Genome-scale spatiotemporal analysis
of Caenorhabditis elegans microRNA promoter activity
Natalia J. Martinez,1,2,3Maria C. Ow,2,3John S. Reece-Hoyes,1,2
M. Inmaculada Barrasa,1,2Victor R. Ambros,2,4and Albertha J.M. Walhout1,2,4
1Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA;
2Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA
The Caenorhabditis elegans genome encodes more than 100 microRNAs (miRNAs). Genetic analyses of miRNA deletion
mutants have only provided limited insights into miRNA function. To gain insight into the function of miRNAs, it is
important to determine their spatiotemporal expression pattern. Here, we use miRNA promoters driving the
expression of GFP as a proxy for miRNA expression. We describe a set of 73 transgenic C. elegans strains, each
expressing GFP under the control of a miRNA promoter. Together, these promoters control the expression of 89
miRNAs (66% of all predicted miRNAs). We find that miRNA promoters drive GFP expression in a variety of
tissues and that, overall, their activity is similar to that of protein-coding gene promoters. However, miRNAs are
expressed later in development, which is consistent with functions after initial body-plan specification. We find that
miRNA members belonging to families are more likely to be expressed in overlapping tissues than miRNAs that do
not belong to the same family, and provide evidence that intronic miRNAs may be controlled by their own, rather
than a host gene promoter. Finally, our data suggest that post-transcriptional mechanisms contribute to differential
miRNA expression. The data and strains described here will provide a valuable guide and resource for the functional
analysis of C. elegans miRNAs.
[Supplemental material is available online at www.genome.org.]
Differential gene expression can be regulated at many levels and
by various trans-acting factors. MicroRNAs (miRNAs) and tran-
scription factors (TFs) are pivotal regulators of metazoans gene
expression. While TFs physically interact with cis-regulatory
DNA elements to activate or repress gene expression, miRNAs
mainly repress gene expression post-transcriptionally by imper-
fect base-pairing to sequences located in the 3?UTR of their target
mRNAs (for review, see Bartel 2004; Walhout 2006). Like TFs,
many miRNAs are highly conserved between related species and
even across phyla. Typically, miRNAs are transcribed by RNA
polymerase II into a primary transcript (pri-miRNA) that is fur-
ther processed by RNASEN (also known as DROSHA) into an ∼60-
nt-long precursor (pre-miRNA), and subsequently by DICER1
(also known as Dicer) into a mature ∼23-nt-long miRNA (for re-
view, see Bartel 2004). The two founding miRNAs, lin-4 and let-7,
were identified genetically as temporal regulators of develop-
ment in the nematode Caenorhabditis elegans (Lee et al. 1993;
Reinhart et al. 2000). MiRNAs regulate a broad range of biological
processes in animals and plants, including patterning of the ner-
vous system, cell death, cell proliferation, and development (Am-
bros 2004; Stefani and Slack 2008). In addition, as for TFs, there
is increasing evidence that mammalian miRNA expression may
also be regulated at the post-transcriptional level (Obernosterer et
al. 2006; Thomson et al. 2006; Wulczyn et al. 2007; Viswanathan
et al. 2008;).
Genome-wide genetic analyses in many organisms have
demonstrated a myriad of critical roles that TFs play in control-
ling gene expression during development, homeostasis, and dis-
ease. For instance, more than 30% of C. elegans TFs confer a
detectable phenotype when knocked down by RNAi (291 out of
940 predicted TFs tested; data obtained from WormBase WS180)
(Vermeirssen et al. 2007b). In contrast, with the exceptions of
lin-4 (Lee et al. 1993), let-7 (Reinhart et al. 2000), lsy-6 (Johnston
and Hobert 2003), and mir-1 (Simon et al. 2008), a single null
mutation does not result in an easily detectable phenotype for
most C. elegans miRNA genes (Miska et al. 2007). The observation
that most C. elegans miRNAs appear to be individually dispens-
able may reflect roles in processes that have not yet been readily
assayable. Alternatively, there may be considerable genetic re-
dundancy between miRNAs and other regulators such as TFs,
other miRNAs, and, in some cases, members of the same miRNA
family. For example, the three related miRNAs lin-58 (hereafter
referred to as mir-48), mir-84, and mir-241 function redundantly
in the control of developmental timing in C. elegans (Abbott et al.
2005). One approach that will help to delineate the biological
function of miRNAs is by determining when and where they are
expressed. The particular pattern of expression of each miRNA
gene should help to identify potential genetic interactors that
exhibit similar expression patterns, and to design experiments
for the delineation of phenotypes of miRNA mutants.
A simple anatomy, invariant cell lineage, transparent body,
and high-quality complete genome sequence make C. elegans a
highly suitable model to study spatiotemporal miRNA expres-
sion. In addition, many biological processes are conserved be-
tween nematodes and higher organisms, so the analysis of
miRNA function in C. elegans may potentially be applicable to
other animals. For instance, it has been demonstrated that mir-1,
a highly conserved miRNA, is expressed and functions in muscle
in diverse organisms such as mice, zebrafish, fruit flies, and nem-
atodes (Sokol and Ambros 2005; Wienholds et al. 2005; Zhao et
3These authors contributed equally to this work.
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al. 2007; Simon et al. 2008). Therefore,
the spatiotemporal expression pattern
and, perhaps, function of many other
miRNAs may also be also conserved.
Previous studies in various organ-
isms have examined miRNA expression
by in situ hybridization (Aboobaker et al.
2005; Wienholds et al. 2005), Northern
blotting (Lau et al. 2001; Lee and Am-
bros 2001), or small RNA library se-
quencing from enriched tissues (Land-
graf et al. 2007; Ruby et al. 2007). Al-
though powerful, such studies can be
limited by a relatively low sensitivity. In
addition, these methods do not enable
the analysis of spatiotemporal expres-
sion patterns in living animals as they
depend on animal fixation (in situ hy-
bridization) or RNA purification (North-
ern blotting, sequencing). Reporter
genes such as that encoding the green
fluorescent protein (GFP) have provid-
ed powerful tools for the analysis of
gene expression in vivo. Indeed,
promoter?gfp fusions in C. elegans have
already been used to analyze more than
350 TFs and ∼1800 other protein-coding
genes (referred to here as “all genes”)
(Hunt-Newbury et al. 2007; Reece-Hoyes
et al. 2007). Importantly, this approach
faithfully recapitulates known gene ex-
pression in the majority of cases exam-
ined (Reece-Hoyes et al. 2007).
Here, we present a collection of 73 transgenic C. elegans
strains (see Note Added in Proof), each containing a miRNA
promoter?gfp fusion construct. We used promoter activity as a
proxy for miRNA expression in vivo. We examined miRNA pro-
moter activity across all developmental stages, and, frequently, to
to that of the TF and “all gene” data sets introduced above. We find
that miRNA promoters are active in all major tissues and cell types.
