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.
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Received July 7, 2008; accepted in revised form September 30, 2008.
Genome-scale C. elegans microRNA promoter activity