Review articles
Control of developmental timing
by small temporal RNAs: a
paradigm for RNA-mediated
regulation of gene expression
Diya Banerjee and Frank Slack*
Summary
Heterochronic genes control the timing of developmental
programs. In C. elegans, two key genes in the heterochronic pathway, lin-4 and let-7, encode small temporally
expressed RNAs (stRNAs) that are not translated into
protein. These stRNAs exert negative post-transcriptional regulation by binding to complementary sequences in the 30 untranslated regions of their target
genes. stRNAs are transcribed as longer precursor RNAs
that are processed by the RNase Dicer/DCR-1 and
members of the RDE-1/AGO1 family of proteins, which
are better known for their roles in RNA interference
(RNAi). However, stRNA function appears unrelated to
RNAi. Both sequence and temporal regulation of let-7
stRNA is conserved in other animal species suggesting
that this is an evolutionarily ancient gene. Indeed,
C. elegans, Drosophila and humans encode at least 86
other RNAs with similar structural features to lin-4 and
let-7. We postulate that other small non-coding RNAs
may function as stRNAs to control temporal identity
during development in C. elegans and other organisms. BioEssays 24:119±129, 2002.
ß 2002 Wiley Periodicals, Inc.; DOI 10.1002/bies.10046
Introduction
The development of multicellular organisms occurs in four
dimensions, the three axes of space and a fourth axis of time.
Spatial patterning is controlled by groups of genes dedicated to
each of the three spatial axes, anterior±posterior, dorsal±
ventral and left±right. For example, the Hox genes direct
pattern formation along the anterior±posterior axis.(1,2) While
a great deal is known about the fundamental mechanisms of
spatial patterning, temporal patterning during development is
not as well understood. However, heterochronic mutations
that alter the relative timing of developmental events reveal
that the temporal dimension of development is explicitly under
genetic control as well. Heterochronic mutations have been
Department of Molecular, Cellular and Developmental Biology, Yale
University.
*Correspondence to: Frank Slack, Department of Molecular, Cellular
and Developmental Biology, Yale University, 266 Whitney Ave,
New Haven, CT 06520, USA. E-mail: frank.slack@yale.edu
BioEssays 24:119±129, ß 2002 Wiley Periodicals, Inc.
identified in a number of organisms including the slime mould
Dictyostelium discoideum,(3) the nematode C. elegans,(4±6)
Drosophila melanogaster,(7) and in plant species such as
Arabidopsis thaliana and Zea mays.(8±10) The genes and
mechanisms of developmental timing have been studied most
extensively in C. elegans, in which a pathway of heterochronic
genes has been found to control the timing of cell fate
determination during postembryonic development. However,
many of the molecules and mechanisms of temporal developmental control first described in C. elegans are conserved
across animal phylogeny, suggesting that similar pathways
are widespread.(11,12)
In this review, we summarize the identities of and interactions among the molecules involved in temporal control of
development in C. elegans. In particular, we focus on two
unusual gene products, the small temporal RNAs (stRNAs) lin4 and let-7.(13,14) which downregulate translation of their
targets by binding to the 30 untranslated regions (UTRs) of their
target mRNAs.(13,15±17) Because stRNAs generate a temporal
cascade of key regulators that are responsible for developmental patterning, they function as molecular ringmasters that
regulate the timing of postembryonic development in C.
elegans. Here we review recent progress in understanding
how stRNAs are regulated and how they exert post-transcriptional regulation on target genes.
Heterochrony in evolution
and development
Heterochrony is an evolutionary term that describes situations
where ancestor and descendant species differ from one
another in the relative timing of developmental events.(18) A
common heterochronic variation is alteration of the time at
which an organism attains sexual maturity compared to its
ancestor. A classic example of this is an aquatic salamander,
the Mexican axolotl, which becomes sexually mature without
undergoing the final metamorphosis to the land-borne adult
form of its ancestor species.(19) Just as heterochrony is
observed in phylogenic variation, mutants can be isolated in C.
elegans that display heterochrony. These mutants express
cell fates, and hence form organs, either too early or too late
relative to wild-type animals.
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For the majority of animals, spatial pattern is laid down over
time and hence spatial identity is often a result of the temporal
sequence of patterning events. The key role that developmental time plays in pattern formation is illustrated in the
exquisite series of heterochronic grafting experiments performed by Summerbell et al.(20) When the tips of young chick
limb buds are grafted onto older limb buds, the limbs develop
with reiterations of limb segments along the proximal±distal
(shoulder to fingers) axis, i.e. these limbs develop with two
consecutive sets of humerus, radius, and ulna bones (Fig. 1).
