golub, T. R., Mootha, V., Lindblad-Toh, K., Lander, E. S. and Kellis, M. (2005). Systematic discovery of regulatory motifs in human promoters and 3' UTRs by comparison of several mammals. Nature 434,338 -345.[CrossRef][Medline]
Xu, P., Vernooy, S. Y., Guo, M. and Hay, B. A. (2003). The Drosophila MicroRNA Mir-14 Suppresses Cell Death and Is Required for Normal Fat Metabolism. Curr. Biol. 13,790 -795.[CrossRef][Medline]
Yekta, S., Shih, I. H. and Bartel, D. P.
(2004). MicroRNA-directed cleavage of HOXB8 mRNA.
Science 304,594
-596.
Yi, R., Qin, Y., Macara, I. G. and Cullen, B. R.
(2003). Exportin-5 mediates the nuclear export of pre-microRNAs
and short hairpin RNAs. Genes Dev.
17,3011
-3016.
Zhao, Y., Samal, E. and Srivastava, D. (2005). Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis. Nature 436,214 -220.[CrossRef][Medline]