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

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

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 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

A Heritable Recombination System for Synthetic Darwinian Evolution in Yeast

Genetic recombination is central to the generation of molecular diversity and enhancement of evolutionary fitness in living systems. Methods such as DNA shuffling that recapitulate this diversity mechanism <i>in vitro</i> are powerful tools for engineering biomolecules with useful new functions by directed evolution. Synthetic biology now brings demand for analogous technologies that enable the controlled recombination of beneficial mutations in living cells. Thus, here we create a Heritable Recombination system centered around a library cassette plasmid that enables inducible mutagenesis <i>via</i> homologous recombination and subsequent combination of beneficial mutations through sexual reproduction in <i>Saccharomyces cerevisiae</i>. Using repair of nonsense codons in auxotrophic markers as a model, Heritable Recombination was optimized to give mutagenesis efficiencies of up to 6% and to allow successive repair of different markers through two cycles of sexual reproduction and recombination. Finally, Heritable Recombination was employed to change the substrate specificity of a biosynthetic enzyme, with beneficial mutations in three different active site loops crossed over three continuous rounds of mutation and selection to cover a total sequence diversity of 10¹³. Heritable Recombination, while at an early stage of development, breaks the transformation barrier to library size and can be immediately applied to combinatorial crossing of beneficial mutations for cell engineering, adding important features to the growing arsenal of next generation molecular biology tools for synthetic biology

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

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

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