In the reciprocal heterochronic graft, old limb buds are grafted
onto young limb buds and the limbs develop with deletion of
segments along the proximal±distal axis, i.e. these limbs develop with a humerus immediately followed by digits, deleting
the radius and ulna. The proximal±distal axis of the limb
develops over time with the proximal elements being produced
first and the distal elements last. Undifferentiated cells in the
progress zone divide under the influence of fibroblast growth
factors (FGFs) produced from the apical epidermal ridge, the
most distal structure in the limb bud. As their daughter cells
move away from the FGF signal, they differentiate into limb
elements.(21±23) The progress zone model proposes that the
Figure 1. Heterochronic mutations are the temporal equivalents of the spatial homeotic mutations. A: The cell lineage pattern of C.
elegans cell T in wild-type animals versus a lin-4 loss-of-function (lf ) mutant. The lin-4 (lf ) mutation results in temporal misregulation of
cell fate patterns, such that cell fates characteristic of the L1 stage (in red) are reiterated in the L2. B: Compared to wild-type Drosophila
body pattern, the ultrabithorax mutant shows a similar case of misregulation of cell fates, but along a spatial rather than temporal axis,
resulting in the structural duplication of the thorax (in red). C: In heterochronic grafting experiments, when the tip of a young chick limb
bud is grafted onto an older bud, the limb develops with a duplication of the humerus (in red), radius and ulna (in blue), compared to
single incidence of the bone pattern during normal chick limb development.
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length of time that a progenitor cell spends in the progress
zone dictates which proximal±distal fates its daughters will
assume. Thus, spatial patterning in the proximal±distal axis
can be thought of as a consequence of temporal patterning
because the specification of each limb element is dependent
on the relative age of the progenitor cell in the progress zone.
Proximal elements are derived from daughters of younger
progenitor cells and distal elements are derived from
daughters of older progenitor cells.
Another example of dependence on time for correct spatial
patterning can be found during anterior±posterior patterning
by Hox genes in vertebrates. Hox genes are arranged in linear
clusters in which the physical order of individual Hox genes
along the DNA correlates with their time of expression as well
as their spatial domains of expression along the anterior±
posterior axis. As cell proliferation progresses in the posteriorly migrating primitive streak, cells that are derived from
developmentally younger progenitors become anteriorly
located and express genes in the Hox cluster that are located
near the 30 end of the cluster. More posteriorly located cells
derived from older progenitors express genes closer to the 50
end of the cluster. This correlative relationship, known as
``colinearity'', emphasizes the intimacy of the relationship
between developmental space and time.(24,25)
The observation of Hox gene colinearity raises the
possibility that temporal and spatial patterning pathways
may share common mechanisms and genes. A first hint of
this possibility is the recent observation that hunchback and
kruppel, two well-known regulators of spatial identity in
Drosophila embryogenesis, are also required for temporal
identity of neurons.(26)
Temporal boundaries and segment identities
Heterochronic genes can be thought of as the temporal
equivalents of the homeotic spatial patterning genes. While
homeotic mutations result in alterations as to where particular
cell fates are expressed, heterochronic mutations result in
temporal transformations of cell fate, that is, changes in when
a particular cell fate is expressed (Fig. 1). Both sets of genes
generate graded levels of morphogens that modify a basic
reiterated pattern of segments. In Drosophila, spatial patterning involves expression of segmentation genes defining the
segment boundaries in the early embryo, followed by specification of segment identity by the homeotic genes. Similarly,
one can define two broad classes of developmental timing
genes, temporal identity genes that affect the fate choices that
a cell makes at a specific time and temporal boundary genes
that set the pace of development, for example, the genes that
control the timing of molting. The C. elegans heterochronic
mutations identified thus far transform temporal cell fate
identity without appreciably affecting the periodicity of progression through the larval stages. These mutations thus
define temporal identity genes. The larval molting cycle is
unaffected by the known heterochronic mutations in C.
elegans, suggesting the existence of separate unidentified
pathways that temporally regulate developmental boundary
formation. In contrast to C. elegans, heterochronic mutations
in Drosophila have almost exclusively defined temporal
boundary genes that are regulated by the steroid hormone
ecdysone. Although heterochronic mutant phenotypes that
are altered for the timing of cell fate decisions have not been
identified in flies, homologs of C. elegans heterochronic genes
exist in the Drosophila genome.(7,12) The function of these
Drosophila homologs in control of developmental timing
remains to be demonstrated.
The role of heterochronic genes
in C. elegans development
C. elegans developmental progression can be grouped into six
stagesÐembryogenesis and the four larval stages (L1 to L4)
that culminate in the reproductively mature adult. The invariant
cell lineages formed during larval development show stagespecific patterns of cell-division and cell-fate determination
(Fig. 2). Mutations in heterochronic genes result in temporal
alterations of these stage-specific patterns of cellular development. In general, heterochronic mutations affect a large
subset of the cells that are involved in morphogenesis during a
particular larval stage, thus affecting a variety of tissues. In
precocious mutants, cells inappropriately express later cell
fates during early stages while, in retarded mutants, cells
reiterate earlier stage fates instead of later wild-type cell fates.
