Activated Ras/MAPK pathway enhances the apoptotic DNA damage response and restores irradiation-induced germ cell death in <i>rpoa-2(op259)</i> mutants.

<p>A) Apoptotic germ cell corpses at 24 hours post irradiation. <i>lip-1(lf)</i> and <i>let-60(gf)</i> increase the IR response of <i>rpoa-2(op259)</i> mutants. <i>lip-1</i> is epistatic to <i>rpoa-2(op259)</i> regarding the IR response; the response in <i>rpoa-2(op259); let-60(gf)</i> is intermediate between the single mutants. Error bars, SD; n≥40 for each condition. B) Simplified model of the EGFR/Ras/MAPK signal axis. Gain-of-function mutations in LET-60 or loss of the phosphatase LIP-1 increase MPK-1 activity. GAP-1 is a GTPase-activating protein acting as a negative regulator of LET-60; <i>gap-1(lf)</i> mutants show synthetic effects on vulval induction with other genes but have no strong phenotype on their own. C) Reduced kinase activity in the Ras/MAPK pathway mutant <i>mpk-1(ga111)</i> or in the p38 MAPK mutant <i>pmk-1(km25)</i> but not the Jnk mutant <i>jnk-1(gk7)</i> reduces germ cell apoptosis. Experiments performed at 20°C; error bars, SD; n = 20 animals per condition. D) <i>rpoa-2(op259)</i> suppresses excessive vulval induction of <i>let-60(n1046gf)</i>. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003943#s4" target="_blank">Methods</a> for assessment of vulval induction index (VI) (reference value is 3.0 for wild type, corresponding to normal induction of one vulva). Error bars, SD of at least 20 animals.</p

The <i>rpoa-2(op259)</i> mutation reduces irradiation-induced germ cell apoptosis but not cell cycle arrest response or DNA damage repair.

<p>A) DIC images of adult worms 24 hours after irradiation. Basal (physiologic) as well as DNA damage-induced germ cell death is confined to the late meiotic pachytene region of the germ line tubes; corpses are visible as cellularised, refractile discs (arrowheads). Size bar, 15 µm. B) Apoptotic cell corpses in the germ lines of X-ray treated adult hermaphrodites. Dashed lines represent basal levels (0 Gy), straight lines the levels following irradiation (60 Gy). At least three independent experiments (with n = 20 animals per condition) were performed with wild-type and <i>rpoa-2(op259)</i> worms, and one with animals mutant for the 9-1-1 complex subunit HUS-1. Error bars, standard deviation (SD) of corpse number per gonad over all experiments. C, D) In contrast to the DNA damage response mutant <i>hus-1(op241)</i>, cell cycle arrest is induced normally in <i>rpoa-2(op259)</i>. C) Percentage of mitotic cells with nuclear enlargement following IR. Error bars, SD from at least 12 gonads (approx. 40–50 total mitotic cells per gonad). D) Gonads were dissected from <i>opIs257[P_rad-54::rad-54::yfp 3′UTR; unc-119(+)]; unc-119(ed3)</i> or <i>rpoa-2(op259)</i>; <i>opIs257; unc-119(ed3)</i> adults, stained with Hoechst, and scored for YFP::RAD-54 foci. Size bar, 10 µm. E, F) A subset of rRNA processing and translation initiation mutants have reduced IR-induced apoptosis. Apoptotic response to IR irradiation in the gonads of the rRNA processing mutants <i>pro-2(na27)</i> and <i>pro-3(ar226)</i> (E) and of the eIF4E homolog mutants <i>ife-1(ok1978)</i> and <i>ife-2(ok306)</i> (F). Dashed lines, 0 Gy, straight lines, 60 Gy; error bars, SD of the number of germ cell corpses per gonad over at least 3 experiments (n = 20 animals per data point and experiment).</p

Baseline levels of activated MPK-1 in the gonad are reduced in <i>rpoa-2(op259)</i> and do not increase upon irradiation.