However, miRNA promoters are active later in development than
protein-coding gene promoters, which is consistent with roles for
miRNAs after the initial specification of body plan, organs, and
tissues (Wienholds et al. 2005; Schier and Giraldez 2006). We cor-
relate promoter activity with previously reported Northern blotting
data and examine two endogenous pri-miRNAs by RT-PCR. Our
data suggest that post-transcriptional regulation of pri-miRNAs
provides an additional layer of differential miRNA expression in
nematodes. The data and transgenic lines that we present pro-
vide a platform for functional miRNA studies to delineate their
roles in the development of the animal and to understand their
function in gene regulatory networks.
Generation of transgenic PmiRNA?gfp C. elegans strains
Of the 134 C. elegans miRNA genes currently available in miRBase
V9.0, 75 reside in intergenic regions, i.e., between protein-coding
genes, and can be assigned to their own promoter (Fig. 1A). An
additional 22 intergenic miRNAs are transcribed in a total of nine
intergenic operons, with a single promoter regulating each op-
eron. Sixteen miRNAs are embedded within the intron of pro-
tein-coding genes in the antisense orientation either as single
genes (seven miRNAs), or as operons (nine miRNAs into two
operons) (Fig. 1A; Supplemental Table S1). Twenty-one miRNAs
are embedded within the intron of a protein-coding gene in the
sense orientation. It has been hypothesized that such miRNAs are
under the control of the host gene promoter (Baskerville and
Bartel 2005), and therefore, we largely focus on the set of 113
miRNAs with presumed independent promoters.
We generated miRNA promoter?gfp (PmiRNA?gfp) fusions
by Gateway cloning (Walhout et al. 2000). We used the PmiRNA
Entry clones we generated previously (Martinez et al. 2008) and
a Gateway-compatible GFP destination vector (Dupuy et al.
2004). Promoter sequences were defined as the intergenic geno-
mic sequence upstream of annotated miRNA genes with a mini-
mum length of 300 bp and a maximum length of 2 kb. It has
been shown previously that upstream sequences defined by these
criteria are often sufficient for rescue of miRNA mutant pheno-
types (Lee et al. 1993; Johnson et al. 2003; Chang et al. 2004)
and/or to recapitulate miRNA expression (Johnson et al. 2005; Li
et al. 2005; Yoo and Greenwald 2005). PmiRNA?gfp constructs
were then used to transform unc-119(ed3) worms by micropar-
ticle bombardment as described (Berezikov et al. 2004; Reece-
Hoyes et al. 2007).
In total, we generated a collection of transgenic lines for 70
PmiRNA?gfp constructs (we will introduce another three below).
These 70 constructs together include upstream sequences for 61
single gene miRNAs and nine miRNA operons, corresponding to
Black boxes indicate protein-coding gene exons; red boxes, miRNA genes. (B) Expression rate of
Pmir?gfp constructs compared with TFs and “all genes.”
(A) Number of miRNA genes and promoters considered according to genome annotation.
Martinez et al.
a total of 86 miRNAs (out of 113 considered, or 76%). On average,
we obtained four independent lines per construct. We observed a
high transmission rate of the PmiRNA?gfp transgene for most of
the lines (data not shown). With only one exception, all inde-
pendent lines for a given construct show similar expression pat-
terns. The exception is the promoter of mir-227-80. While one
line shows mosaic expression in excretory cells, vulva, body wall
muscle, and head neurons, two other independent lines show
expression in the pharynx and head neurons. All strains were
genotyped to verify the presence of Pmir-227-80, and both ex-
pression patterns are provided in our EDGEdb database (Barrasa
et al. 2007).
Characterization of miRNA expression patterns
We examined the activity of miRNA promoters throughout the
whole organism and across all developmental stages in living
animals, and, when feasible, to the level of individual cells. Spe-
cifically, for each transgenic line we examined GFP expression in
a mixed stage population of hermaphrodites. We only recorded
the expression pattern of a given PmiRNA?gfp reporter strain
that was observed consistently in each of the independent
PmiRNA?gfp transgenic lines (data not shown). Detailed descrip-
tions and representative images can be found in Supplemental
Table S2 and in our publicly available EDGEdb database.
In total, 90% of the miRNA promoters confer GFP expres-
sion (63 out of 70) (Supplemental Table S3). The expression rate
of PmiRNA?gfp fusions is comparable to that of TFs (91%) (Reece-
Hoyes et al. 2007) and “all genes” (79%) (Fig. 1B; Hunt-Newbury
et al. 2007). This demonstrates that the chosen genomic se-
quences upstream of miRNAs indeed function as promoters. The
promoters of seven miRNAs did not drive detectable GFP expres-
sion in vivo. Two of these miRNAs are conserved in the related
nematode Caenorhabditis briggsae: lsy-6, a well-characterized
miRNA involved in neuronal specification (Johnston and Hobert
2003), and mir-77, for which a phenotype has not been described
but which has been detected in large-scale sequencing analyses
(Ruby et al. 2006). The fact that we did not observe GFP expres-
sion for these promoters may be because they lack elements re-
quired for expression or because the transgene is present at a low
copy number, which may not suffice for the detection of GFP
expression. The other five miRNAs for which we did not detect
promoter activity, mir-257, mir-258, mir-261, mir-267, and mir-
271, are not conserved in C. briggsae and have not been detected
by large-scale sequencing (Ruby et al. 2006). We found a signif-
icant correlation between GFP expression and conservation
(Fisher exact test, P-value < 0.05) and between GFP expression
and detection by sequencing (Fisher exact test, P-value < 0.05).
Based on these observations, it is possible that some or all of
mir-257, mir-258, mir-261, mir-267, and mir-271 are not genuine
miRNAs and/or are not transcribed under normal culture condi-
Temporal PmiRNA?gfp activity correlates with Northern blot
Northern blots have been extensively used to determine the tem-
poral expression of miRNAs in C. elegans (Lau et al. 2001; Lee and
Ambros 2001; Ambros et al. 2003; Lim et al. 2003). We searched
WormBase (WS180) for information regarding temporal miRNA
expression. Of the 81 miRNAs for which we had temporal infor-
mation based on PmiRNA?gfp transgenic lines, equivalent infor-
mation was available for 58 (Supplemental Table S4). We found
that the observed temporal GFP expression pattern agrees with
the pattern detected by developmental Northern blots in most of
the cases. Four PmiRNA?gfp strains did not match the temporal
expression pattern. For instance, we only detected mir-82 pro-
moter activity in the L4 and adult stage, while Northern blotting
detected mature miRNA in all developmental stages (Fig. 2A, see
also below). These discrepancies may be due to a lack of regula-
tory elements in the chosen genomic DNA fragment. In other
cases, the temporal pattern partially agrees with previously re-
ported patterns (Supplemental Table S4). Twelve PmiRNA?gfp
strains exhibited earlier expression than reported previously by
Northern blotting. There are several explanations for this differ-
ence. For instance, the DNA fragments used as promoters may
lack transcriptional elements that are required for repression of
miRNA expression in early developmental stages. Also, GFP
transgenics may be more sensitive for detecting spatially re-
stricted miRNA expression in early stages of development. For
instance, mature mir-237 was detected from L3 to adult stages;
however, we observed GFP expression in Pmir-237?gfp animals
as early as the first larval stage (see also Esquela-Kerscher et al.