For example, lin-41 mutations cause the precocious expression of adult fates in the L4 stage, while let-7 mutations cause
the opposite phenotype, where L4 fates are expressed
inappropriately at the adult stage (Fig. 2).
The stRNAs of the heterochronic pathway
The C. elegans heterochronic pathway encompasses a
diverse range of gene products (Table 1). The genetic and
molecular interactions among these various genes have not
been fully worked out, but epistasis analysis has ordered the
better-characterized heterochronic genes into the pathway
proposed in Fig. 3A. The detailed genetic interactions among
these heterochronic genes have been reviewed elsewhere.(5,6) Here we focus on two key components of the
pathway, the small temporal RNA genes lin-4, which controls
cell fate transitions through early stages of larval development
(L1/L2), and let-7, which controls later transitions (L4/adult).
lin-4 and let-7 produce precursor transcripts of around 70
nucleotides that are predicted to form stem±loop structures
(Fig. 4). The mature 21 nucleotide, single-stranded, stRNAs
are processed from these larger precursors.(11,13,14,27)
stRNAs are not translated and function to repress their target
genes, which are lin-14 and lin-28 for lin-4, and lin-41 for let-7.
Post-transcriptional repression is achieved by binding of
the stRNA to complementary 30 UTR sites on the target
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Figure 2. Cell-lineage pattern defects associated with
C. elegans heterochronic mutations. One convenient
tissue with which to examine the role of heterochronic
genes is the epidermal layer or hypodermis, which is
responsible for synthesizing and secreting the cuticle.
The cell-lineage pattern of one representative hypodermal cell (V1) in wild-type and heterochronic loss-offunction (lf ) mutant animals is shown. The lateral
hypodermal seam cells divide with a stem-cell-like
pattern during the larval stages and then terminally
differentiate during the transition from L4 to adult
(represented by the three horizontal bars). The dashed
line represents continued reiterations of the lineage
pattern. The vertical axis represents time. Similar
alterations in specificity of temporal identity occur
coordinately in somatic cell lineages all over the animal.
However, neither embryonic development nor development of the post-embryonic reproductive germline
appear to be affected by mutations in the known
heterochronic genes.
transcripts.(12,13,15,16) Based on RNA and protein expression
data, the temporal patterning of the stRNAs and their targets
may be modeled as shown in Fig. 3B. The temporal identity for
each stage appears to be determined by the relative levels of
these stRNAs and their targets. For example, L1 stage fates
appear to be maintained by low levels of lin-4 stRNA and high
levels of LIN-14 protein, a repressor of L2 fates. Acquisition of
L2 fates is triggered by LIN-14 levels decreasing below a
threshold level, due to its post-transcriptional inhibition by
rising levels of lin-4 stRNA. The L1 reiteration caused by lin-4
loss-of-function mutations is due to continued expression of
LIN-14 in the absence of lin-4 stRNA. Thus, L2 fates remain
repressed by the presence of LIN-14 and L1 fates are
reiterated. Conversely, the precocious L2 phenotype of lin14 loss-of-function is due to the premature loss of LIN-14
repression of L2 fates. Similarly, the correct transition from L4
to adult fates appears to be triggered by falling levels of LIN-41
protein, due to its inhibition by rising levels of let-7 stRNA. Cells
appear to read out the levels of these key regulators allowing
them to behave in a stage-appropriate manner. We hypothesize that additional, as yet unidentified, small non-coding
RNAs may function in the pathway to specify the temporal
identity of other developmental stages, such as cell fate
transitions of the embryonic to L1 or L3 to L4 stages (Fig. 3).
Mechanism of post-transcriptional
repression by stRNAs
lin-4 and let-7 stRNAs do not share similarity in either their
transcribed or upstream sequences. Their target complementary sequences also vary. However, one commonality
Table 1. C. elegans heterochronic genes
Gene
LCE*
LCS**
alg-1
alg-2
daf-12
dcr-1
None
None
None
None
2
None
3
None
let-7
lin-4
lin-14
lin-28
lin-29
lin-41
lin-42
n/a
n/a
7
1
None
1
None
n/a
n/a
3
1
None
2
None
Product
PIWI and PAZ domain proteins, homologues of C. elegans gene rde-1
required for RNA interference(27)
NHR (Nuclear Hormone Receptor) transcription factor(55)
Rnase III, PAZ domain containing homologue of Drosophila gene Dicer required for RNA
interference(27)
21 nucleotide small temporal RNA(14)
21 nucleotide small temporal RNA(13)(56)
Novel nuclear factor(57)(58)
Probable RNA-binding protein(16)
Zinc ®nger transcription factor(59)(60)
RBCC (RING-B-Box-Coiled-coil) protein(12)
PAS domain protein, homologue of Drosophila circadian timing gene period(61)
*LCE, number of lin-4 complementary elements in 30 UTR of indicated gene.