<p>A) Gonads were extruded from non-irradiated (0 Gy) and irradiated (60 Gy) adult worms 4 hours after treatment, fixed, and immunostained for the doubly phosphorylated (activated) MPK-1. MPK-1 activity is high in the late meiotic pachytene region (white dashed lines) and is further increased following IR in wild-type worms, but not in <i>rpoa-2(op259)</i> mutants. Control staining included total MPK-1 with anti-ERK, and anti-dsDNA to detect an epitope other than protein for normalisation of fluorescence intensity. Size bar, 40 µm. B) Quantification of the anti-ppMPK signal intensity along the central axis (white streak) of the late meiotic pachytene region, starting distally to the first oocyte (white dashed reference line) and extending into the mid-pachytene region. Profiles were plotted for at least 8 worms per condition (thin grey lines for individual wild-type worms, thin green lines for individual <i>rpoa-2(op259)</i> mutants) and a spline was calculated from their overlay. Size bar, 25 µm.</p

Basal apoptosis in <i>rpoa-2(op259)</i> mutant animals depends on CEP-1/p53.

<p>A) <i>rpoa-2(op259)</i> mutants show increased basal expression of CEP-1 target genes. qRT-PCR measurements of EGL-1 and CED-13 transcript levels in total worm RNA extracts 200 min post treatment. Error bars, SD of normalised transcript levels (wild type and <i>rpoa-2(op259)</i>, n>11 independent samples per condition; <i>cep-1(gk138)</i> and <i>cep-1 rpoa-2(op259)</i>, n = 2). B) Apoptotic corpses (arrowheads) are abundant in <i>rpoa-2(op259); ced-6(n1813)</i> double mutants, but rare in <i>cep-1 rpoa-2; ced-6</i> triple mutants. Distal gonad arm is at the top, proximal arm with oocytes at the bottom; size bar, 25 µm. C) Germ cell corpse accumulation in the engulfment defective mutant background <i>ced-6(n1813)</i>. Apoptotic corpses persist in the gonad instead of being swiftly removed by the surrounding sheath cells, which allows for assessing the cumulative number of cells having undergone apoptosis. Note that an immense number of germ cell corpses are found in irradiated <i>ced-6(n1813)</i> animals already at 24 hours after irradiation, but not in <i>rpoa-2(op259)</i> or <i>cep-1(lf)</i>. Basal levels of apoptosis are observed in <i>rpoa-2(op259)</i> and <i>cep-1</i> single mutants, but not in the <i>cep-1 rpoa-2</i> double mutant. Dashed lines, 0 Gy, straight lines, 60 Gy; error bars, SD over at least 3 experiments (n = 20 animals per data point and experiment).</p

<i>rpoa-2(op259)</i> mutants have altered ribosome synthesis.

