Helge Großhans ¹, Ted Johnson ², Kristy L. Reinert ¹, Mark Gerstein ², and Frank J. Slack ¹ *

¹ Department of Molecular, Cellular, and Developmental Biology, Yale University, 266 Whitney Avenue, New Haven, Connecticut 06520
² Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, Connecticut 06520

Correspondence:  Frank J. Slack   Phone (203) 432-3492;    Fax: (203) 432-6161
E-mail:  frank.slack@yale.edu

Abstract:

The let-7 microRNA is phylogenetically conserved and temporally expressed in many animals. C. elegans let-7 controls terminal differentiation in a stem cell–like lineage in the hypodermis, while human let-7 has been
implicated in lung cancer. To elucidate let-7’s role in temporal control of nematode development, we used
sequence analysis and reverse genetics to identify candidate let-7 target genes. We show that the nuclear
hormone receptor daf-12 is a let-7 target in seam cells, while the forkhead transcription factor pha-4 is a target
in the intestine. Additional likely targets are the zinc finger protein die-1 and the putative chromatin remodeling
factor lss-4. Together with the previous identification of the hunchback ortholog hbl-1 as a let-7 target in the
ventral nerve cord, our findings show that let-7 acts in at least three tissues to regulate different transcription
factors, raising the possibility of let-7 as a master temporal regulator.

MicroRNAs (miRNAs) constitute a large family of genomically encoded, regulatory RNAs found in plants and
animals (Lagos-Quintana et al., 2001; Lau et al., 2001; Lee et al., 2001; Lee et al., 1993; Llave et al., 2002; Reinhart et al., 2000; Reinhart et al., 2002). These RNAs are transcribed as longer precursor transcripts, from which the ca. 22 nucleotide long, mature miRNAs are derived through endonucleolytic processing (reviewed by Bartel, 2004). Animal miRNAs regulate the expression of their respective target genes by binding to partially complementary sequences in the 3' untranslated regions (3' UTRs) of target mRNAs, apparently resulting in inhibition of translation (Bartel, 2004). Some miRNAs, such as let-7, are perfectly conserved in species as divergent as nematodes and humans, indicating important developmental roles (Pasquinelli et al., 2000). However, for the vast majority of miRNAs, functions remain unknown. Similarly, although recent sequence analyses have allowed the prediction of hundreds of miRNA targets in fly, fish, mouse, and humans (Enright et al., 2003; John et al., 2004; Lewis et al., 2003; Stark et al., 2003), only a subset of these targets has been experimentally validated, and the significance of the predicted interactions in vivo is usually not known.

The founding members of the miRNA family, lin-4 and let-7, were initially identified in genetic screens for C.
elegans mutants exhibiting heterochronic defects, such as abnormalities in the pattern of temporal development
(Lee et al., 1993; Reinhart et al., 2000). In heterochronic mutants, cells adopt fates usually found at earlier
or later developmental times, resulting in retarded or precocious phenotypes, respectively (Rougvie, 2001).
lin-4 expression can first be detected in the first (L1) larval stage during C. elegans development, when it
directs downregulation of its target lin-14, thus promoting progression from the L1 to the L2 stage (Wightman
et al., 1993). The subsequent downregulation of a second lin-4 target, lin-28, is required for the execution of
the L3 larval stage (Moss et al., 1997). let-7, by contrast, is expressed later in development and is required
for the execution of the larval to adult (L/A) switch, which occurs at the end of the L4 stage (Reinhart et al.,
2000). This switch involves major changes in many tissues including the hypodermis (Reinhart et al., 2000),
but little is known about how let-7 mediates this developmental transition.

Two targets of let-7 have been identified: lin-41 prevents precocious for seam cell fusion and alae formation,
while hbl-1, the worm hunchback ortholog, appears to be a target of let-7 in the ventral nerve cord (Abrahante
et al., 2003; Lin et al., 2003; Slack et al., 2000). However, let-7 may regulate additional, partially redundant targets, as lin-41 or hbl-1 mutations cause only partially penetrant heterochronic and let-7 suppression phenotypes.

While few data are available on temporal patterning pathways in vertebrates (Rougvie, 2001), the strong
conservation of many C. elegans heterochronic genes in vertebrates suggests that a similar pathway might
function there. Indeed, a large family of let-7 homologs, including three with perfect sequence conservation in
the mature miRNA, have been identified in humans (Lagos-Quintana et al., 2001; Lim et al., 2003; Pasquinelli et al., 2000), and recent work revealed deletion or poor expression of let-7 in lung cancer tissues (Calin et al., 2004; Takamizawa et al., 2004).

To understand let-7’s function in the C. elegans L/A switch, as well as its potential role in vertebrates and
cancer, we sought genes that are regulated by let-7. Here, we identify nine novel potential let-7 targets through a sequence-based approach and subsequent genetic interactions. Using reporter assays, we validate the nuclear hormone receptor DAF-12, an important regulator of temporal patterning as well as aging and dauer formation, and the forkhead transcription factor and FOXA/HNF3 ortholog PHA-4 as let-7 targets. The zinc finger transcription factor die-1 and lss-4, a putative chromatin remodeling factor, are likely to be additional let-7 targets. Our results demonstrate that let-7 regulates a number of different transcription factors in different tissues at the larval to adult transition.

