Decreased LIN-35/Rb contributes to the RNAi hypersensitivity of <i>mir-35-41(gk262)</i> worms.

<p>(A) Northern blot analyses of <i>lin-35/Rb</i> mRNA levels in WT, <i>mir-35-41(gk262)</i> and <i>lin-35(n745)</i> embryos. After normalization to actin mRNA the average and standard deviation from 3 independent experiments was calculated with wild type levels set to one. (B) LIN-35 protein is decreased in <i>mir-35-41(gk262)</i> embryos compared to wild type, as shown by western blotting. After normalization to tubulin the average and standard deviation from 4 independent experiments was calculated with wild type levels set to one (Student's t-test *p<4×10⁻⁵). (C) PAGE Northern blot analysis of RNA from wild type and <i>lin-35(n475)</i> embryos shows similar levels of pre- and mature miR-35 expression. The rRNAs are shown as loading controls. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002536#s2" target="_blank">Results</a> are representative of 3 independent experiments. (D) The indicated worm strains were grown on bacteria expressing <i>unc-22</i> dsRNA. <i>Ex[lin-35; sur-5::GFP]</i> is an extrachromosomal array that expresses <i>lin-35</i> in GFP positive (+) worms. <i>Int[sur-5::GFP]</i> is an integrated array that expresses GFP in all worms and <i>Ex[myo-2::GFP]</i> is an extrachromosomal array that expresses GFP in worms that inherit the array. Phenotype was scored as percent of twitching or paralyzed worms after 28 h of exposure to RNAi from the L4 to adult stage. Error bars represent the standard error of the mean (s.e.m) for at least two independent experiments.</p

Mis-regulation of the E01G4.5 endo–siRNA target in <i>mir-35-41</i> mutants.

<p>RT-qPCR of E01G4.5 pre-mRNA (A) and mature mRNA (B) normalized to 18S rRNA and compared to wild type levels (mean ± s.e.m., n = 3, *, P<0.05).</p

Deletion of the <i>mir-35-41</i> miRNA cluster results in RNAi hypersensitivity.

<p>(A) L4 stage wild type, <i>mir-35-41(gk262)</i> and <i>mir-35-41(gk262)</i>; <i>apEx160 [mir-35-41]</i> strains were grown on bacteria expressing <i>unc-22</i> dsRNA (open columns) or the empty RNAi vector L4440 (black columns). The percentages of paralyzed worms after 28 h of exposure to RNAi conditions are graphed as the mean and standard deviation from three independent experiments. (B) PAGE Northern blot analysis of RNA from wild type, <i>mir-35-41(gk262)</i> and <i>mir-35-41(gk262)</i>; apEx160 [mir-35-41] embryos shows restored expression of precursor and mature <i>mir-35</i> miRNA in the transgenic strain. 5.8S rRNA levels are shown as loading controls.</p

Maternal rescue of RNAi hypersensitivity in <i>mir-35-41</i> and <i>lin-35/Rb</i> mutants.

<p>Histogram representing the percent of paralyzed worms for the indicated strains fed <i>unc-22</i> dsRNA for 28 hours from the L4 to adult stage. Wild type and the mutants <i>mir-35-41(gk262)</i> and <i>lin-35(n745)</i> (maternal⁻, zygotic⁻: m⁻/z⁻) were crossed to wild type males containing a GFP expressing transgene to generate green heterozygous F1 progeny (m⁻/z⁺), which were transferred to <i>unc-22</i> RNAi and scored for paralysis. To test for maternal rescue of RNAi sensitivity, heterozygous F1s were singled and allowed to self fertilize. The F2 progeny were transferred to <i>unc-22</i> RNAi and each worm was scored for paralysis and then genotyped. Error bars represent the standard error of the mean (s.e.m) for two independent experiments.</p

The RNAi sensitivity of <i>mir-35-41(gk262)</i> mutant worms is dependent on core RNAi factors.

<p>L4 staged worms of each strain were transferred to <i>unc-22</i> RNAi and the percentage of unaffected, twitching or paralyzed adult worms was scored 28 hours later. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002536#s2" target="_blank">Results</a> are mean ± standard deviation (average of at least 2 experiments). Numbers in parentheses following the genotype represent the total number of worms scored.</p

The <i>mir-35-41(gk262)</i> mutant worms show enhanced RNAi in multiple tissues and stages of development.

<p>Worm strains of the indicated genotype were grown on bacteria expressing dsRNA against the indicated genes at 20°C. RNAi phenotype results are mean ± standard deviation (average of 3 independent experiments). Numbers in parentheses represent the total number of adult worms or embryos scored.</p>*<p>L4 staged worms were transferred to RNAi and the percentage of paralyzed (%Prl) adult worms was scored 28 hours later.</p>†<p>L1 staged worms were plated on RNAi and the percentage of adult worms showing multivulva (%Muv) or roller (% Rol) phenotypes was scored.</p>††<p>L4 staged worms were transferred to RNAi, allowed to lay embryos and adult worms were removed 28 hours later. Percent embryonic lethal (%Emb) was calculated as the number of embryos that did not hatch.</p

The RNAi hypersensitivity of <i>mir-35-41</i>(<i>gk26</i>2) is comparable to that of <i>lin-35(n745)</i>.

<p>L4 staged worms of each strain were transferred to <i>unc-22</i> RNAi and the percentage of unaffected, twitching or paralyzed adult worms was scored 28 hours later. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002536#s2" target="_blank">Results</a> are mean ± standard deviation (average of at least 2 experiments). Numbers in parentheses represent the total number of worms scored.</p

The RNAi hypersensitivity of <i>mir-35-41</i> mutants is independent of <i>nrde-3</i> activity.

<p>(A) Fluorescent microscopy showing subcellular localization of GFP::NRDE-3 in seam cells of the indicated genotypes. Pictures are representative of 50 worms analyzed for each strain. (B) Histogram representing the percentages of paralyzed worms for the indicated strains fed <i>unc-22</i> dsRNA for 28 hours from the L4 to adult stage. The means and standard deviations from three independent experiments are graphed.</p