**LCS, number of let-7 complementary sites in 30 UTR of indicated gene.
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Figure 3. Model of the heterochronic pathway and temporal expression of the stRNAs and the protein products of their target genes.
A: A proposed heterochronic pathway based on genetic interactions among C. elegans temporal identity genes and on their mutant
phenotypes. The heterochronic pathway has been superimposed upon a time scale. The boundaries between postembryonic
developmental phases are marked by moults. Arrows represent positive regulation, (\) represents negative regulation. For example, lin4 negatively regulates both lin-14 and lin-28, and lin-14 negatively regulates L2 stage fates. It is likely that lin-41 represses adult fates,
although this has not been rigorously established. Based on interactions among defined heterochronic genes in the L1, L2 and L4
stages, we postulate the presence of at least two other heterochronic genes (indicated by ?s) that would function to define L3 identity.
These unidentified heterochronic genes would likely negatively regulate genes that function downstream from them in the heterochronic
pathway. B: The expression pattern of lin-4 and let-7 stRNAs and the protein products of their target genes. The vertical axis represents
the levels of stRNA or protein, and the horizontal axis represents time. Rising levels of lin-4 and let-7 stRNA, and falling levels of LIN-14
and LIN-41 permit cell fate transitions from L1 to L2, and from L4 to adult stages respectively. LIN-14 and LIN-41 are expressed in
embryos but, as indicated by the broken red and orange lines, it is not clear when expression of these two proteins begins during the
embryonic stage. It has also not been determined how long lin-4 and let-7 expression is maintained after transition to the adult stage, as
indicated by the broken green and blue lines. We hypothesize the existence of different stRNAs in addition to lin-4 and let-7, which would
function to specify the temporal identities of the other stages. The presence and expression of these undiscovered stRNAs is indicated
by the black broken lines at the appropriate developmental stages (i.e embryo, L2 and L3).
between the stRNAs is that complementary sequences to
each stRNA are found in the 30 UTRs of their target genes. lin14 contains seven lin-4 stRNA complementary elements
(LCE) in its 30 UTR and lin-41 contains two let-7 stRNA
complementary sites (LCS) in its 30 UTR (Table 1 and Fig. 5).
Reporter genes linked to the lin-14 or lin-41 30 UTRs are
regulated like endogenous genes, that is, reporter expression
is downregulated at the appropriate larval stage in a lin-4- or
let-7-dependent manner. This regulation is lost when the
stRNA complementary sites are deleted, underscoring the
functional importance of the 30 UTR sites in regulation
by stRNAs.(12,14,28) Some of the duplexes that are predicted
to form between stRNAs and their complementary sites are
shown in Fig. 5. These RNA hybrids form imperfect duplexes
with unpaired nucleotides forming bulges. Interestingly, sitedirected mutagenesis has defined the single bulged cytosine
(C) residue on lin-4 as being essential for post-transcriptional
regulation of lin-14 mRNA by lin-4.(28) The two most stable
forms of the predicted let-7/lin-41 duplexes contain adenine-
uridine (AU) bulges that we hypothesize may be similarly
important for let-7 function.(12) The nucleotide bulges may
provide recognition sequences for RNA-binding proteins that
function in translational control. While the formation of stRNA/
mRNA duplexes can be demonstrated in vitro (Ref. 28; E. Choi
and F. Slack, unpublished data), their formation remains to be
experimentally verified in vivo. lin-4 stRNA complementary
sites have also been found in lin-41, and let-7 stRNA
complementary sites have been found in lin-14, lin-28 and
daf-12, but the relevance of these sites is unclear.(14) No
changes in LIN-14 and LIN-28 levels are detectable in a let-7
loss-of-function mutant suggesting that the level of regulation
by let-7 stRNA is subtle, if present at all.(14)
While the presence of the 30 UTR complementary sites
immediately suggests that the stRNAs exert their effect by
binding to and inhibiting their target genes, the exact
mechanism of repression is not known. Regulatory control
by stRNAs is thought to occur at the level of translation rather
than RNA stability, because the mRNA levels of lin-14 and
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Figure 4. Secondary structures of lin-4 and let-7 stRNA
precursor transcripts.Both lin-4 and let-7 stRNAs are transcribed as 70 nucleotide precursor transcripts that are
predicted to adopt the secondary structures shown. The
sequences of the mature 21 nucleotide lin-4 and let-7 stRNAs
are shown shaded. lin-4 stRNA is shown as being 21
nucliotides long based on the findings of Lau et al. (Ref 33).