Results:

Computational Sequence Analysis Reveals Potential let-7 Target Genes

To find novel let-7 targets, we sought genes with let-7 family complementary sites (LCSs) in their 3' UTRs.
We first identified conserved elements in known and putative LCSs of five different heterochronic genes with
such sites in their 3' UTRs: lin-14, lin-28, lin-41, hbl-1, and daf-12 (Abrahante et al., 2003; Lin et al., 2003; Reinhart et al., 2000; Slack et al., 2000). An alignment of the training set of 16 LCSs present in these five genes showed greatest similarity at the LCS 3' ends (Supplemental Figure S1A). In contrast, the mature miRNA sequences from 15 let-7 family members from C. elegans and vertebrates (Supplemental Figure S1B;
Lagos-Quintana et al., 2001; Lagos-Quintana et al., 2002; Lau et al., 2001; Lim et al., 2003; Lim et al., 2003) are most similar at their 5' ends. Together, these two sets of alignments strongly suggest that the 5' end of the let-7 miRNA makes conserved base pairs with the 3' end of an LCS. The sequence similarity between members of the let-7 family of miRNAs also suggests that the previously predicted but unvalidated let-7 targets used in our training set, lin-28, daf-12, and lin-14, might be targets of one of these other let-7 miRNA family members rather than let-7 itself. Interestingly, vertebrate Lin-28 also appears to be regulated by the slightly divergent let-7 homolog let-7b rather than let-7a, which constitutes the ortholog of C. eleganslet-7 and is identical in sequence to it (Kiriakidou et al., 2004; Moss et al., 2003).

A pattern of conservation similar to what we observed for let-7was also seen for lin-4 family members and lin-4 targets (Supplemental Figures S1C and S1D), suggesting that it might constitute a general feature of target recognition by miRNAs. Indeed, computer analysis of Drosophila miRNAs and their putative targets among the Brd box and K box families also led to the suggestion that the 5' end of an miRNA might have an important role in target recognition or function (Lai, 2002). More recently, this phenomenon has also been observed in cell culture experiments and used as a basis for computational screens for miRNA targets in organisms as diverse as flies, fish, mice, and humans (Doench et al., 2004; Enright et al., 2003; Lewis et al., 2003; Stark et al., 2003).

We used MicroTarget (http://www.gersteinlab.org/proj/microrna; T.J. and M.G., unpublished data) to scan the
9,418 annotated 3'UTRs in the C. elegans Wormbase database for the presence of LCS characteristics shared by the known LCSs of our training set as detailed in the Experimental Procedures. We identified 1,529 3'UTRs, representing 1,280 genes, with at least one strong LCS site. This list included our training set genes except for lin-14, whose 3'UTR was not present in the original Wormbase 3'UTR data set. When we supplied the published sequence of lin-14 3' UTR, our program readily identified six LCSs and six lin-4 complementary elements (LCEs). From the complete set of computationally identified target 3'UTRs, 95 (representing 83 genes) contained more than two LCSs, or two LCSs and one or more LCE, a condition found in all the known published let-7 target genes. This short list of potential let-7 targets (Supplemental Table S1) contained the entire training set except for lin-28, whose 3'UTR contains only one LCS and one LCE (Moss et al., 1997; Reinhart et al., 2000).

Genetic Validation of Predicted let-7 Targets

let-7(n2853ts) mutant worms behave like genetic null mutants at the restrictive temperature (25°C) and die as
young adults by bursting through the vulva (Reinhart et al., 2000). This phenotype can be partially suppressed, and some viability thus restored, by knocking down expression of the known let-7 targets lin-41 and hbl-1 through RNA interference (RNAi) (Abrahante et al., 2003; Lin et al., 2003; Slack et al., 2000). This demonstrates that the let-7 mutant phenotype is at least partially caused by combined overexpression of lin-41 and hbl-1.

We reasoned that other true let-7 targets could also be overexpressed in a let-7 mutant and contribute to the
inviability phenotype, so that reducing their expression might similarly suppress let-7 lethality. Using an RNAi
library (Fraser et al., 2000; Kamath et al., 2003) and additional RNAi constructs prepared for this study, we examined 73 of the 83 short-listed genes for their ability to suppress the lethality caused by the let-7(n2853)
mutation. We found that in addition to lin-14, lin-41, and hbl-1, 9 of the 73 tested genes partially suppressed
let-7(n2853ts) lethality at nonpermissive temperatures, 20°C and 25°C (Table 1). While our list of 83 genes is
thus likely to contain a substantial number of false positives, we believe that additional predicted genes may be
true let-7 targets that do not suppress let-7(n2853) lethality because their levels may not be sufficiently reduced
by RNAi or because they do not contribute to this particular let-7 mutant phenotype. Some predicted targets may also be regulated by let-7 family members rather than by let-7 itself.

