Effect of the genetic background and RNAi silencing on <i>C</i>. <i>elegans</i> lifespan at 20°C.

<p>Effect of the genetic background and RNAi silencing on <i>C</i>. <i>elegans</i> lifespan at 20°C.</p

Expression of UPR^mt markers in <i>mttu-1</i>, <i>mtcu-</i>1 and <i>mtcu-2</i> mutant strains.

<p><b>(A and B)</b> Expression of the <i>hsp-6</i>_<i>p</i>::GFP or <i>hsp-60</i>_<i>p</i>::GFP reporters in the indicated strains. Representative fluorescence micrographs of wild-type and single mutants harbouring <i>hsp-6</i>_<i>p</i>::GFP or <i>hsp-60</i>_<i>p</i>::GFP transgenes at L4 stage of development are shown in (A). Quantification is shown in (B) (n>20). * denotes p<0.05, **, p<0.01 and ***, p<0.001. <b>(C)</b> Fluorescence micrographs of wild-type and single mutants harbouring <i>hsp-6</i>_<i>p</i>::GFP or <i>hsp-60</i>_<i>p</i>::GFP transgenes at L4 stage of development after <i>cyc-1</i>(RNAi) from the L1 stage. <b>(D)</b> Fluorescence micrographs of one-day old, adult wild type and single mutants harbouring <i>hsp-6</i>_<i>p</i>::GFP transgene exposed to 1 mM paraquat for 24 h. Note that <i>cyc-1</i>(RNAi) (C) and paraquat treatment (D) cause marked increases in GFP expression. <b>(E)</b> Quantitation of <i>clpp-1</i> and <i>ubl-5</i> mRNA levels by qRT-PCR in the indicated strains at L4 stage (n≥3). The mRNA levels were normalized to those of <i>act-1</i> in the wild-type strain. Statistical significance was evaluated with Student’s unpaired t-test. Error bars indicate standard deviation (SD). *, p<0.05.</p

Modification of the wobble uridine (U₃₄) in mitochondrial and bacterial tRNAs.

<p>Schema of the U₃₄ modification pathways in human and yeast mt-tRNAs and <i>Escherichia coli</i> tRNAs <b>(A)</b> and <i>A</i>. <i>suum</i> mt-tRNAs <b>(B)</b>. In A, proteins GTPBP3, MTO1, and TRMU (also named MTU1) from humans, and MSS1, MTO1, and MTU1, from yeast, are orthologous of the bacterial MnmE, MnmG and MnmA proteins, respectively. Taurine (humans) and glycine (<i>E</i>. <i>coli</i> and yeast) are used to introduce the τm and cmnm groups into position 5 of U₃₄. In B, MTCU-1, MTCU-2 and MTTU-1 are the nematode orthologues of GTPBP3, MTO1, and TRMU, respectively, and their roles are inferred from studies of the <i>E</i>. <i>coli</i> and yeast proteins. The fractions of <i>A</i>. <i>suum</i> modified mt-tRNAs, according to the study by Sakurai <i>et al</i>. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006921#pgen.1006921.ref028" target="_blank">28</a>], are indicated below the schema. Note that mt-tRNA^Glu, mt-tRNA^Lys and mt-tRNA^Gln are fully modified at position 5 but lack thiolation at position 2, whereas most (~90%) is modified at both positions (2 and 5), and about 50% of mt-tRNA^Trp molecules do not contain any modification at the U₃₄.</p

Effect of the <i>mttu-1</i>, <i>mtcu-1</i> and <i>mtcu-2</i> mutations on the modification status and steady-state levels of mt-tRNAs.

<p>(<b>A and B</b>) Analysis of the 2-thiolation status of (A) and mt-tRNA^Gln (B) by APM-Northern blotting. Total small RNAs obtained from mixed-stage populations of liquid-cultured worms were purified from wild-type (N2) and <i>mttu-1</i>, <i>mtcu-1</i> or <i>mtcu-2</i> strains, and analyzed in 10% polyacrylamide/8 M urea gels with (+) or without (-) 0.01 mg/ml APM. At least three replicates were performed. #Total small RNA stained with methylene blue. <b>(C-F)</b> Northern blot analysis of molecules after digestion with angiogenin <i>in vitro</i>. Three μg of total small RNA obtained from mixed-stage populations of liquid-cultured worms from N2 and <i>mtcu-2</i> (C) or <i>mtcu-1</i> (E) strains were digested with 12.5 μg/ml of angiogenin for the indicated times and and cyt-tRNA^Lys were detected with a specific probe. Quantification of at least two independent assays similar to those shown in C and E is given in D and F, respectively. <b>(G)</b> Quantification of the steady-state levels of , mt-tRNA^Gln and 5S rRNA in <i>mttu-1</i>, <i>mtcu-1</i> and <i>mtcu-2</i> single mutants in comparison to the steady-state levels in wild-type strain (n≥3). <b>(H)</b> Quantification of mtDNA/nDNA ratio by qPCR. Four L4 worms grown at 20°C were used to quantify the mtDNA/nDNA ratio in each strain (n = 3). Error bars indicate ± SD (standard deviation). Statistical significance was evaluated with Student’s unpaired t-test. ** and *** denote p<0.01 and p<0.001, respectively.</p

Simultaneous inactivation of MTTU-1 and MTCU-2 leads to lifespan extension in <i>C</i>. <i>elegans</i>.