lin-28 remain unchanged while their protein levels steadily
decrease with increasing levels of lin-4.(17) lin-14 mRNA
remains associated with polyribosomes suggesting that
regulation occurs after the initiation of translation.(17) One
uninvestigated possibility is that stRNAs may modify their
mRNA targets in a manner similar to that of small nucleolar
RNAs (snoRNAs), which post-transcriptionally regulate their
targets by directing methylation and pseudouridylation.(29)
Conservation of let-7 stRNA
While the mode of action of stRNAs is unclear, recent data
indicate that stRNA-mediated regulation might be widespread.
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Genome sequence comparisons and expression analyses
have revealed that let-7 is conserved across a wide range of
animal species, although it is not found in plants or unicellular
organisms.(11) Drosophila has one exact match to the
C. elegans let-7 stRNA while humans have at least three
exact matches, as well as several sequences with just
one nucleotide difference from the C. elegans let-7 stRNA.
Furthermore, the predicted Drosophila and human let-7 precursor sequences can form stem±loop structures similar to
the C. elegans nascent transcript. As in C. elegans, the mature
21 nucleotide transcripts are easily detected in human and
Drosophila, while the longer pre-let-7 RNAs are rare.(11)
The remarkable level of let-7 sequence conservation
across evolutionarily distant phyla places it among the most
highly conserved molecules, with similar levels of conservation found only in ribosomal RNA and small nucleolar RNAs.(30)
We hypothesize that the striking degree of conservation is due
to multiple levels of constraints on sequence divergence away
from the let-7 stRNA consensus. Such constraints include the
need for complementarity to its cognate stem partner sequence, the need for complementarity to its target sequence, and
for maintenance of recognition motifs for both processing
factors and other cofactors that may function with the mature
let-7 stRNA. Alternatively, the sequence could encode the only
solution to some catalytic function of the RNA.
Not only let-7 stRNA sequence but also temporal regulation
of its expression is widely conserved.(11) In C. elegans, let-7
expression begins during L3, rises and plateaus in L4 and is
maintained through the adult stage. Similarly, in Drosophila
let-7 stRNA is absent until just before metamorphosis and then
increases during early pupal stages, after which its level is
maintained until adulthood. Even in vertebrates, which do not
undergo larval development, let-7 displays temporal regulation.
For example, in zebrafish let-7 expression begins 24 to 48 hours
after fertilization and continues through the adult stages. let-7
stRNA may thus regulate the timing of later stages of animal
development. Consistent with this pattern, the lowest level of
let-7 expression in human tissues was found in bone marrow,
which largely consists of immature and undifferentiated cells.(11)
In addition to the widespread conservation of let-7
sequence and temporal expression pattern, the sequence of
its target, lin-41, is also conserved in Drosophila, zebrafish and
mouse.(12) The Drosophila and zebrafish lin-41 homologs
carry let-7 complementary sites (LCS) in their 30 UTRs.(11)
Conservation of the larger precursor transcripts, temporal
patterns of expression, and lin-41 sequence conservation and
presence of LCSs in the 30 UTRs together indicate that the
regulatory pathways involving stRNAs in C. elegans are
evolutionarily ancient. Furthermore, it strongly suggests that
let-7 homologs are involved in temporal patterning across
metazoansÐa tantalizing idea that remains to be tested.
Just as there may remain unidentified stRNAs that function
in the C. elegans heterochronic pathway (Fig. 3), there may
Review articles
Figure 5. Predicted duplexes formed between stRNAs
and their target sequences. Examples of duplexes
predicted to form between lin-4 and let-7 stRNAs and
their target mRNAs and their relative positions in the 30
UTRs are shown. The bulged C that is essential for lin-4
function is shown in red. The AU bulges that are
predicted to form on pairing of let-7 with its target mRNA
are also shown in red. The lin-4 bulged C and let-7
bulged AU are found in many of the duplexes.
also be additional stRNAs that are involved in specifying the
temporal identities of various developmental stages in other
animals (by a combination of approaches such as comparative
genomics and molecular biology). Numerous small noncoding RNAs with unassigned function have been discovered
in C. elegans, Drosophila and vertebrate species.(31 ±34) These
are excellent candidates for new stRNAs. Indeed, a subset of
these non-coding RNAs, called micro RNAs (miRNA), share
some of the characteristics of stRNAs. For example, each
appears to be expressed as a pre-miRNA that forms a stemloop structure, which is then processed to a 20- to 24nucleotide mature RNA. Additionally, transcription of some of
the miRNAs is temporally regulated.