Table 1. Suppressors of let-7(n2853) Lethality

[a] Gene loci names according to Wormbase, Release 104. ORF, open reading frame; lss, letsevensuppressor.
[b] Dm, Drosophila melanogaster; Hs, Homo sapiens.
[c] LCS: Number of let-7 complementary sites identified in the 3'UTR of the indicated gene. In daf-12, six LCSs were identified computationally; an additional two sites were found by visual inspection.
[d] RNAi suppression of let-7(n2853ts) lethality at the indicated, nonpermissive temperature displayed as % viable adult animals. Over 100 let-7(n2853ts) animals were scored in each of two separate experiments for each RNAi construct. Under these conditions, control animals fed on bacteria carrying the empty vector showed 11% and 6% survival at 20 and 25°C, respectively.
[e] RNAi suppression of lethality associated with let-7(mn112) null allele. >50 animals were examined for each
construct. (+): >25% viable adults. (+): Only ~15% of let-7(mn112)let-60(RNAi) animals survived but most of
these had eggs. As a control, 0% of animals survived when fed the empty vector.

The majority of the novel let-7 interactors encode transcription factors (Table 1). Of the remaining four genes,
three encode known or predicted signal transduction molecules. LET-60 is the C. elegans RAS ortholog (Han et al., 1990); PAR-5 is a 14-3-3 protein involved in early embryonic cell polarity (Morton et al., 2002); and LSS-18 is homologous to MO25, which is involved in binding and activating various mammalian kinases (Boudeau et al., 2003). C48B6.6 is related to the human PIK kinase hSMG-1, which has a role in nonsense-mediated decay (Yamashita et al., 2001).

Some of the novel suppressors, notably daf-12, pha-4, die-1, and lss-4 (let-seven suppressor), were very efficient and could also partially suppress the let-7(mn112) null mutant allele (Table 1). To determine whether the extent of suppression correlated with the efficiency of RNAi, we examined the effect of RNAi on the mRNA levels of the weak suppressors lin-59 and let-60, and of the strong suppressors lss-4, die-1, and pha-4 (Figure 1). We found comparable levels of reduction of these mRNAs, typically by ~80%, suggesting that inefficient RNAi might not be a major reason for weaker suppression by some of the genes. It is possible that weaker suppression reflects a less important contribution to the lethality of let-7 mutations by smg-1, lin-59, let-60, and lss-18. We focused our subsequent analysis on the strong suppressors.

Figure 1. RNAi of Both Weak and Strong let-7(n2853) Suppressors Causes a Significant Reduction of the Levels of the Respective Target mRNAs

Figure 1. RNAi of Both Weak and Strong let-7(n2853) Suppressors Causes a Significant Reduction of the Levels of the Respective Target mRNAs

Aliquots of undiluted or 1:5 diluted templates were used for RT-PCR as indicated to determine whether target mRNA levels were reduced compared to the control eft-2 mRNA; “-” indicates the control reaction without reverse transcriptase.

daf-12(lf) Suppresses the Heterochronic Phenotypes of let-7 Mutants

Our combined sequence and reverse genetic analysis identified the nuclear hormone receptor DAF-12 as a
potential let-7 target. DAF-12 is an important regulator of development in C. elegans, where it controls
developmental timing, diapause, gonad migration, and lifespan (Antebi et al., 1998; Antebi et al., 2000; Ao et al., 2004; Gems et al., 1998; Larsen et al., 1995). Although daf-12 had been previously identified as a heterochronic gene with putative LCSs (Reinhart et al., 2000) and was thus included in our training set, no interaction with let-7 had so far been demonstrated and was not actually anticipated. This is because the alleles of daf-12 with heterochronic phenotypes such as daf-12(rh61) cause a retarded seam cell phenotype (Antebi et al., 1998), thus acting in the same direction as let-7 mutations. By contrast, the opposite, precocious seam cell phenotype is observed when the known let-7 targets lin-41 or hbl-1 are mutated (Abrahante et al., 2003; Lin et al., 2003; Slack et al., 2000). Moreover, a mutation in the early temporal patterning gene lin-28 is epistatic to daf-12(rh61) (Antebi et al., 1998), suggesting a function of daf-12 early in the pathway before let-7 is expressed. Based on the fact that daf-12(rh61) mutant animals display a delayed onset of let-7 transcription, it has been suggested that DAF-12 activates the let-7 gene (Johnson et al., 2003). Taken together, these findings indicated that DAF-12 acted upstream of let-7. The daf-12LCSs thus appeared to be more likely to mediate regulation by let-7 family members such as mir-84 or mir-48, which are expressed early in development (A. Kerscher and F.J.S., unpublished data; Lau et al., 2001), than by the later expressed let-7 itself. However, our new work shows that daf-12(RNAi), using an RNAi construct that targets all three daf-12 splice variants, suppresses let-7 mutations, suggesting that daf-12 is a likely downstream target of let-7.

To confirm that daf-12(RNAi) did not suppress the let-7(n2853ts) mutation through off-target effects, we
constructed genetic let-7 daf-12 double mutants. daf-12 mutant alleles fall into six different classes depending
on their phenotypes (Antebi et al., 2000). daf-12(rh61) belongs to a small class with retarded heterochronic
phenotypes. Strikingly, the daf-12(rh61) phenotypes are not observed with more severe alleles, and rh61 is
considered a recessive gain-of-function (gf) allele (Antebi et al., 2000). We therefore chose the daf-12(m20) loss-of-function (lf) allele, which is not known to cause heterochronic phenotypes. This allele, which is designated the daf-12 reference allele by Wormbase, has a nonsense mutation in the DNA binding domain, which is shared by two of the three daf-12 isoforms; the third isoform does not encode this domain (Antebi et al., 2000).