2005). We performed additional Northern blotting using StarFire
probes to detect the temporal expression of nine miRNAs: mir-
241, mir-84, mir-48, let-7, mir-83, mir-230, mir-240, mir-82, and
mir-85. This allowed for the more sensitive detection of low levels
of mature miRNAs than traditional oligonucleotide probes
(Behlke et al. 2000; Abbott et al. 2005; Ow et al. 2008). For four
of these miRNAs (mir-241, mir-84, mir-48, and mir-83), we de-
tected a weak miRNA signal at earlier stages than previously re-
ported, consistent with our PmiRNA?gfp expression data
(Supplemental Table S4; Fig. 2A). We also detected the temporal
expression of two additional miRNAs (mir-59 and mir-90) for which
there was no information in WormBase, and found that it was
consistent with promoter activity (Supplemental Table S4; Fig. 2A).
We have also observed cases in which mature miRNAs were
only detected early by Northern blotting, while we detected con-
tinuous PmiRNA?gfp activity in later stages. This was the case for
two miRNA operons: mir-42-44 and mir-35-41 (a total of nine
miRNAs). In addition to the aforementioned reasons, the differ-
ences observed between mature miRNA expression and miRNA
promoter activity may be due to post-transcriptional mecha-
nisms that may regulate transcript stability or processing of ei-
ther the pri-miRNA, the pre-miRNA, or the mature miRNA (see
Taken together, the temporal expression in Pmir?gfp ani-
mals was consistent with the expression determined by Northern
blotting for 65% (39/60) of the miRNAs in our data set. For only
7% (4/60) of the miRNAs, the expression determined by North-
ern blotting does not agree with promoter activity, while the
remaining 28% (17/60) partially agrees (Fig. 2B).
Post-transcriptional mechanisms contribute to differential
Our data suggest that post-transcriptional mechanisms affect
transcript processing or stability of several miRNAs. For instance,
while mir-61 is detected by Northern blotting in all developmen-
tal stages, mir-250 is only detected starting from the L1 stage (Lee
and Ambros 2001; Lim et al. 2003), even though both miRNAs
are likely transcribed from the same promoter (Pmir-61-250). This
suggests that post-transcriptional mechanisms may either pre-
vent processing of the pre-mir-250 transcript or affect mature miR-
250 stability. Similarly, three other miRNAs that are expressed
Genome-scale C. elegans microRNA promoter activity
from a single operon and are thus controlled by one promoter,
mir-42, mir-43, and mir-44, are differentially expressed: Mature
mir-42 and mir-43 are only detected in embryos, while mir-44 is
detected not only in embryos but also in larval and adult stages
(Lau et al. 2001). Consistent with the expression of mature mir-
44, we observed promoter activity from Pmir-42-44 in all devel-
opmental stages, suggesting that mir-42 and mir-43 might be sub-
ject to post-transcriptional regulation. Lau et al. (2001) only de-
tected mature miRNAs from the mir-35-41 operon in the embryo
by Northern blotting. However, they detected the precursor of
miR-35 (pre-mir-35) both in the embryo and at the L4 stage, sug-
gesting that it is down-regulated between those stages. We de-
tected Pmir-35-41 activity (by GFP fluorescence) not only in em-
bryos and L4 stages but also in the other larval stages and in
adults (EDGEdb) (Supplemental Table S3). To test whether this
may be the result of GFP stability rather than promoter activity,
we used RT-PCR to detect the endogenous mir-35-41 primary
transcript (pri-mir-35-41). We observed pri-mir-35-41 in embryos
and L4, where mature and pre-miRNAs are detected, as well as in
L1, L2, and L3 stages, where neither mature nor pre-miRNAs
from this cluster were detected. This suggests that post-
transcriptional mechanisms regulate the processing or stability of
the mir-35-41 primary transcript, pre-miRNAs, or mature miRNAs
during L1 to L4 stages (Fig. 2C). We also compared the expression
of the let-7 primary transcript to the expression of mature let-7 as
described previously (Bracht et al. 2004). We detected mature
let-7 by Northern blotting starting at the L3 stage, which is in
agreement with previous observations (Fig. 2A; Reinhart et al.
2000). However, we detected pri-let-7 by RT-PCR as early as the
embryonic stage, consistent with the GFP expression observed in
miRNA expression. (A) Northern blot analyses using StarFire probes detect temporal expression of mature miRNAs. E indicates embryo; L, larvae; *L1,
starved L1; A, adult. Probe against the U6 snRNA was used as control. (B) Comparison between miRNA expression determined by Northern blotting and
promoter?gfp reporters. (C) Detection of mir-35-41 and let-7 primary transcripts by RT-PCR. As control, we used primers to amplify a protein-coding
mRNA (fat-4). Total RNA from N2 embryos, L1, L2, L3, and L4 stages and total RNA from VC514 mir-35-41 mutant embryos (mut) were subjected to
reverse transcription (+RT, lanes 2,4,6,8,10,12,15,17,19,21,23,25,28,30,32,34,36,38,40,42,44,46) or mock reactions (?RT, lanes
3,5,7,9,11,13,16,18,20,22,24,26,29,31,33,35,37,39,41,43,45,47). Genomic DNA was used as size marker (g, lanes 1,14,27,48). Cartoons indicate the
predicted size of PCR amplicons from mir-35-41 primary transcript, let-7 primary transcript, and fat-4 mRNA and indicate the primers that were used.
Note that fat-4 L and R primers amplify a product of different size when genomic DNA (lane 14) or cDNA (lanes 15–26) was used as template.
Temporal PmiRNA?gfp activity correlates with Northern blot analysis and uncovers possible post-transcriptional mechanisms that control
Martinez et al.
Plet-7?gfp strains (EDGEdb) (Fig. 2C). Similar observations have
been made for let-7 in mammalian systems, where let-7 process-
ing is selectively blocked in embryonic stem cells (Wulczyn et al.
2007; Viswanathan et al. 2008). Our results suggest that post-
transcriptional mechanisms likely regulate pri-let-7 processing at
early stages (Fig. 2C).
Taken together, our results show that miRNA promoter ac-
tivity largely overlaps with mature miRNA expression and that
post-transcriptional mechanisms likely contribute to differences
in primary and mature miRNA expression.
The promoters of miRNAs are active later in development
We detected GFP expression conferred by miRNA promoters in
all developmental stages. Representative examples of embryonic
promoter activity are shown in Figure 3A. We compared the tem-
poral expression conferred by promoter of miRNAs to that of TFs
and “all genes” and noticed that miRNA promoters overall tend
to be less active in embryos (P-value < 0.05) (Fig. 3B). In addition,
the majority of miRNA promoters that confer embryonic expres-
sion tend to do so at later embryonic stages, on average, than do
promoters of TFs (this analysis could not be done for the “all
genes” data set as analogous temporal expression information
was not available) (Fig. 3C). This observation is in agreement
with previous studies in other organisms that suggest that
miRNAs are involved in tissue differentiation and maintenance
rather than the establishment of body plan, organs, and tissues
(Wienholds et al. 2005; Schier and Giraldez 2006).
