Figure 1.
Summary of experimental design and results.
Shown is a flowchart outlining the experiments and analyses leading to the discovery of 3 new potential let-7 targets.
Table 1.
Genes up-regulated more than 2-fold in let-7(n2853) compared to wild-type.
Table 2.
Genes down-regulated more than 2-fold in let-7(n2853) compared to wild-type.
Figure 2.
Novel suppressors of vulval rupture in let-7 null mutants.
(A) Null let-7(mn112) 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 let-7(mn112); Ex[let-7(+);myo-2::GFP] 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 (daf-12) or a novel candidate (sox-2) allows let-7(mn112) 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.
Table 3.
Phenotypic suppressors of let-7 mutants.
Figure 3.
Suppression of supernumerary seam cell nuclei in let-7 mutants.
(A) While wild-type worms have 16 seam cell nuclei, let-7(n2853) worms have significantly more (∼23) [82]. To score for suppression of the extra seam cell phenotype, let-7 mutants expressing nuclear GFP in seam cells (let-7(n2853);Int[scm::GFP]) were grown at the restrictive temperature (25°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 let-7(n2853) (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.
Figure 4.
Differential effects of let-7 target candidates on vulva formation.
(A) Micrographs of the protruding multiple vulva (pmuv) phenotype in lin-28(n719) and the suppression to a single protruding vulva (pvul) when combined with let-7(mn112). White arrowheads point to protruding vulvas in the mutants. (B) To screen for changes in the pmuv phenotype, 50–100 lin-28(n719) 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 lin-28(n719);let-7(mn112) 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.
Figure 5.
Argonaute associates with targets in a let-7–dependent manner.
(A) Sequences in the indicated genes were detected by semi-quantitative PCR of cDNA from ALG-1 immunoprecipitation assays from L4 staged WT and let-7(n2853) strains. Based on enrichment in the WT compared to let-7 RIP from 4 independent experiments, three new targets were identified, T27D12.1, prmt-1, and opt-2. (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 let-7(n2853) cDNA from L4 staged worms. Targets were normalized to 18S ribosomal RNA. Shown is the average and standard deviation from 3 independent experiments.