Like their daf-12(RNAi); let-7(n2853) mutant counterparts, most daf-12(m20) let-7(n2853) double mutant
animals were viable and fertile at 20°C and 25°C (Figure 2A–2C; Table 2), suggesting that the daf-12(RNAi)
phenotype is indeed specific. daf-12(m20) not only suppressed the let-7(n2853ts) allele but also the let-7(mn112) null allele, albeit to a lesser extent (Figure 2D). While let-7(mn112) mutant worms died as young adults by bursting through the vulva (<2% survival, n = 322), more than 75% of let-7(mn112) daf-12(m20) double mutant worms survived into adulthood (n = 738) and produced eggs (0–27 eggs with a mean of 8.6, n = 40). The fact that the let-7 null mutant phenotype depends on a wild-type copy of daf-12 supports the idea that daf-12 acts downstream of let-7.

Figure 2. daf-12 Interacts with let-7 and Affects the Timing of Seam Cell Fusion

Figure 2. daf-12 Interacts with let-7 and Affects the Timing of Seam Cell Fusion

(A) let-7(n2853) mutants die at the L4 molt by bursting through the vulva (arrowhead), while (B) daf-12(m20) let-7(n2853) double mutant adult animals survive into adulthood, as indicated by the presence of oocytes (asterisk) and a functional vulva (arrowhead) (B). (C) These animals produce viable embryos. (D) daf-12(m20) let-7(mn112) adults are viable and fertile (arrows, embryos). (E and F) Alae are absent from (E) let-7(n2853) single mutant worms but present (arrows) in (F) let-7(n2853) daf-12(m20) double mutant worms grown at 20°C. (G) Wild-type N2; wIs79 animals have unfused seam cells at the early L4 stage as indicated by AJM-1/GFP signal at the cell junctions (arrowheads). (H) daf-12(RNAi) animals occasionally display precocious seam cell fusion as indicated by lack of GFP signal at cell junctions. Arrows in lower DIC panels in (G) and (H) point to distal gonad tips at a different part of the animals, confirming appropriate developmental stage. (I) Precocious seam cell fusion is strongly enhanced in lin-41(ma104); daf-12(RNAi) double mutant animals at the L3 molt. Magnification in (A)–(D) is 400× except for the left panel in (C), which was observed at 100×; in (E)–(H) it is 630×. Anterior of each animal is left, ventral down.

Table 2. daf-12(m20) Suppresses let-7(n2853) Phenotypes

daf-12(lf) Mutations Can Promote Precocious Development

In addition to the lethality phenotype, let-7 mutations cause retarded development of seam cells (Reinhart et al., 2000; Slack et al., 2000). In let-7(n2853) adults, seam cells fail to terminally differentiate and instead divide (Reinhart et al., 2000). The resulting absence of alae in young adult let-7(n2853) animals (Figure 2E) was reversed in let-7(n2853ts) daf-12(m20) double mutant animals, which almost always displayed complete alae (Table 2; Figure 2F). This finding suggests that the restoration of viability in let-7 mutant animals through loss of DAF-12 activity may be linked to the function of both genes in the heterochronic pathway. Importantly, and in contrast to previous observations, these data also indicate that daf-12 mutations can actually promote, rather than delay, execution of later developmental cell fates.

To test this latter idea directly, we examined the effect of daf-12(RNAi) on seam cell terminal differentiation and alae formation. In agreement with earlier findings (Antebi et al., 1998), daf-12(RNAi) failed to elicit a precocious alae phenotype, and only a small percentage of animals displayed precocious seam cell fusion (Figure 2G–2I and data not shown). However, in the sensitized heterochronic lin-41(RNAi) background, daf-12(RNAi) greatly enhanced precocious seam cell fusion (Figure 2I). We conclude that wild-type DAF-12 is required to prevent premature execution of adult developmental fates, consistent with the roles of other known let-7 targets (Abrahante et al., 2003; Lin et al., 2003; Slack et al., 2000)

The daf-12 3' UTR Can Confer Posttranscriptional Gene Repression

Eight LCSs in the daf-12 3' UTR have the potential to base pair with let-7 (Figure 3A; Supplemental Figure S2). When we aligned the genomic DNA sequence from the C. elegansdaf-12 3' UTR with that from a related nematode, C. briggsae (Stein et al., 2003), we found that four of the LCSs (LCS 3, 5, 7, and 8; Figure 3A; Supplemental Figure S2) are highly conserved. This suggests that these sites in particular may be important for daf-12 regulation and that daf-12 is likely to be a conserved target of let-7. Strikingly, all nucleotide changes observed between the two species in two of the daf-12 LCSs retain base pairing by substituting G:U base pairs with A:U base pairs (Figure 3A). This may indicate that base pairing in these parts of the duplex is important for LCS function and that G:U and A:U base pairs can, to some degree, act interchangeably.