Most miRNA promoters drive GFP expression
in a tissue-specific manner
We found that miRNA promoters drive expression in all major
tissues and cell types, except the germline (Fig. 4A; Supplemental
Table S3). Representative examples of miRNA promoter activity
in various parts of the somatic gonad and neuronal cells are
shown in Figure 4B and C. Microparticle bombardment has been
reported to be the method of choice when germline expression is
desired (Praitis et al. 2001). However, none of the miRNA pro-
moters are able to direct GFP expression in the germinal gonad.
Thus, it is possible that the miRNAs assayed here are exclusively
expressed in somatic tissues. However, promoters of protein-
coding genes also generally fail to drive GFP reporter expression
in the germline (Hunt-Newbury et al. 2007; Reece-Hoyes et al.
2007). Thus, we think that it is more likely that the GFP trans-
gene is silenced in the germinal gonad. Future studies that use
germline-specific deep sequencing of miRNAs will reveal whether
any of the miRNAs are expressed in this tissue.
To enable the comparison at a tissue level between miRNAs
and protein-coding genes, we reannotated the TF and “all genes”
data sets according to a systematic spatiotemporal expression
scheme that we devised. We defined 23 categories (hereafter re-
ferred to as “tissues”), including intestine, vulva, head neurons,
etc. (for precise definitions, see Methods and Supplemental Table
S5). Some of these are highly specific (e.g., distal tip cells), and
others are broader (e.g., head neurons). We observed that most
miRNA promoters confer GFP expression in only a few tissues or
cell types. For instance, ∼50% of the promoters confer expression
in three or fewer tissues, while only less than 5% of promoters
confer ubiquitous somatic expression (lin-4, let-7, and mir-53). A
high degree of tissue specificity has also been observed for
miRNAs in other organisms, including chicken and zebrafish
(Wienholds et al. 2005; Xu et al. 2006). The promoters of TFs and
“all genes” drive GFP expression with a similar degree of tissue
specificity (Fig. 4D). We recently obtained a genome-scale
miRNA transcriptional network (Martinez et al. 2008) that re-
veals a similar overall network architecture as protein-coding
gene networks (Deplancke et al. 2006; Vermeirssen et al. 2007a;
Martinez et al. 2008). Together, these observations indicate that
the regulation of miRNA gene promoters is not fundamentally
different from that of protein-coding gene promoters.
ages of miRNA promoters that drive GFP expression in the embryo. (Left)
DIC images; (right) GFP fluorescence. Additional images can be found in
the EDGEdb database (Barrasa et al. 2007). (B) miRNAs tend to be ex-
pressed later in development compared to TFs and “all genes.” The as-
terisk indicates a significant difference (P < 0.05). (C) Percentage of
miRNA and TF promoters that drive expression in the embryonic stage:
embryo only, early, mid, and late embryonic stages.
Temporal miRNA promoter activity. (A) Representative im-
Genome-scale C. elegans microRNA promoter activity
Members of miRNA families can be
expressed in distinct or overlapping
MiRNAs can be classified into families
according to sequence similarities (Bar-
tel 2004). Sixty percent of C. elegans
miRNAs (78 out of 134) can be classified
into 24 families, each containing be-
tween two and eight members (Ruby et
al. 2006). Members of a given family are
predicted to share target mRNAs and
may function redundantly (Abbott et al.
2005; Miska et al. 2007). For instance,
the let-7 family members mir-48, mir-84,
and mir-241 function together to regu-
late the L2 to L3 cell fate transitions in
the hypodermis (Abbott et al. 2005). Re-
dundancy among miRNAs from the
same family can occur if miRNA family
members are (partially) coexpressed. We
examined the extent to which spatio-
temporal promoter activity of members
of a miRNA family overlap. We were able
to compare the expression patterns for
10 complete miRNA families, as well as
for two families for which we have ex-
pression patterns for most, but not all of
the members (Fig. 5A). Interestingly, we
observed that some families are ex-
pressed with a high degree of overlap,
whereas other families exhibit largely
nonoverlapping spatiotemporal expres-
sion. For instance, miRNAs from the mir-
35 family (mir-35-36-37-38-39-40-41
cluster and mir-42) are expressed
throughout all stages and in overlapping
tissues, including the vulva, seam cells,
head neurons, and the rectum (Fig. 5B).
Similarly, members of the lin-4 (lin-4,
mir-237) and mir-46 (mir-46, mir-47)
families are expressed in overlapping tis-
sues (Supplemental Table S3). In con-
trast, miRNAs from the mir-75 family
(mir-75 and mir-79) are expressed in dif-
ferent tissues. While Pmir-75 confers
GFP expression exclusively in the intes-
tine, Pmir-79 drives expression in the hy-
podermis (Fig. 5C). Similarly, members
of the mir-232 (mir-232, mir-357) and
mir-251 (mir-251, mir-252) families ex-
hibit distinct expression patterns
(Supplemental Table S3).
We introduce a “tissue overlap co-
efficient” (TsOC) as the number of tis-
sues shared between two miRNAs di-
vided by the smallest of the total num-
ber of tissues where either miRNA is
expressed (Fig. 5D). This coefficient is
similar to the topological overlap coeffi-
cient (TOC) that is used for network
modularity analysis (Vermeirssen et al.
in a survey of tissues. (B) Multiple miRNA promoters drive expression in various parts of the somatic
gonad, including gonadal sheath, vulva, and uterus. (Top) DIC images; (bottom) GFP fluorescence. (C)
Multiple miRNA promoters drive GFP expression in the nervous system. (Top) DIC images; (bottom)
GFP fluorescence. Additional images can be found in the EDGEdb database (Barrasa et al. 2007). (D)
Most miRNAs as well as TFs and “all genes” promoters confer GFP expression in a tissue-specific
Spatial miRNA promoter activity. (A) Percentage of miRNA promoters that drive expression
Martinez et al.
2010 Genome Research
2007a). We used TsOC as a measure to
determine if the overlap in expression
between miRNAs from the same family
is different than the overlap in expres-
sion between miRNAs from distinct
families. We calculated TsOCs for all
pairs of miRNAs from distinct families,
as well as pairs of miRNAs from the same
family (see Methods). We found that
the distribution of TsOCs for pairs of
miRNAs from the same family is signifi-
cantly different than the distribution of
TsOCs for pairs of miRNAs from distinct
families (Fisher Freeman Halton test P-
value < 0.001) (Fig. 5E). Pairs of miRNAs
from the same family tend to have a
higher TsOC compared with pairs of
miRNAs from distinct families. Taken to-
gether, the degree of overlapping expres-
sion varies per miRNA family; however,
miRNAs from the same family do tend to
exhibit overlapping expression patterns.