As a caveat to the hypothesis of stRNA conservation in
developmental timing pathways, it should be noted that
homologs of neither lin-4 nor its target lin-14 have been found
in non-nematode species, either by sequence or expression
analysis.(11) Although the lack of lin-4 homologs may indicate
that lin-4 stRNA is unique to nematode species, it is also
possible that lin-4 in other species has diverged substantially
from the C. elegans molecule but retains functional homology,
perhaps through a conserved secondary structure.
Searching for non-coding RNAs
Small non-coding RNAs have been difficult to identify because
most genomic and genetic screens for new genes are biased
against their discovery. These genes are very small and thus
present a difficult target for mutagenesis. Additionally, they are
immune to frame-shift or nonsense mutations, and are
sometimes present in multiple redundant copies.(30) Moreover, using primary alignment methods such as BLAST is
often not informative, since many small RNAs have conserved
secondary structure rather than primary sequence, as illustrated by the lack of sequence similarity between lin-4 and let-7.
Given these limitations, one powerful approach to searching
for other small miRNAs that might be involved in developmental temporal patterning involves sophisticated algorithms
that look for stem±loop structures of approximately 60 to 70
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nucleotides, where sequence of one arm of the stem is complementary to a sequence in the UTR of another gene. Comparative genome analysis is another approach. In genomes
that have diverged sufficiently, alignments show that functional regions stand out as islands of conservation, thus
revealing regions that are under selective pressure, such as
stRNA genes.(30,34) Model organisms in different phyla are too
far diverged to be useful for such comparative genome analysis, but completion of the C. briggsae and mouse genomes in
the near future will prove invaluable for comparison to C.
elegans and human genomes, respectively. Biochemical
approaches using fractionation to isolate small RNAs have
also proven very effective, leading to the identification
of approximately a hundred small non-coding RNAs in C.
elegans and other animals.(31±34)
Intersection of the RNAi and
the heterochronic pathways through Dicer
The C. elegans heterochronic pathway is not saturated by
mutagenesis. Traditional forward and reverse genetic screens,
as well as identification of interactors and downstream targets
by microarray analysis, are fleshing out the basic pathway. In
addition to these strategies, some researchers have sought to
understand the regulation and mechanism of stRNAs by
looking for conserved mechanisms with other biological
phenomena that involve post-transcriptional repression by
small non-coding RNAs, such as the phenomenon of RNA
interference in C. elegans. Indeed, recent findings, detailed
below, point to a tight connection between stRNA and RNAi
molecular machineries.
RNA interference (RNAi) is a form of post-transcriptional
gene silencing (PTGS) in which introduced double-stranded
RNA can cause sequence-specific silencing.(35) The mechanistic paradigm for PTGS is that the double-stranded RNA is
processed to small 21 to 23 nucleotide guide RNAs, which
direct an RNA±protein complex to the complementary target
mRNA sequence, leading to degradation of the mRNA.(36±39)
In Drosophila, the ribonuclease III-like enzyme Dicer appears
to process longer double-stranded RNA into the small RNAs
that guide mRNA destruction.(38,39) These 21- to 23-nucleotide
double-stranded guide RNAs, called small interfering RNAs
(siRNAs), are similar in size to the lin-4 and let-7 stRNAs,
which also negatively regulate their targets, albiet through
translational repression. The structural and possible mechanistic similarities between siRNAs in RNAi and the stRNAs in
developmental timing suggest the possibility that RNAi pathway components may be involved in the heterochronic
pathway. Lending credence to this possibility is the observation that the Arabidopsis ortholog of Dicer, SIN-1/CARPEL
FACTORY (sin1/caf-1), is required for normal floral development.(40) Similarities between the mechanisms of RNAi and of
developmental timing have now been reported by several
groups.
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Grishok et al.(27) followed by Ketting et al.(41) demonstrated
that the C. elegans ortholog of Drosophila Dicer, dcr-1, plays a
role in both RNAi and heterochronic pathways. RNAi of dcr-1
results in reduced RNAi of a second targeted gene, implicating
dcr-1 in the RNAi pathway in C. elegans. RNAi of dcr-1 also
results in a retarded heterochronic phenotype resembling lin-4
and let-7 loss-of-function mutations. Both research groups
tested whether dcr-1 is involved in the heterochronic pathway
as an essential player upstream of the lin-4 and let-7 stRNAs.
They showed that processing of both lin-4 and let-7 stRNAs
from their larger precursor transcripts is dependent on dcr-1
activity. These findings in C. elegans have been corroborated
by Hutvagner et al.(42) who have shown that the human
ortholog of Dicer, Helicase-MOI, is required for processing of
the let-7 RNA precursor in cultured human cells. Thus, the
regulatory interactions defined in C. elegans appear to be
conserved in higher animals.