Figure 3. The daf-12 3'UTR Mediates Posttranscriptional, let-7-Dependent Control of Gene Expression

Figure 3. The daf-12 3'UTR Mediates Posttranscriptional, let-7-Dependent Control of Gene Expression

(A) Examples of LCS:let-7 duplexes predicted for daf-12; let-7 are shown in gray. Arrows indicate where the sequence of C. briggsae daf-12 (uppercase) differs from C. elegansdaf-12. (B) Diagram of the col-10::lacZ::daf-12 reporter gene construct. (C) The reporter gene is efficiently expressed in early larval (L1–L3 stage) animals but downregulated in L4 and adult animals. Deletion of the first four (DLCS1–4) or the last four (DLCS5–8) LCSs impairs downregulation (*p < 0.05, **p < 0.01, paired Student’s t test). (D) Repression is relieved in the let-7(n2853) mutant background (four lines) (*p < 0.05). Error bars indicate standard deviations.

Intestinal Expression of pha-4 Is Temporally Regulated by Its 3'UTR and let-7

pha-4 is a forkhead transcription factor with a role in pharynx organogenesis during embryogenesis (Horner et al., 1998; Kalb et al., 1998), where it regulates >100 genes, many of them directly (Gaudet et al., 2002). Interestingly, the onset of expression of the direct target genes appears to depend on the affinity of PHA-4 to the PHA-4 binding sites in the target promoter, suggesting a mechanism by which this transcription factor can direct different temporal transcription patterns in the same target tissue (Gaudet et al., 2002). PHA-4 is also important for normal postembryonic development, although its exact function remains unclear, and it is required for exit from diapause (Ao et al., 2004; Gaudet et al., 2002).

To determine whether pha-4 exhibits a let-7-dependent temporal expression pattern, we examined the
expression of a PHA-4/GFP fusion protein transcribed from a transgene containing the pha-4 3'UTR (S.
Mango, personal communication). We observed fluorescent signal in a number of tissues during development,
including several head neurons and the intestine but not the hypodermis (Figure 4B and data not shown). The
intestinal GFP signal was reduced in all adult animals (n > 20) compared to L1, L2, and L3 larval animals
(Figure 4B and 4D), demonstrating that pha-4::gfp is downregulated during the larval to adult transition.
Together with the previous observations that the development of the intestine is under the control of
heterochronic genes and that let-7 is expressed in the intestine (Antebi et al., 1998; Johnson et al., 2003),
our finding suggests that let-7 might regulate pha-4 expression in the intestine by binding to its 3'UTR.
Consistent with this notion, downregulation of PHA-4/GFP was eliminated (n > 20) when the pha-4 3'UTR
was replaced by the 3'UTR of the nontemporally regulated unc-54 gene (S. Mango, personal communication;
Figure 4C). Because the two PHA-4 fusion constructs differ slightly, with pha-4::gfp::unc-54 encoding only a
partial PHA-4 protein, it is possible that additional factors, such as PHA-4 sequences determining protein
stability, might contribute to the observed difference in expression pattern between the two constructs.
However, when we examined adult pha-4::gfp::pha-4_3'UTR expression in a let-7(n2853) mutant background
at the permissive temperature, we observed persistence of strong intestinal GFP expression in all living adult
animals (n > 20; Figure 4E), indicating that the pha-4::gfp expression pattern requires active let-7. These data
and our sequence and genetic analyses thus support the idea that pha-4 is a target of let-7 in the intestine.

Figure 4. The pha-4 3'UTR Regulates let-7-Dependent Downregulation of Intestinal pha-4::gfp Expression

Figure 4. The pha-4 3'UTR Regulates let-7-Dependent Downregulation of Intestinal pha-4::gfp Expression

(A) Examples of LCS:let-7 duplexes predicted for pha-4. (B and D) A pha-4::gfp::pha-4 3'UTR transgene is highly expressed in the intestinal cells of early stage (L3) larvae but is only weakly visible in L4 and adult animals. (C) Strong intestinal expression persists in adult animals when the pha-4 3'UTR of the transgene is replaced by the unc-54 3'UTR. (E) let-7(n2853) mutant animals continue to express the pha-4::gfp::pha_3'UTR transgene as adults. Images in (D) and (E) were taken for the same exposure time and processed identically.

In addition to daf-12 and pha-4, another four of our candidate let-7 target genes have a known or predicted role in transcription (Table 1). We tested the 3'UTRs of two of these, lss-4 and die-1, in our reporter assay (Figure 5). lss-4 encodes a putative chromatin remodeling factor (Simon et al., 2002), while die-1 codes for a zinc finger protein involved in hypodermal cell rearrangements in embryogenesis (Heid et al., 2001). Interestingly, die-1 activity is also required for expression of the lsy-6 miRNA during neuronal development, and neuronal die-1 expression itself is regulated by the mir-273 miRNA (Chang et al., 2004). While the die-1 and lss-4 3'UTRs conferred less robust downregulation to the hypodermally expressed reporter genes than did the daf-12 3'UTR, the observed effect was statistically significant and let-7 dependent, suggesting that die-1 and lss-4 also constitute valid let-7 targets. These findings may thus indicate a bias of let-7 toward regulating transcription factors.