Thus, it is likely that the lack of pheno-
types for individual miRNAs can be ex-
plained (at least partly) by familial re-
dundancy and that, in addition, many
miRNAs may have a synthetic genetic
interaction with other miRNAs, or per-
haps with protein-coding genes.
MiRNA genes that are located within the
intron of a protein-coding gene in the
sense orientation are thought to be un-
der the control of the host gene pro-
moter (Baskerville and Bartel 2005). We
generated PmiRNA?gfp constructs using
the immediate upstream sequence of
three of these intragenic miRNAs: mir-
58, mir-2, and mir-82, which are embed-
ded in the intron of Y67D8A.1, ppfr-1,
and T07D1.2, respectively (Fig. 6; also
see Note Added in Proof). We found that
the region upstream of mir-58 does not
confer GFP expression (data not shown).
Surprisingly, however, sequences up-
stream of both mir-82 and mir-2 drive
tissue-specific GFP expression (Fig. 6). In
addition, the annotation of lin-4 has re-
cently changed; rather than being lo-
cated in an intergenic region (WS140), it
is now annotated to be located in an in-
tron of F59G1.4 (WS180). We and others
have shown that the genomic fragment
immediately upstream of lin-4 does
function as a promoter (Esquela-
Kerscher et al. 2005; Ow et al. 2008; this
study). It has been previously shown
that internal promoters in operons are a
common feature in the C. elegans ge-
nome (Huang et al. 2007). It is tempting
to speculate that internal miRNA pro-
expression patterns. (A) Cartoon depicting expression patterns of 10 complete and two incomplete
(let-7 and mir-80 families shown at the bottom) miRNA families. Each color represents a family. Spa-
tiotemporal expression is as in Supplemental Table S3. (B) miRNAs from the mir-35 family are expressed
in overlapping tissues/cell types. Pmir35-41?gfp and Pmir-42-44?gfp are shown. (C) miRNAs from the
mir-75 family are expressed in different tissues/cell types. (Top) DIC images; (bottom) GFP fluorescence.
(D) Definition and example of TsOC between any two miRNAs. (E) Distribution of TsOC among miRNA
pairs from the same or different family.
miRNAs from a given family can have overlapping as well as different spatiotemporal
Genome-scale C. elegans microRNA promoter activity
moters located in the introns of protein-coding genes might be
common as well. In contrast to C. elegans miRNAs, most human
miRNAs are located within introns (Rodriguez et al. 2004). It will
be interesting to see if the genomic sequences upstream of these
miRNAs can function as promoters in mammals or whether this
is specific to nematodes.
We present here the generation and analysis of transgenic ani-
mals for 73 PmiRNA?gfp constructs that represent the expression
of 89 C. elegans miRNAs. Several lines of evidence indicate that
the majority of these transgenic animals likely recapitulate en-
dogenous miRNA transcription. First, it has been demonstrated
previously that a 2-kb fragment upstream of the translational
start site of protein-coding genes accurately drives gene expres-
sion in the majority of cases examined (Reece-Hoyes et al. 2007).
Second, the majority of PmiRNA?gfp lines completely or partially
recapitulate previously reported temporal expression of miRNAs
detected by Northern blotting (Lau et al. 2001; Lee and Ambros
2001; Ambros et al. 2003; Lim et al. 2003). Third, for a handful of
miRNAs it has been shown that such a fragment is sufficient for
miRNA rescue and in other expression experiments (Lee et al.
1993; Johnston and Hobert 2003; Chang et al. 2004; Johnston Jr.
et al. 2005; Li et al. 2005; Yoo and Greenwald 2005).
We compared miRNA promoter activity to mature miRNA
expression determined by Northern blotting. While in the ma-
jority of cases promoter activity exactly agrees with mature
miRNA expression, there are cases in which they only partially
agree. We have shown in the case of let-7 and the mir-35-41
operon that this partial agreement is likely due to post-
transcriptional mechanisms that contribute to differential
miRNA expression. Such mechanisms can in principle control
pri-miRNA, pre-miRNA, or mature
miRNA stability and/or processing. It
has been shown previously that mam-
malian miRNAs can be regulated post-
transcriptionally (Obernosterer et al.
2006). Viswanathan et al. (2008) have
identified LIN-28 as a developmentally
regulated RNA-binding protein that se-
lectively blocks the processing of pri-let-7
in embryos. In the future, it will be im-
portant to dissect the factors that play a
role in post-transcriptional regulation of
C. elegans miRNAs.
We found that miRNAs are ex-
pressed in a variety of tissues. In ze-
brafish and fruit flies, previous studies
have also shown a broad expression for
many miRNAs (Aboobaker et al. 2005;
Wienholds et al. 2005). We also found
that miRNAs are expressed relatively late
in development, which is in agreement
with results obtained in zebrafish and
likely reflects a function of miRNAs in
tissue differentiation and maintenance,
rather than in tissue establishment
(Wienholds et al. 2005).
Most miRNAs do not confer a de-
tectable phenotype when deleted (Miska
et al. 2007). It is likely that the lack of
phenotypes for individual miRNAs can be explained not only
by familial redundancy but also by genetic interactions with
miRNAs from other families, or perhaps by interactions with pro-
tein-coding genes, such as TFs. The spatiotemporal miRNA ex-
pression patterns will provide an important tool for the identifi-
cation of genes with which they may act redundantly and,
hence, will be an important tool that can be used toward under-
standing the cellular functions of each miRNA.
Our study provides some important advantages over other
studies of miRNA expression. First, our method is noninvasive,
which means that expression can be studied in living animals.
Second, in contrast to methods such as Northern blotting or
sequencing, we can frequently annotate miRNA expression to
the single cell level. Third, we will provide all the strains to the C.
elegans community, which should help to delineate the expres-
sion patterns at greater levels of resolution. Fourth, the trans-
genic lines will enable the study of miRNA expression under dif-
ferent (experimental) conditions, including dauer, stress, etc.,
and in males. And finally, the transgenic lines will be available
for other studies. For example, they can be used to identify or
validate upstream regulators of miRNA expression. We recently
mapped a genome-scale miRNA regulatory network by high-
throughput yeast one-hybrid assays (Deplancke et al. 2004, 2006)
and used several of the transgenic lines described here for in vivo
validation of the interactions obtained (Martinez et al. 2008; Ow
et al. 2008).
Generation of Pmir?gfp constructs
For our network study (Martinez et al. 2008) we used the 115
miRNA predictions available in WormBase WS130 (http://
drives expression in the nerve ring (left) and ventral nerve cord and tail neurons (right). (B) Pmir-82
drives expression in pharyngeal muscle and head neurons (left), and developing spermatheca (right).
(Top) DIC images; (bottom) GFP fluorescence. Arrows indicate expression. Dotted arrows indicate
sequence used as promoter.
