Functional genomic analysis of the let-7 regulatory network in Caenorhabditis elegans.

The let-7 microRNA (miRNA) regulates cellular differentiation across many animal species. Loss of let-7 activity causes abnormal development in Caenorhabditis elegans and unchecked cellular proliferation in human cells, which contributes to tumorigenesis. These defects are due to improper expression of protein-coding genes normally under let-7 regulation. While some direct targets of let-7 have been identified, the genome-wide effect of let-7 insufficiency in a developing animal has not been fully investigated. Here we report the results of molecular and genetic assays aimed at determining the global network of genes regulated by let-7 in C. elegans. By screening for mis-regulated genes that also contribute to let-7 mutant phenotypes, we derived a list of physiologically relevant potential targets of let-7 regulation. Twenty new suppressors of the rupturing vulva or extra seam cell division phenotypes characteristic of let-7 mutants emerged. Three of these genes, opt-2, prmt-1, and T27D12.1, were found to associate with Argonaute in a let-7-dependent manner and are likely novel direct targets of this miRNA. Overall, a complex network of genes with various activities is subject to let-7 regulation to coordinate developmental timing across tissues during worm development

Genes down-regulated more than 2-fold in <i>let-7(n2853)</i> compared to wild-type.

1<p>Sequence names from WormBase (<a href="http://www.wormbase.org" target="_blank">http://www.wormbase.org</a>).</p>2<p>FDR corrected.</p>3<p>W = mirWIP <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Hammell1" target="_blank">[41]</a>, P = PITA <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Kertesz1" target="_blank">[40]</a>, Y = (this study), T = TargetScan <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Lewis2" target="_blank">[69]</a>, R = RNA22 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Miranda1" target="_blank">[39]</a>, G = MicroTarget <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Grosshans1" target="_blank">[11]</a>, M = Miranda <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Enright1" target="_blank">[36]</a>, C = PicTar <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Lall1" target="_blank">[38]</a>.</p

Phenotypic suppressors of let-7 mutants.

1<p>Sequence names from WormBase (<a href="http://www.wormbase.org" target="_blank">http://www.wormbase.org</a>).</p>2<p>W = mirWIP <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Hammell1" target="_blank">[41]</a>, P = PITA <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Kertesz1" target="_blank">[40]</a>, Y = (this paper), T = TargetScan <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Lewis2" target="_blank">[69]</a>, R = RNA22 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Miranda1" target="_blank">[39]</a>, G = MicroTarget <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Grosshans1" target="_blank">[11]</a>, M = Miranda <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Enright1" target="_blank">[36]</a>, C = PicTar <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Lall1" target="_blank">[38]</a>.</p>3<p>R = Suppression of rupturing phenotype (% non-rupture), S = Suppression of the extra seam cell nuclei phenotype (significance level.</p>4<p>Locations of ALG-1 binding sites C =  coding region, I = intron, 3 = 3' UTR <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Zisoulis1" target="_blank">[66]</a>.</p

Novel suppressors of vulval rupture in <i>let-7</i> null mutants.

<p>(A) Null <i>let-7(mn112)</i> worms were maintained with an extrachromosomal rescuing transgene (let-7(+)) co-expressing a pharyngeal GFP marker (myo-2::GFP). Progeny that lack the transgene rupture from the vulva and die. (B) The <i>let-7(mn112); Ex[let-7(+);myo-2::GFP]</i> strain was grown on bacteria expressing dsRNA corresponding to candidate targets and the empty vector control. The percent rupture of non-rescued (non-GFP) animals was determined for each RNAi clone. (C) The vector control RNAi fails to suppress vulval rupturing, while knockdown of a known target (<i>daf-12</i>) or a novel candidate (<i>sox-2</i>) allows <i>let-7(mn112)</i> animals to survive to adulthood. (D) The rate of vulval rupture was plotted for each RNAi clone tested. Green points indicate clones that reduced the rupture rate to below 75% in 2/2 experiments (n>50 worms/experiment). Purple points indicate RNAi clones depicted in (C). Red points indicate clones that failed to reproducibly meet the 75% cut-off. The vector negative controls are shown in black.</p

Suppression of supernumerary seam cell nuclei in <i>let-7</i> mutants.