Figure 5. The 3'UTRs of Additional Predicted Transcription Factor Targets Confer let-7-Dependent Regulation

Figure 5. The 3'UTRs of Additional Predicted Transcription Factor Targets Confer let-7-Dependent Regulation

(A and B) Examples of LCS:let-7 duplexes predicted for the indicated transcription factors; the let-7 sequence is shown in gray. (C) The expression of the col-10::lacZ reporter construct is downregulated in wild-type (N2) adult animals when the reporter contains the lss-4 or die-1 3'UTRs (*p < 0.05, **p < 0.01, paired Student’s t test; error bars are standard deviations between independent lines). (D and E) In contrast, expression of the reporter genes persists in adult let-7(n2853) mutant animals (right columns). Four let-7(n2853); col-10::lacZ::lss-4 and seven let-7(n2853); col-10::lacZ::die-1 lines, respectively, were assayed along with their respective isogenic parent (*p < 0.05, **p < 0.01, paired Student’s t test).

In C. elegans, let-7 controls cell fate determination during the transition from larva into adult in hypodermal
cells, but little is known about how this switch occurs. To understand this important temporal transition, we
sought genes that are regulated by let-7. In this study, we used a bioinformatics approach to identify potential
let-7 target genes as those with multiple let-7 complementary sites in their 3'UTRs. We have shown that RNAi
of 12 of these potential let-7 targets suppresses a let-7 mutation in a manner consistent with valid targets of this
miRNA. The C. briggsae orthologs of these 12 genes all contain putative LCSs in their 3'UTRs, though not
always in a conserved position (Figure 3 and data not shown), suggesting possible conservation of function.
Reporter gene analyses provided further evidence that some of the let-7 suppressors are indeed regulated by
let-7 (this work; Johnson et al., 2005), and are therefore likely to be direct targets of let-7. Interestingly, two of
the validated targets, DAF-12 and PHA-4, have human orthologs, VDR and FOXA1/HNF3A, respectively, that are linked to cancer (Vandewalle et al., 1994; Yasui et al., 2001), and these genes also have LCSs in
their 3'UTRs (H.G. and F.J.S., unpublished data). It is thus an intriguing possibility, to be addressed in future
experiments, that misregulation of these genes contributes to let-7’s recently discovered involvement in lung
cancer (Calin et al., 2004; Abrahante et al., 2003; Takamizawa et al., 2004 ).

let-7 Regulates daf-12 through the daf-12 3'UTR

Among the genes identified and confirmed as let-7 targets was daf-12, whose 3'UTR contains multiple conserved let-7 complementary sites. DAF-12 is a transcription factor with many roles in C. elegans
development including temporal patterning, dauer formation, and aging (Antebi et al., 1998; Antebi et al., 2000; Ao et al., 2004; Gems et al., 1998; Larsen et al., 1995). DAF-12 was previously suggested to act upstream of let-7 in the heterochronic pathway (Antebi et al., 1998), but our work presents multiple lines of evidence suggesting that DAF-12 is also a true let-7 target. Firstly, daf-12(lf) mutants partially suppress let-7 mutant phenotypes and potentiate precocious heterochronic phenotypes. Second, a reporter gene carrying the daf-12 3'UTR is downregulated concomitant with the onset of let-7 expression, and so is a daf-12::gfp::daf-12_3'UTR translational fusion (Antebi et al., 2000). Third, downregulation of the lacZ reporter is impaired in a let-7(n2853) mutant background or if a subset of LCSs is deleted from the daf-12 3'UTR. We note that we were unable to create a let-7(n2853) mutant strain expressing a functional DAF-12A/GFP fusion using a previously published, healthy daf-12A::gfp parental strain (Antebi et al., 2000). It thus appears possible, although unproven, that daf-12 overexpression or expression at later stages is lethal. Certainly, wild-type daf-12 contributes to the inviability of let-7 mutant animals, and we conclude that one important role for let-7 in the larval to adult transition is to downregulate daf-12 expression. Based on our data and previous reports (Antebi et al., 1998; Johnson et al., 2003), we propose a feedback loop between let-7 and daf-12, where DAF-12 activity is initially required to turn on, but not to sustain, let-7 transcription. Once let-7 miRNA has sufficiently accumulated, it may bind to the daf-12 3'UTR and repress DAF-12 production.

let-7 as a Master Temporal Regulator

Along with daf-12, we identified three additional transcription factor targets of let-7, namely pha-4, lss-4, and
die-1. Another two of the candidate target genes that have not yet been investigated by reporter gene assays
also have a known or predicted role in transcription (Table 1). Together with previous work that identified the
hbl-1 transcription factor as a let-7 target (Abrahante et al., 2003; Lin et al., 2003), these findings may
indicate a bias of let-7 toward regulating transcription factors. Downregulation of transcription factors might
allow the cell to connect miRNA-mediated translational control, with its advantage of rapid onset, to
transcriptional control of a larger number of more downstream targets in the pathway.