Upstream sequences of two intragenic miRNAs can drive GFP expression in vivo. (A) Pmir-2
Martinez et al.
www.wormbase.org) and miRNA registry V4.0 (http://microrna.
sanger.ac.uk) (Ambros et al. 2003; Lim et al. 2003; Griffiths-Jones
et al. 2006). We completed these with 19 recently discovered
miRNAs (WormBase WS175 and miRBase V9.0). A miRNA pro-
moter is defined as the intergenic region upstream of the pre-
dicted stem-loop sequence or from the mature miRNA as anno-
tated in miRBase V4.0 (Supplemental Table S1). We used a mini-
mal length of 300 bp and a maximal length of 2 kb. In total, 93
promoters (that control 113 miRNAs) were selected. Seventy-
three promoters (controlling 89 miRNAs) were successfully
cloned into pDEST-DD04 by Gateway cloning as described (Wal-
hout et al. 2000; Dupuy et al. 2004). Constructs were verified by
DNA sequencing using either GFP Fw (5?-TTCTACTTCTTTTAC
TGAACG) or GFP Rv (5?-CTCCACTGACAGAAAATTTG) primers.
The following PmiRNA?gfp constructs were generated by
conventional restriction enzyme-based cloning into the
pPD97.75 vector (for information on restriction sites used, see
Supplemental Table S1): Pmir-257, Pmir-51, Pmir-2, Pmir-228,
Pmir-54, Pmir-81, Pmir-235, Pmir-227-80 and Pmir-234, Plet-7,
Plin-4, Pmir-48, Pmir-237, Pmir-241, Pmir-84.
C. elegans strains
Routine C. elegans maintenance and culture were done as de-
scribed (Brenner 1974). The DP38 strain (unc-119(ed3)) was cul-
tured in liquid media for microparticle bombardment as de-
scribed (Reece-Hoyes et al. 2007) or in egg plates (Wood 1988).
Transformation of C. elegans by microparticle bombardment
Transgenic PmiRNA?gfp animals were generated as described pre-
viously (Berezikov et al. 2004; Reece-Hoyes et al. 2007).
The genotype of each transgenic line was confirmed by single
animal PCR (Williams et al. 1992) using GFP Fw and GFP Rv
primers (see above) as described, followed by DNA sequencing to
confirm the identity of the miRNA promoter in the PmiRNA?gfp
Characterization of GFP expression patterns
Mixed populations of hermaphrodites were examined by fluores-
cence microscopy using a Zeiss Axioskop 2 plus microscope
equipped with a FITC filter. We recorded the expression pattern
conferred by each miRNA promoter that was consistent in each
of the independently derived transgenic lines (except for Pmir-
227-80, see main text). Fluorescence photographs representative
of each expression pattern were taken using a Hamamatsu
Orca-ER/1394 video camera and Axiovision Rel. 4.5 software and
stored in the EDGEdb database (Barrasa et al. 2007). For each
genotype, we stored up to three independent lines into frozen
stocks. These lines were chosen based on highest transmission
level and/or GFP expression (data not shown). These lines will be
made available through the CGC.
PmiRNA?gfp expression pattern annotation
We devised a standarized temporal and spatial annotation to
record the expression pattern of each PmiRNA?gfp. Temporal
expression patterns were classified into eight stages: early, mid,
and late embryo; all four larval stages; and adult stage. We de-
fined early embryo as the pre-comma stage, mid-embryo as
comma stage, and late embryo as two and threefold embryos.
Spatial expression patterns were classified into 23 categories that
correspond to tissues, cell types, organs, and, when feasible, to
individual cells (i.e., coelomocytes and distal tip cells) (Supple-
mental Table S5). For GFP expression analysis purposes, temporal
and spatial expression was standardized into a binary code,
where 1 represents expression detected and 0 represents no ex-
pression detected (Supplemental Table S3).
Other data sets
GFP expression patterns driven by “TFs” and other protein-
coding gene (“all genes”) promoters where obtained from Reece-
Hoyes et al. (2007) and Hunt-Newbury et al. (2007), and con-
verted into our binary annotation scheme. Specifically, “all
genes” patterns were classified as follows: BM (body wall muscle);
BN (body neurons, lateral nerve cords/commissures, ventral
nerve cord); C (coelomocytes); DTC (distal tip cell); GS (gonad
sheath cells); HH (hypodermis); HN (amphids, dorsal nerve cord,
head neurons, labial sensilla, nerve ring, pharyngeal neurons); I
(intestinal, intestinal muscle); O (other: amphid socket cells, de-
veloping gonad, head mesodermal cell, mechanosensory neu-
rons, pvt interneuron, unidentified body, unidentified cells, un-
identified tail, unidentified head, uterine-seam cell, other);
P (arcade cells, pharynx); PG (pharyngeal gland cells); PIV (pha-
ryngeal-intestinal valve); R (anal depressor muscle, anal sphinc-
ter, rectal epithelium, rectal gland cells); S (developing sper-
matheca, spermatheca); SC (seam cells); TN (phasmids, tail neu-
rons); U (developing uterus, uterine muscle, uterus); USV
(spermatheca-uterine valve); V (developing vulva, vulva other,
vulval muscle); and X (excretory cells, excretory gland cells). For
comparison analyses, several tissues/systems were fused in one or
more of the data sets to allow the same category types in all three
data sets: HH and BH categories were fused into one category, H
(hypodermis); HM and BM categories were fused into one M
(muscle); PG and P were fused into P (pharynx); and I and PI were
fused into I (intestinal).
Northern blot analyses
Total RNA was extracted using TRIzol reagent (Invitrogen) and
analyzed by Northern blotting using 5 µg of RNA from each stage
as described before (Ow et al. 2008).
Total RNA was extracted as above and digested with RNase-Free
DNase Set (Qiagen) following the manufacturer’s recommenda-
tions. First strand cDNA synthesis was performed using 2.5 µg of
total RNA, random primers, and SuperScript II (Invitrogen) fol-
lowing manufacturer’s recommendations.
Primer sequences used in the PCR reactions were as follows:
fat-4 L: 5?-TGTTTCTATCTTGTTGGAGG
fat-4 R: 5?-GGTAAACCATTTGCTGCTGC
Primers used to detect the let-7 primary transcript are A62, A127,
and A63 (Bracht et al. 2004).
The TsOC between any two miRNAs was defined as the number
of tissues where both miRNAs are expressed divided by the small-
est of the total number of tissues in which either miRNA is ex-
pressed (see Fig. 5D). In case of operons, where several miRNAs
are expressed from a single promoter, the same expression pat-
tern was assigned to all miRNAs in the operon. We calculated a
TsOC for all individual pairs of miRNAs from different families
(3160 total pairs) and all pairs of miRNAs from the same families
(80 total pairs). We grouped the TsOCs into four bins
(0 < TsOC ? 0.25, 0.25 < TsOC ? 0.5, 0.5 < TsOC ? 0.75, and
0.75 > TsOC ? 1) and calculated if the distribution of TsOCs
Genome-scale C. elegans microRNA promoter activity
within families was significantly different from the distribution
between families using a Fisher Freeman Halton test for
4-rows ? 2-columns tables (Freeman and Halton 1951).
We thank the members of the Walhout and Ambros laboratories
and Job Dekker for advice and critical reading of the manuscript.