<p><b>(A)</b> Survival of the wild-type strain and the <i>mttu-1</i>, <i>mtcu-1</i> and <i>mtcu-2</i> single mutants at 20°C (n = 4). <b>(B)</b> Survival of the wild-type strain and the <i>mtcu-2; mttu-1</i> double mutant at 20°C (n = 3). <b>(C-J)</b> <i>aak-1</i> (n = 2) (C), <i>aak-2</i> (n = 2) (D), <i>daf-2</i> (n = 1) (E), <i>rict-1</i> (n = 1) (F), <i>daf-16</i> (n = 2) (G), <i>kri-1</i> (n = 2) (H), <i>daf-9</i> (n = 2) (I), and <i>daf-12</i> (n = 2) (J) silencing effect on the survival of the N2 and <i>mtcu-2; mttu-1</i> strains at 20°C. The empty vector L4440 was used as a negative control. Animals used for controls were of the same age as the experimental animals. To avoid disturbing embryonic development, silencing was started at the L4 stage (pointed with and arrow and a dashed line). Statistical significance was evaluated with Log-rank (Mantel Cox test) and Gehan-Breslow-Wilcoxon test and statistics are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006921#pgen.1006921.t001" target="_blank">Table 1</a>.</p

Simultaneous lack of mitochondrial MTTU-1 and MTCU-2 proteins is associated with embryonic lethality, developmental defects and sterility.

<p><b>(A)</b> Silencing of <i>mttu-1</i> in the <i>mtcu-2</i> mutant (from L4 stage onwards) at 25°C produces a slower rate of development and sterility of their progeny. <b>(B)</b> and <b>(C)</b> Silencing of <i>mtcu-2</i> in the <i>mttu-1</i> mutant (from L4 stage onwards) at 25°C causes arrest of development at the L1-L2 stages (B) and embryonic lethality (C). The total number of eggs and the number that failed to hatch were quantified (n≥5). **p<0.01, ***p<0.001. Statistical significance was evaluated with Student’s unpaired t-test. Error bars indicate standard deviation (SD).</p

Effect of the <i>mttu-1</i>, <i>mtcu-1</i> and <i>mtcu-2</i> mutations on fecundity and reproductive cycle length.

<p><b>(A, B)</b> Fertility of the wild-type (N2), <i>mttu-1</i>, <i>mtcu-1</i> and <i>mtcu-2</i> strains was measured by the number of progeny laid by adult hermaphrodite worms at 20°C (A) and 25°C (B) (n≥2). <b>(C, D)</b> Length of the reproductive cycle in the wild-type, <i>mttu-1</i>, <i>mtcu-1</i> and <i>mtcu-2</i> strains at 20°C (C) and 25°C (D) (n≥3). *** denotes p<0.001. Statistical significance was evaluated with Student’s unpaired t-test.</p

<i>mttu-1</i>, <i>mtcu-1</i> and <i>mtcu-2</i> mutants display mild mitochondrial defects.

<p><b>(A)</b> Representative western blot of protein extracts from young adults worms (day 1 of adulthood) probed with antibodies to NUO-2 (complex I), CTC-1(COX-1) (complex IV), ATP-2 (complex V) and actin. <b>(B)</b> Densitometric analysis of blots obtained from at least three independent experiments. The steady state levels of the OXPHOS subunits were normalized with respect to actin and are represented as the equivalent amounts in wild type. Error bars indicate standard deviation (SD). * denotes p<0.05, ***, p = 0.0001. <b>(C, D)</b> Quantitation of (C) TMRE staining (n>4) (D) and MitoTracker Red staining (n>9) in L4 worms during 16 h. *p<0.05, ***p = 0.0002. <b>(E)</b> Graph showing the AMP/ATP ratio in the indicated strains at L4 stage. The wild-type strain (N2) treated for 2 h with 1 mM sodium azide (SA), which blocks ATP production, was included in the analysis as a positive control (n = 3). ***p<0.001. Statistical significance was evaluated with Student’s unpaired t-test. Data is represented as mean ± SD.</p

Mitochondrial respiratory capacity of the <i>mttu-1</i>, <i>mtcu-1</i>, <i>mtcu-2</i> and <i>mtcu-2; mttu-1</i> mutants.

<p><b>(A-C)</b> Basal (A) and maximal (B) oxygen consumption rates and spare respiratory capacity (C) of worms grown at 20°C. Statistical significance for the double mutant was evaluated with unpaired T-test with Welch’s correction. *** denotes p<0.0001. <b>(D-F)</b> Basal (D) and maximal (E) oxygen consumption rates and spare respiratory capacity (F) of worms grown at 25°C. Statistical significance for the single mutants was evaluated with one-way ANOVA with a Dunnett’s post hoc test for multiple comparisons. *, ** and *** denote p<0.05, p<0.01 and p<0.001, respectively. Data is represented as mean ± SD.</p

The single mutants exhibit higher sensitivity to inhibitors of complex I and II and mild antioxidant response.

<p><b>(A, B)</b> Graphs showing survival of L1 worms after a 4-day exposure to different concentrations of the complex I inhibitor, rotenone (A) or the complex II inhibitor, TTFA (B) (n = 3). <b>(C)</b> Percentage of worms at different developmental stages and dead animals after a 4-day exposure to different concentrations of the complex II inhibitor TTFA. Treatment was initiated in synchronized L1 populations. <b>(D, E)</b> Quantitation of <i>sod-3</i>, <i>sod-1</i>, <i>ctl-2</i>, <i>gst-4</i>, <i>gcs-1</i> and <i>cts-1</i> mRNA levels by qRT-PCR in the indicated strains at L4 stage of development (n≥3). The mRNA levels were normalized to <i>act-1</i> and the wild-type strain. * denotes p<0.05, **, p<0.01 and ***, p<0.001. Statistical significance was evaluated with Student’s unpaired t-test and data is represented as mean ± SD.</p