Interestingly, let-7 appears to target these factors in at least three different tissues, namely the hypodermis
(daf-12), the intestine (pha-4), and the ventral nerve cord (hbl-1) (Abrahante et al., 2003; Lin et al., 2003). In addition, the lss-4 and die-1 3'UTRs can mediate let-7-dependent reporter gene repression in the hypodermis, where die-1 is active during embryonic development (Heid et al., 2001), but we do not know whether these genes are normally expressed in this tissue during postembryonic development. Based on these observations, we propose that let-7 may be a master temporal regulator of late larval development in C. elegans. This idea appears particularly intriguing in the wake of the finding that let-7 is perfectly conserved in sequence across phylogeny and temporally expressed in a number of animals (Pasquinelli et al., 2000). The presence of a substantial number of direct let-7 targets in the genome of an organism would constrain sequence divergence by necessitating coevolution of the miRNA and its target, particularly if the targets themselves are important developmental regulators.

Experimental Procedures:

Bioinformatics Analysis

Initial putative binding sites were identified by comparing the miRNA sequence to the annotated 3'UTRs in
AceDB version 4_9p using ssearch34, an implementation of the Smith-Waterman algorithm contained in the fasta package (Pearson, 1991; Smith et al., 1981), followed by iterative sequence masking to reveal
additional potential sites in a given 3'UTR. Gap penalties were reduced to -5 for gap opening and -2 for gap
extension to reflect the observation that known biologically active sites frequently contain bulges with minimal
anchor sequence to either side. Alphabetic substitution and an appropriately altered scoring matrix were used to
allow for G:U base pairing. To identify LCSs and LCEs, respectively, these raw alignments were then tested
against the following criteria derived from our training set (see Results): (a) six or more matches in the last eight bases of the complementary site. Seven or more matches in the first 10 nucleotides indicated a “strong” site. Here, a “match” includes G:U base pairs, except that no G:U pairs were allowed for the CCTC of an LCS, and the TCAGG of an LCE; (b) 15 or more exact matches over the entire LCS sequence, 12 or more over an
LCE. (c) We eliminated all sequences with a maximum contiguous match of less than six. “Contiguous” is
defined as having one or fewer mismatches per every four matches. This criterion ensures that rather than having mismatches spread evenly throughout the duplex, they are restricted to fewer regions, such as loops, so that the longer stretches of matching sequence allow efficient duplex formation.

RNAi Experiments

Gene knockdown was achieved through RNAi by feeding using previously published RNAi constructs and
protocols (Fraser et al., 2000; Kamath et al., 2003; Timmons et al., 1998). Media supplements were used at the following concentrations: ampicillin, 100 µg/ml; tetracycline, 12.5 µg/ml; isopropyl-b-D-thiogalactopyranoside, 1 mM. The initial validation of predicted let-7 targets was performed in 12- or 24-well multiwell plates with ~100 synchronized let-7(n2853) L1 larvae per well, at both 20°C and 25°C. Retesting was performed at 20°C and 25°C on 6 cm diameter plates with >100 synchronized L1 larvae per plate. Clones were considered positive when at least 25% of the animals were viable as adults at least at one temperature, 20°C or 25°C. Control animals fed bacteria carrying the empty vector showed 11% and 6% survival under these respective conditions; these are somewhat higher levels of survival than in the presence of OP50 bacterial food on standard NGM plates, where <3% survive at either temperature (Table 2). We designed primers to amplify and clone the following additional genes into the L4440 RNAi vector: nhr-28, R10E4.2, and F54A5.3. These clones, as well as all clones producing a let-7 suppressor phenotype, were sequence verified.

For double-RNAi experiments, bacteria carrying a daf-12(RNAi) construct were mixed with an equal number
of cells (determined by optical density at 600 nm) carrying a lin-41(RNAi) construct. To control for potential
dilution effects, the single-RNAi control experiments (daf-12(RNAi) alone or lin-41(RNAi) alone) were
similarly mixed with an equal number of bacteria carrying an empty vector. Synchronized N2; wIs79
[ajm-1::gfp; scm::gfp] L1 larvae were added to the plates and grown at 25°C.