We thank the Caenorhabditis Genetics Center (CGC), which is
funded by the NIH National Center for Research Resources
(NCRR), for providing the unc-119(ed3) strain. We thank A. Fire
for providing pPD95.75. M.C.O. was supported in part by NIH
postdoctoral fellowship GM070118-02. This work was supported
by NIH grants DK068429 to A.J.M.W. and GM348642 to V.R.A.
Note added in proof
During the review of this paper, we have generated an additional
C. elegans transgenic strain containing Pmir-71::gfp. This strain
was not included in the analysis, but information regarding GFP
expression is available in Supplemental Tables and EDGEdb. mir-
71 is an intragenic miRNA, annotated in the intron of ppfr-1, the
same intron where mir-2 is annotated. Sequence upstream of mir-
71 drives GFP expression in vivo.
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:
Aboobaker, A.A., Tomancak, P., Patel, N.H., Rubin, G.M., and Lai, E.C.
2005. Drosophila microRNAs exhibit diverse spatial expression
patterns during embryonic development. Proc. Natl. Acad. Sci. 102:
Ambros, V. 2004. The functions of animal microRNAs. Nature 431:
Ambros, V., Lee, R.C., Lavanway, A., Williams, P.T., and Jewell, D. 2003.
MicroRNAs and other tiny endogenous RNAs in C. elegans. Curr. Biol.
Barrasa, M.I., Vaglio, P., Cavasino, F., Jacotot, L., and Walhout, A.J.M.
2007. EDGEdb: A transcription factor-DNA interaction database for
the analysis of C. elegans differential gene expression. BMC Genomics
8: 21. doi: 10.1186/1471-2164-8-21.
Bartel, D.P. 2004. MicroRNAs: Genomics, biogenesis, mechanism, and
function. Cell 116: 281–297.
Baskerville, S. and Bartel, D.P. 2005. Microarray profiling of microRNAs
reveals frequent coexpression with neighboring miRNAs and host
genes. RNA 11: 241–247.
Behlke, M., Dames, S.A., McDonald, W.H., Gould, K.L., Devor, E.J., and
Walder, J.A. 2000. Use of high specific activity StarFire
oligonucleotide probes to visualize low-abundance pre-mRNA
splicing intermediates in S. pombe. Biotechniques 29: 892–897.
Berezikov, E., Bargmann, C.I., and Plasterk, R.H. 2004. Homologous
gene targeting in Caenorhabditis elegans by biolistic transformation.
Nucleic Acids Res. 32: e40. doi: 10.1093/nar/gnh033.
Bracht, J., Hunter, S., Eachus, R., Weeks, P., and Pasquinelli, A.E. 2004.
Trans-splicing and polyadenylation of let-7 microRNA primary
transcripts. RNA 10: 1586–1594.
Brenner, S. 1974. The genetics of Caenorhabditis elegans. Genetics 77:
Chang, S., Johnston Jr., R.J., 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.
Deplancke, B., Dupuy, D., Vidal, M., and Walhout, A.J.M. 2004. A
Gateway-compatible yeast one-hybrid system. Genome Res. 14:
Deplancke, B., Mukhopadhyay, A., Ao, W., Elewa, A.M., Grove, C.A.,
Martinez, N.J., Sequerra, R., Doucette-Stam, L., Reece-Hoyes, J.S.,
Hope, I.A., et al. 2006. A gene-centered C. elegans protein–DNA
interaction network. Cell 125: 1193–1205.
Dupuy, D., Li, Q., Deplancke, B., Boxem, M., Hao, T., Lamesch, P.,
Sequerra, R., Bosak, S., Doucette-Stam, L., Hope, I.A., et al. 2004. A
first version of the Caenorhabditis elegans promoterome. Genome Res.
Esquela-Kerscher, A., Johnson, S.M., Bai, L., Saito, K., Partridge, J.,
Reinert, K.L., and Slack, F.J. 2005. Post-embryonic expression of C.
elegans microRNAs beloning to the lin-4 and let-7 families in the
hypodermis and reproductive system. Dev. Dyn. 234: 868–877.
Freeman, G.H. and Halton, J.H. 1951. Note on an exact treatment of
contingency, goodness of fit and other problems of significance.
Biometrika 38: 141–149.
Griffiths-Jones, S., Grocock, R.J., van Dongen, S., Bateman, A., and
Enright, A.J. 2006. miRBase: MicroRNA sequences, targets and gene
nomenclature. Nucleic Acids Res. 34: D140–D144.
Huang, P., Pleasance, E.D., Maydan, J.S., Hunt-Newbury, R., O’Neil, N.J.,
Mah, A., Baillie, D.L., Marra, M.A., Moerman, D.G., and Jones, S.J.
2007. Identification and analysis of internal promoters in
Caenorhabditis elegans operons. Genome Res. 17: 1478–1485.
Hunt-Newbury, R., Viveiros, R., Johnsen, R., Mah, A., Anastas, D., Fang,
L., Halfnight, E., Lee, D., Lin, J., Lorch, A., et al. 2007.
High-throughput in vivo analysis of gene expression in
Caenorhabditis elegans. PLoS Biol. 5: e237. doi:
Johnson, S.M., Lin, S.-Y., and Slack, F.J. 2003. The time of appearance of
the C. elegans let-7 microRNA is transcriptionally controlled utilizing
a temporal regulatory element in its promoter. Dev. Biol. 259:
Johnson, S.M., Grosshans, H., Shingara, J., Byrom, M., Jarvis, R., Cheng,
A., Labourier, E., Reinert, K.L., Brown, D., and Slack, F. 2005. RAS is
regulated by the let-7 microRNA family. Cell 120: 635–647.
Johnston, R.J. and Hobert, O. 2003. A microRNA controlling left/right
neuronal asymmetry in Caenorhabditis elegans. Nature 426: 845–849.
Johnston Jr., R.J., Chang, S., Etchberger, J.F., Ortiz, C.O., and Hobert, O.
2005. MicroRNAs acting in a double-negative feedback loop to
control a neuronal cell fate decision. Proc. Natl. Acad. Sci. 102:
Landgraf, P., Rusu, M., Sheridan, R., Sewer, A., Iovino, N., Aravin, A.,
Pfeffer, S., Rice, A., Kamphorst, A.O., Landthaler, M., et al. 2007. A
mammalian microRNA expression atlas based on small RNA library
sequencing. Cell 129: 1401–1414.
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.
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.
Li, M., Jones-Rhoades, M.W., Lau, N.C., Bartel, D.P., and Rougvie, A.E.
2005. Regulatory mutations of mir-48, a C. elegans let-7 family
microRNA, cause developmental timing defects. Dev. Cell 9:
Lim, L.P., Lau, N.C., Weinstein, E.G., Abdelhakim, A., Yekta, S.,
Rhoades, M.W., Burge, C.B., and Bartel, D.P. 2003. The microRNAs
of Caenorhabditis elegans. Genes & Dev. 17: 991–1008.