Grishok et al.(27) have further shown that expression of a
reporter gene linked to the lin-14 30 UTR failed to be downregulated when dcr-1 expression was inhibited by RNAi.
Additionally, lin-14 and lin-41 mutations suppressed the
retarded heterochronic phenotype caused by inhibition of
dcr-1, just as the retarded heterochronic phenotype caused by
lin-4 and let-7 loss-of-function mutations can be suppressed
by lin-14 and lin-41 mutations. In similar experiments, Ketting
et al.(41) found that lin-41 is epistatic to dcr-1. Taken together
these results support the idea that dcr-1 is required for
generation of the mature lin-4 and let-7 stRNAs that regulate
target genes to control temporal development.
Different RDE-1/AGO1 family members function
in heterochronic development versus RNAi
Members of the rde-1 (for RNAi defective)/ago1 (argonaute)
gene family function in RNAi and other forms of gene silencing.
Recent findings have shown that members of this family are
also involved in the stRNA pathway. In C. elegans, null
mutations of rde-1 result in insensitivity to RNAi but no other
discernable phenotype, indicating that rde-1 is an essential
gene for RNAi.(43) rde-1 belongs to a large gene family whose
products are well conserved in structure, as well as genesilencing function, in various eukaryotic organisms.(44,45) One
family member, Drosophila argonaute2 (ago2), is a subunit of
the enzyme complex, RISC (RNA-induced silencing complex),
which degrades targeted mRNAs.(46) Many of the rde-1 family
of genes also act in developmental pathways and commonly
function to regulate germ-cell and stem-cell function. For
example, in Drosophila, a close relative of ago2, argonaute1
(ago1), is required for embryogenesis,(47) piwi is required for
maintenance of the germline stem-cell population,(48) and
aubergine/sting is required for proper expression of the germline determinant Oskar.(49,50) In Arabidopsis, argonaute (ago1)
which is required for PTGS,(45) is also required along with
pinhead/zwille for stem-cell patterning of the plant meristem.(51)
Review articles
C. elegans has 24 rde-1 homologs, of which 14 were
examined by Grishok et al.(27) for a role in developmental
pathways using RNAi. Two C. elegans rde-1 homologs, alg-1
and alg-2 (for argonaute like genes) were found to cause
retarded heterochronic phenotypes like those of lin-4 and let-7
loss-of-function mutations and by RNAi of dcr-1. Stagespecific downregulation of a reporter gene linked to the 30
UTR of lin-41 was prevented by inhibition of alg-1 and alg-2.
Moreover, the heterochronic mutations caused by alg-1 and
alg-2 were suppressed by lin-14 and lin-41 mutations,
suggesting roles for alg-1 and alg-2 in the heterochronic
pathway. Additionally, alg-1 and alg-2 were shown to be
essential for accumulation of normal levels of mature lin-4 and
let-7 stRNAs. However, unlike the case with dcr-1, RNAi is not
dependent on alg-1 or alg-2 function in C. elegans. Interestingly,
we have identified two potential let-7 complementary sites in the
30 UTR of alg-1, suggesting that it may be a target for let-7.
RDE-1 family proteins as specificity factors
Despite the common involvement of small RNAs, Dicer and
RDE-1/AGO1 family members in both RNAi and heterochronic
pathways, there are obvious differences between the pathways. The most patent difference is in regards to the output of
each pathway, namely that of RNA degradation with
siRNAs(52) versus translational inhibition with stRNAs.(17) It
has been proposed that distinct members of the large RDE-1
family in C. elegans may provide specificity for RNAi versus
stRNA pathways(27) (Fig. 6). For example, Dicer may be
targeted to double-stranded RNA by RDE-1 in the RNAi
pathway, or to stRNA precursors via ALG-1 and ALG-2 in the
heterochronic pathway. The RDE-1 family molecules may
associate with small RNAs and provide specificity to insure that
they are targeted to the appropriate downstream complex,
mediating the decision between mRNA destruction and translation inhibition. Furthermore, since 21 nucleotide single-strand
RNAs that are transcribed in vitro are unstable and rapidly
degraded,(42) RDE-1 molecules may also function to stabilize
and prevent degradation of the 21 nucleotide stRNAs in vivo.
Another significant difference between RNAi and heterochronic pathways is that only one strand of the RNA duplex is
stable after stRNA processing. Maturation of let-7 is asymmetric, resulting in only let-7 stRNA and no complementary
RNA fragments, i.e antisense let-7.(11,42) In contrast, processing of precursor RNA in the RNAi pathway is symmetric,
yielding 21- to 23-nucleotide double-stranded RNA.