RT-PCR

We used RT-PCR analysis to verify gene knockdown. Synchronized L1 stage N2 animals were plated on
L4440 (empty vector/control), lss-4(RNAi), lin-59(RNAi), let-60(RNAi), pha-4(RNAi), or die-1(RNAi)
plates. Animals were harvested at the L4 molt (43 hr at 20°C after plating) and flash-frozen in liquid nitrogen.
Frozen worms were ground in a mortar and RNA extracted using Trizol reagent (Invitrogen, Carlsbad, CA)
according to the manufacturer’s instructions. DNA was removed from samples using DNAfree (Ambion,
Austin, TX) according to the manufacturer’s instructions, and integrity of the resulting RNA was confirmed by
agarose gel electrophoresis. Total RNA (1 µg) was used for reverse transcription with Ambion’s RetroScript kit
according to the manufacturer’s instructions; control reactions without reverse transcriptase were assembled in
parallel. To avoid amplification of potential dsRNA intermediates of RNAs targeted for RNAi-mediated
degradation, reverse transcription was performed with oligo(dT) primers. PCR reactions were run with 1 µl of
undiluted or 1:5 diluted reverse transcribed template for 28 (eft-2) or 30 cycles (all other RNAs). Undiluted
template was used for the reaction without reverse transcriptase. The following primers were used:
DIE-1F1:  5'-GACGGAGATTCAACGGCAGCC-3',
DIE-1R1:  5'-GATCGGACAGGCAGTTGAGCAAG-3',
LET-60F1:  5'-TCGACATATTGGATACCGCC-3',
LET-60R1:  5'-TGACGCTCACGATGCTTGC-3',
LIN-59F1:   5'-CTTCTACGCGTGGCCTCAT-3',
LIN-59R2:   5'-GAGATGGATTCGATGGATCG-3',
LSS-4F1:    5'-ACGGTCTGCTGCTGCAC-3',
LSS-4R1:   5'-GCAACGGCTGTCGTCAGAG-3',
PHA-4F1:   5'-TCTTGGCCTAATTGACCCATCG-3',
PHA-4R1:   5'-TAGGTTGGCGGCCGAGTT-3'.
All primers were chosen such that potentially remaining genomic DNA contamination would have resulted in larger amplification products that would have been easily distinguishable from reverse transcribed products on the 2% agarose gel; however, no contaminating bands were observed in any sample. The control primers against the eft-2 housekeeping gene were adapted from Bracht et al., 2004);
EFT-2F:    5'-GTCAACTTCACGGTCGATG-3',
EFT-2R:   5'-TCCGAGCTTCTCAACAAGAGC-3'.
Twenty microliter aliquots of PCR reactions were separated on 2% agarose gels. Exposure times for image acquisitions were selected so that no signal saturation occurred. Image levels were adjusted using Adobe Photoshop as necessary while taking care that no information was discarded and without changing g; this processing step was only performed on entire panels (that is, mock and specific RNAi were affected equally).

X-Gal Assay and Transgenic Lines

X-Gal staining of animals expressing the lacZ gene transcribed under the control of the constitutive, seam
cell–specific col-10 promoter and fused to the 3'UTR of interest was performed as described (Wightman et
al., 1993). To obtain pFS1051, pFS1048, and pFS1039, the entire 3'UTRs of daf-12, die-1, and lss-4,
respectively, were amplified from genomic DNA using PCR and cloned into plasmid B29 (Ha et al., 1996),
while in pHG16 and pHG18 the full-length daf-12 3'UTR of pFS1051 was replaced with truncated
sequences covering nt 160–1388 and nt 1–502, respectively (Supplemental Figure S2). Constructs were
injected into N2 wild-type animals at 5 ng/µl with rol-6(su1006) as a coinjection marker at 80 ng/µl. We
examined the following number of independent lines, grown at 20°C: four (daf-12), five (die-1;
daf-12DLCS1-4; daf-12DLCS5-8), and seven (lss-4). One representative transgenic line for each full-length
3'UTR construct was crossed into a let-7(n2853) mutant strain. In these experiments, the respective parental
wild-type line was used as a control and tested in two (daf-12), five (lss-4), and six (die-1) independent
experiments. Both the mutant and the wild-type lines were grown at the let-7(n2853ts) permissive temperature,
15°C. A one-tailed, paired Student’s t test was used to determine whether the observed downregulation of
expression in L4 or adult animals was significant, except in the case of N2; pFS1051 in Figure 3D, where the
unpaired test was used to accommodate the small sample number.

pha-4::gfp Strains

Strains SM141 (pxIs1 [pha-4::gfp::pha-4_3'UTR, rol-6(su1006)]) and SM469 (pxIs6 [pha-4::gfp::unc-54_3'UTR; rol-6(su1006)]) were kindly provided by Dr. Susan Mango (Alder et al., 2003). In SM141, GFP has been fused to the C terminus of PHA-4, while SM469 encodes only a partial PHA-4 protein; GFP starts after the second intron.

Miscellaneous

Microscopy images were acquired using a Zeiss Axioplan II microscope equipped with a Zeisscam CCD camera and Axiovision software (Zeiss, Thornwood, NY).

Acknowledgments:

We thank Dr. Susan Mango for the pha-4::gfp strains, Dr. Adam Antebi for the daf-12A::gfp strain, Dr. Aurora Kerscher for assisting with microinjections, and Dr. Kerscher and Dr. Diya Banerjee for comments on this manuscript. Additional nematode strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR). H.G. was supported by
a postdoctoral fellowship from the International Human Frontier Science Program Organisation. F.J.S. was
supported by NIH R01 grant GM62594. M.G. was supported by the Yale Center of Excellence in Genomic
Sciences.

Supplemental Data:

Supplemental Document S1.
   Supplementary Table 1.
   Supplementary Figure 1.
   Supplementary Figure 2.
http://www.developmentalcell.com/cgi/content/full/8/3/321/DC1/

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   This detailed and exciting new study by Helge Großhans, Ted Johnson, Kristy Reinert, Mark Gerstein, and Frank Slack reveals some of the profound effects produced by microRNA let-7 during the maturation phase from larva to adult in C. elegans. It also reveals the conservation of almost-identical let-7 sequences from nematode and Drosophila through the vertebrates to humans, the possible role played by let-7 sequences in human embryological stem cell differentiation, and the fact of a pathological reduction of let-7 expression within some human neoplasms. They confirm the reality of let-7 as a master temporal regulator gene.

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