<p>(A) While wild-type worms have 16 seam cell nuclei, <i>let-7(n2853)</i> worms have significantly more (∼23) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Hayes1" target="_blank">[82]</a>. To score for suppression of the extra seam cell phenotype, <i>let-7</i> mutants expressing nuclear GFP in seam cells (<i>let-7(n2853)</i>;<i>Int[scm::GFP]</i>) were grown at the restrictive temperature (25<b>°</b>C) on bacteria expressing dsRNA against candidate targets and the vector control. The number of seam cell nuclei was counted in a population of 20 worms evaluated against the same size population concurrently grown on the empty vector control. RNAi clones that resulted in worm populations with significantly lower seam cell numbers (p<0.05) were retested and scored using a population of at least 20 worms. (B) Suppressors of the extra seam cell nuclei phenotype in <i>let-7(n2853)</i> (p-value<0.05) are shown by bubble plot. Each bubble indicates the number of seam cell nuclei per worm for a population (n≥20) and the size of each bubble is proportional to the number of the animals in the population with a given seam cell number. * p<0.05, ** p<0.01, *** p<0.0001 in two independent trials.</p

Argonaute associates with targets in a <i>let-7</i>–dependent manner.

<p>(A) Sequences in the indicated genes were detected by semi-quantitative PCR of cDNA from ALG-1 immunoprecipitation assays from L4 staged WT and <i>let-7(n2853)</i> strains. Based on enrichment in the WT compared to <i>let-7</i> RIP from 4 independent experiments, three new targets were identified, <i>T27D12.1</i>, <i>prmt-1</i>, and <i>opt-2</i>. (B) let-7 complementary sites (LCS) are present in each of the newly identified targets. Each LCS is within an ALG-1 binding site. (C) qPCR analysis of WT and <i>let-7(n2853)</i> cDNA from L4 staged worms. Targets were normalized to 18S ribosomal RNA. Shown is the average and standard deviation from 3 independent experiments.</p

Differential effects of <i>let-7</i> target candidates on vulva formation.

<p>(A) Micrographs of the protruding multiple vulva (pmuv) phenotype in <i>lin-28(n719)</i> and the suppression to a single protruding vulva (pvul) when combined with <i>let-7(mn112)</i>. White arrowheads point to protruding vulvas in the mutants. (B) To screen for changes in the pmuv phenotype, 50–100 <i>lin-28(n719)</i> worms were grown to adulthood on vector control or gene specific RNAi plates (x-axis) and scored for percentage of pmuv (y-axis). The bar graphs represent the average percent of pmuv worms as determined from 5 independent experiments. Error bars represent the standard deviation from the mean and the * points to clones that resulted in significant enhancement or suppression in the % of pmuv worms when compared to the control (empty vector), *P<0.05. (C) To screen for changes in the pvul phenotype, 50–100 <i>lin-28(n719);let-7(mn112)</i> worms were grown to adulthood on vector control or gene specific RNAi plates (x-axis) and scored for percentage of pvul (y-axis). The bar graphs represent the average percent of pvul worms as determined from 4 independent experiments. Error bars represent the standard deviation from the mean and the * points to clones that resulted in significant suppression in the % of pvul worms when compared to the control (empty vector), *P<0.05.</p

Summary of experimental design and results.

<p>Shown is a flowchart outlining the experiments and analyses leading to the discovery of 3 new potential <i>let-7</i> targets.</p

Genes up-regulated more than 2-fold in <i>let-7(n2853)</i> compared to wild-type.

1<p>Sequence names from WormBase (<a href="http://www.wormbase.org" target="_blank">http://www.wormbase.org</a>).</p>2<p>FDR corrected.</p>3<p>W = mirWIP <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Hammell1" target="_blank">[41]</a>, P = PITA <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Kertesz1" target="_blank">[40]</a>, Y = (this study), T = TargetScan <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Lewis2" target="_blank">[69]</a>, R = RNA22 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Miranda1" target="_blank">[39]</a>, G = MicroTarget <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Grosshans1" target="_blank">[11]</a>, M = Miranda <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Enright1" target="_blank">[36]</a>, C = PicTar <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Lall1" target="_blank">[38]</a>.</p