Finally, siRNAs can target complementary sequences
anywhere in the mature mRNA, while stRNAs pair with specific
sites in the 30 UTRs of their target gene transcripts. Grishok
et al.(27) propose that flanking sequences for stRNA-binding
sites could provide for a context-specific modification that
allows inhibition of translation rather than mRNA destruction.
The bulged C in the theoretical lin-4/lin-14 duplex could be
critical for either duplex stability or translational inhibition.(28)
The predicted stRNA/mRNA duplexes form bulged structures
Figure 6. Model showing the involvement of the
Dicer homologue, DCR-1, and RDE-1/AGO-1 family
proteins in developmental temporal patterning and
RNAi pathways in C. elegans. DCR-1 activity is
required for processing of both double-stranded RNA
and the 70 nucleotide stRNA precursors into the 21
to 23 nucleotide small interfering or small temporal
RNAs, respectively. The RDE-1/AGO1 family of
proteins is represented by filled ovals (green for those
specific to developmental temporal patterning pathways and blue for those specific to RNAi pathways).
The RDE-1 family of proteins is postulated to confer
specificity in the two pathways in a number of possible
ways: RDE-1 and ALG-1/ALG-2 may function to
recognize the similarly structured precursor RNA
molecules and guide them to DCR-1. RDE-1 and
ALG-1/ALG-2 or other members of the family also may
associate with the mature RNAs and prevent their
degradation and/or guide them to their specific mRNA
targets. The choice between translational inhibition
and degradation could be directed by specific factors,
possibly RDE-1/AGO1 family members, that recognize
the divergent structures formed by stRNAs and
siRNAs binding to their target mRNAs.
BioEssays 24.2
127
Review articles
due to imperfect stRNA complementarity to the target sequence, and could thus allow access to sequence-specific
RNA-binding proteins (stRNPs, perhaps members of the
RDE-1 family) or might reduce the affinity with which a
nuclease could cleave the mRNA/stRNA hybrid.
The hypothesis that RDE-1 family molecules act to provide
specificity in different pathways has been put forward based on
the pleiotropic phenotypes of loss-of-function mutations of
rde-1/ago1 family members.(27,45,49 ±51) This pleiotropy could
be due to multiple regulatory functions of rde-1/ago1 family
members in numerous developmental pathways. Alternatively, pleiotropy could also be due to a more general misregulation of silencing mechanisms that are necessary to
ensure proper stem-cell maintenance and differentiation.
RDE-1-related proteins might associate with different small
RNA-encoding genes analogous to lin-4 and let-7. We suspect
that further investigation in other animal systems will reveal a
requirement for the RDE-1/AGO1 family of genes by let-7
homologues and by other small miRNAs.
Conclusions and future perspectives
The discovery that small non-coding RNAs are involved in gene
regulation during temporal developmental patterning supports
the idea that RNAs can perform many of the same functions as
proteins. The stRNAs appear to be representatives of a large
class of microRNAs and thus provide a paradigm for the study
of gene regulation by small RNAs. This regulation by small
RNAs may represent a primitive but common form of genetic
control, perhaps a vestige of a time before proteins.(53,54) The
discovery of some 86 miRNAs, as well as the common function
of Dicer in both RNAi and development, suggests to us that
RNAi may have evolved from an ancient mechanism whose
purpose is to process miRNAs, rather than having evolved
specifically as a defense against viral invaders. With the recent
identification of hundreds of miRNAs in C. elegans and other
animals, it seems that we are on the verge of an explosion of
knowledge in this area.
Further investigation should allow us to determine how
many miRNA genes exist, how ancient they are, and how they
function in development and other biological processes.
Identification of these additional small regulatory RNAs will
present us with patterns that should help us discover more of
these genes and their regulatory sequences. Many challenges
remain. How is expression of the stRNAs controlled? What is
the basis of specificity between stRNA and its target? What
structural and/or sequence motifs of pre-stRNAs determine
asymmetric cleavage of the stRNAs? What is the level of
interplay between RNAi and developmental pathways? Future
research will no doubt uncover layers of complexity along with
unifying themes as to how stRNAs control developmental
timing across phylogeny. This is indeed an exciting time to
explore this newly identified mechanism of gene control that
we believe is widespread in metazoan development.
128
BioEssays 24.2
Acknowledgments
The authors would like to thank A. Pasquinelli for critical
reading of the manuscript and V. Ambros, D. Bartel, J. Brosius,
A. Grishok, C. Mello, A. Pasquinelli, G. Ruvkun, T. Tuschl and
P. Zamore for sharing results and ideas prior to publication.
We also gratefully acknowledge members of our laboratory for
advice and support during preparation of the manuscript.
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