<p>A) Most cells in <i>C. elegans</i> exhibit a single but often very prominent nucleolus. In germ cells of the meiotic pachytene stage, the nucleoli (inner white dashed circle) impose as central spheres of approximately half the diameter of the containing nuclei (outer white dashed circle). In <i>rpoa-2(op259)</i>, the nucleolar diameter is reduced. Many of the germ cell nucleoli show a substructure by DIC that is enlarged in <i>rpoa-2(op259)</i> mutants (black arrows). Size bar, 10 µm. B) Nucleolar volume calculated from measures of the diameter in DIC images of the late meiotic pachytene region of wild-type, <i>hus-1(op241)</i>, <i>cep-1(gk138)</i>, <i>ced-3(n717)</i>, <i>rpoa-2(op259)</i>, <i>ncl-1(e1865)</i>, and <i>rpoa-2(op259); ncl-1(e1865)</i> gonads. Error bars, SD of nucleolar size over all animals tested (12 animals, 5 nucleoli per animal). C) Quantification of ribosomal RNA in whole worm extracts by qRT-PCR. The mean levels of the mature rRNAs (competimer strategy) and of transcribed spacer sequences of the pre-rRNA were normalised by the internal controls in each sample (Pol II transcripts) and compared to N2 wild type. At least 9 or 4 samples per strain were probed for rRNAs or pre-rRNAs, respectively; error bars, 95% CI of the mean. D) Ribosomal proteins represent a smaller fraction of total proteins in <i>rpoa-2(op259)</i> mutants compared to wild-type animals. Individual protein abundance was determined by spectral counting and a quantitative value was assigned by the Scaffold 3 software <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003943#pgen.1003943-Searle1" target="_blank">[109]</a>. Ribosomal proteins were grouped as small or large ribosomal subunit proteins, or a small group of acidic ribosomal proteins. A subset of structural proteins is included as a control group. Similar numbers of proteins were identified in each group for both samples (numbers inside circle). Mean ratio of protein abundance in <i>rpoa-2(op259)</i> over wild type is indicated. E) Ethidium bromide-stained denaturing agarose gel with 800 ng total RNA from adult wild-type and <i>rpoa-2(op259)</i> mutant worms. An approximately 2.8 kb distinct band running between the 26S and 18S ribosomal RNAs is more prominent in <i>rpoa-2(op259)</i> (arrow). Characterisation of its sequence and its endings (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003943#pgen.1003943.s023" target="_blank">Text S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003943#pgen.1003943.s011" target="_blank">Fig. S11</a>) revealed a precise truncation of the mature 26S rRNA and no addition of a poly-A tail. The base numbers indicate the position in the reference sequence of the rDNA repeat unit (top) or along the 26S rRNA (bottom), respectively. F) Relative abundance of the 26S-short rRNA in <i>rpoa-2(op259)</i>, rRNA processing mutants (<i>pro-2</i>, <i>pro-3</i>), translation initiation factor mutants (<i>ife-1</i>, <i>ife-2</i>), apoptosis mutants (<i>cep-1</i>, <i>ced-3</i>), an exosome mutant (<i>crn-3</i>), and of double mutant combinations with <i>rpoa-2(op259)</i>. On each northern blot membrane, the ratio of the 26S-short to 26S rRNA signals was arbitrarily set to 1.0 for the wild-type controls. Mean relative ratios, 95% CI of the mean, and number of samples analysed per strain.</p

Model for the role of RPOA-2 and Ras/MAPK in determining apoptosis.

<p>A) Model for the effect of <i>rpoa-2(op259)</i> on the apoptotic network via the Ras/MAPK pathway. CEP-1/p53 activity is induced by irradiation, and increased at baseline in <i>rpoa-2(op259)</i> by an unknown mechanism, but this results in only little germ cell apoptosis. <i>rpoa-2(op259)</i> thus has an additional inhibitory effect on apoptosis downstream of CEP-1 activation, at the level of the core apoptotic machinery (possibly at the level of CED-9). <i>rpoa-2(op259)</i> leads to reduced levels of activated MPK-1 and genetic overactivation of Ras/MAPK restores IR-induced germ cell apoptosis levels in <i>rpoa-2(op259)</i> animals, suggesting that this pathway provides the link. <i>rpoa-2(op259)</i> might influence Ras/MAPK activity by affecting the phosphatase LIP-1. B) Model for the role of the Ras/MAPK pathway as a central and general modulator of germ cell apoptosis levels. Classically, DNA damage-induced and constitutive ‘physiological’ germ cell death have been distinguished, genetically converging only at the level of the core apoptotic machinery. We and others see that the level of Ras/MAPK pathway activity is decisive for the extent of germ cell death, both at standard growth conditions and following ionising irradiation. Furthermore, activated MPK-1 levels are increased soon after irradiation treatment. For constitutive cell death, it remains to be resolved whether Ras/MAPK pathway activity is simply permissive or whether it is a direct part of the induction signalling. Overall, MAP kinase pathways could be acting as a master lever for the sensitivity of germ cells towards further pro-apoptotic stimuli.</p

Triggering of RNA Interference with RNA–RNA, RNA–DNA, and DNA–RNA Nanoparticles

Control over cellular delivery of different functionalities and their synchronized activation is a challenging task. We report several RNA and RNA/DNA-based nanoparticles designed to conditionally activate the RNA interference in various human cells. These nanoparticles allow precise control over their formulation, stability in blood serum, and activation of multiple functionalities. Importantly, interferon and pro-inflammatory cytokine activation assays indicate the significantly lower responses for DNA nanoparticles compared to the RNA counterparts, suggesting greater potential of these molecules for therapeutic use