Martinez, N.J., Ow, M.C., Barrasa, M.I., Hammell, M., Sequerra, R.,
Doucette-Stamm, L., Roth, F.P., Ambros, V., and Walhout, A.J.M.
2008. A genome-scale microRNA regulatory network in C. elegans
reveals composite feedback motifs that provide high information
flow. Genes & Dev. 22: 2535–2549.
Miska, E.A., Alvarez-Saavedra, E., Abbott, A.L., Lau, N.C., Hellman, A.B.,
McGonagle, S.M., Bartel, D.P., Ambros, V.R., and Horvitz, H.R. 2007.
Most Caenorhabditis elegans microRNAs are individually not essential
for development or viability. PLoS Genet. 3: e215. doi:
Obernosterer, G., Leuschner, P.J., Alenius, M., and Martinez, J. 2006.
Post-transcriptional regulation of microRNA expression. RNA 12:
Ow, M.C., Martinez, N.J., Olsen, P., Silverman, S., Barrasa, M.I.,
Conradt, B., Walhout, A.J.M., and Ambros, V.R. 2008. The FLYWCH
transcription factors FLH-1, FLH-2 and FLH-3 repress embryonic
expression of microRNA genes in C. elegans. Genes & Dev. 22:
Praitis, V., Casey, E., Collar, D., and Austin, J. 2001. Creation of
low-copy integrated transgenic lines in Caenorhabditis elegans.
Genetics 157: 1217–1226.
Reece-Hoyes, J.S., Shingles, J., Dupuy, D., Grove, C.A., Walhout, A.J.,
Vidal, M., and Hope, I.A. 2007. Insight into transcription factor gene
duplication from Caenorhabditis elegans Promoterome-driven
expression patterns. BMC Genomics 8: 27. doi:
Reinhart, B.J., Slack, F.J., Basson, M., Pasquinelli, A.E., Bettinger, J.C.,
Martinez et al.
2014 Genome Research
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.
Rodriguez, A., Griffith-Jones, S., Ashurst, J.L., and Bradley, A. 2004.
Identification of mammalian microRNA host genes and transcription
units. Genome Res. 14: 1902–1910.
Ruby, J.G., Jan, C., Player, C., Axtell, M.J., Lee, W., Nusbaum, C., Ge, H.,
and Bartel, D.P. 2006. Large-scale sequencing reveals 21U-RNAs and
additional microRNAs and endogenous siRNAs in C. elegans. Cell
Ruby, J.G., Stark, A., Johnston, W.K., Kellis, M., Bartel, D.P., and Lai,
E.C. 2007. Evolution, biogenesis, expression, and target predictions
of a substantially expanded set of Drosophila microRNAs. Genome
Res. 17: 1850–1864.
Schier, A.F. and Giraldez, A.J. 2006. MicroRNA function and
mechanism: Insights from zebrafish. Cold Spring Harb. Symp. Quant.
Biol. 71: 195–203.
Simon, D.J., Madison, J.M., Conery, A.L., Thompson-Peer, K.L., Soskis,
M., Ruvkun, G.B., Kaplan, J.M., and Kim, J.K. 2008. The microRNA
miR-1 regulates a MEF-2-dependent retrograde signal at
neuromuscular junctions. Cell 133: 891–902.
Sokol, N.S. and Ambros, V. 2005. Mesodermally expressed Drosophila
microRNA-1 is regulated by Twist and is required in muscles during
larval growth. Genes & Dev. 19: 2343–2354.
Stefani, G. and Slack, F.J. 2008. Small non-coding RNAs in animal
development. Nat. Rev. Mol. Cell Biol. 9: 219–230.
Thomson, J.M., Newman, M., Parker, J.S., Morin-Kensicki, E.M., Wright,
T., and Hammond, S.M. 2006. Extensive post-transcriptional
regulation of microRNAs and its implications for cancer. Genes &
Dev. 20: 2202–2207.
Vermeirssen, V., Barrasa, M.I., Hidalgo, C., Babon, J.A.B., Sequerra, R.,
Doucette-Stam, L., Barabasi, A.L., and Walhout, A.J.M. 2007a.
Transcription factor modularity in a gene-centered C. elegans core
neuronal protein-DNA interaction network. Genome Res. 17:
Vermeirssen, V., Deplancke, B., Barrasa, M.I., Reece-Hoyes, J.S., Arda,
H.E., Grove, C.A., Martinez, N.J., Sequerra, R., Doucette-Stamm, L.,
Brent, M., et al. 2007b. Matrix and Steiner-triple-system smart
pooling assays for high-performance transcription regulatory
network mapping. Nat. Methods 4: 659–664.
Viswanathan, S.R., Daley, G.Q., and Gregory, R.I. 2008. Selective
blockade of microRNA processing by Lin28. Science 320: 97–100.
Walhout, A.J.M. 2006. Unraveling transcription regulatory networks by
protein-DNA and protein–protein interaction mapping. Genome Res.
Walhout, A.J.M., Temple, G.F., Brasch, M.A., Hartley, J.L., Lorson, M.A.,
van den Heuvel, S., and Vidal, M. 2000. GATEWAY recombinational
cloning: Application to the cloning of large numbers of open
reading frames or ORFeomes. Methods Enzymol. 328: 575–592.
Wienholds, E., Kloosterman, W.P., Miska, E., Alvarez-Saavedra, E.,
Berezikov, E., de Bruijn, E., Horvitz, H.R., Kauppinen, S., and
Plasterk, R.H. 2005. MicroRNA expression in zebrafish embryonic
development. Science 309: 310–311.
Williams, B.D., Schrank, B., Huynh, C., Shownkeen, R., and Waterston,
R.H. 1992. A genetic mapping system in Caenorhabditis elegans based
on polymorphic sequence-tagged sites. Genetics 131: 609–624.
Wood, W.B. 1988. The nematode Caenorhabditis elegans. Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY.
Wulczyn, F.G., Smirnova, L., Rybak, A., Brandt, C., Kwidzinski, E.,
Ninnemann, O., Strehle, M., Seiler, A., Schumacher, S., and Nitsch,
R. 2007. Post-transcriptional regulation of the let-7 microRNA during
neural cell specification. FASEB J. 21: 415–426.
Xu, H., Wang, X., Du, Z., and Li, N. 2006. Identification of microRNAs
from different tissues of chicken embryo and adult chicken. FEBS
Lett. 508: 3610–3616.
Yoo, A.S. and Greenwald, I. 2005. LIN-12/Notch activation leads to
microRNA-mediated down-regulation of Vav in C. elegans. Science
Zhao, Y., Ransom, J.F., Li, A., Vedantham, V., von Drehle, M., Muth,
A.N., Tsuchihashi, T., McManus, M.T., Schwartz, R.J., and Srivastava,
D. 2007. Dysregulation of cardiogenesis, cardiac conduction, and cell
cycle in mice lacking miRNA-1-2. Cell 129: 303–317.
Received July 7, 2008; accepted in revised form September 30, 2008.
Genome-scale C. elegans microRNA promoter activity