Analysis of Genetic Code Ambiguity Arising from Nematode-Specific Misacylated tRNAs

<div><p>The faithful translation of the genetic code requires the highly accurate aminoacylation of transfer RNAs (tRNAs). However, it has been shown that nematode-specific V-arm-containing tRNAs (nev-tRNAs) are misacylated with leucine <i>in vitro</i> in a manner that transgresses the genetic code. nev-tRNA^Gly (CCC) and nev-tRNA^Ile (UAU), which are the major nev-tRNA isotypes, could theoretically decode the glycine (GGG) codon and isoleucine (AUA) codon as leucine, causing GGG and AUA codon ambiguity in nematode cells. To test this hypothesis, we investigated the functionality of nev-tRNAs and their impact on the proteome of <i>Caenorhabditis elegans</i>. Analysis of the nucleotide sequences in the 3’ end regions of the nev-tRNAs showed that they had matured correctly, with the addition of CCA, which is a crucial posttranscriptional modification required for tRNA aminoacylation. The nuclear export of nev-tRNAs was confirmed with an analysis of their subcellular localization. These results show that nev-tRNAs are processed to their mature forms like common tRNAs and are available for translation. However, a whole-cell proteome analysis found no detectable level of nev-tRNA-induced mistranslation in <i>C. elegans</i> cells, suggesting that the genetic code is not ambiguous, at least under normal growth conditions. Our findings indicate that the translational fidelity of the nematode genetic code is strictly maintained, contrary to our expectations, although deviant tRNAs with misacylation properties are highly conserved in the nematode genome.</p></div

Detection of the 3′ CCA end sequences of nev-tRNAs.

<p>(A) PCR scheme for the detection of the 3′ ends of mature tRNAs: nev-tRNA^Gly (CCC) and nev-tRNA^Ile (UAU) and their cognates, tRNA^Gly (UCC) and tRNA^Ile (UAU), respectively. Numbers indicate the nucleotide positions relative to the 5′ end of each tRNA. (B) RT–PCR amplification of the 3′ end of each tRNA. PCR products of the expected sizes are shown as red dots. (C) Nucleotide sequence chromatograms of the 3′ end region of each tRNA.</p

Subcellular localization of nev-tRNAs in <i>C. elegans</i>.

<p>RNA was isolated from each fraction of <i>C. elegans</i>: whole cell (W), nuclear (N), or cytoplasmic (C). RT–PCR analysis was used to detect snU6 and snoU3 RNAs (nuclear markers), tRNA^iMet (cytoplasmic marker), and four tRNAs (nev-tRNA^Gly and nev-tRNA^Ile, and their cognate tRNAs). 5S rRNA expression is shown as the loading control. Band densities were evaluated semiquantitatively with densitometry.</p

Summary of life spans.

<p>Life spans (<b>LS</b>±SEM, standard error of the mean, at 25°C, if not stated otherwise) under different experimental conditions in WT, two different alleles of <i>vang-1</i> (<i>tm1422</i> and <i>ok1142</i>), the intestine-specific RNAi strain OLB11 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032183#pone.0032183-Pilipiuk1" target="_blank">[60]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032183#pone.0032183-McGhee1" target="_blank">[61]</a>, germline-specific RNAi strain NL2098 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032183#pone.0032183-Sijen1" target="_blank">[62]</a> and the neuron-enhanced and neuron-specific strains TU3311 and TU3401 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032183#pone.0032183-Calixto1" target="_blank">[64]</a>. OP50 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032183#pone.0032183-Brenner1" target="_blank">[75]</a> and RNAi HT115 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032183#pone.0032183-Kamath1" target="_blank">[77]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032183#pone.0032183-Timmons1" target="_blank">[78]</a> indicate standard and RNAi <i>E. coli</i> strains, respectively. Comparison of significant results are indicated by *(p<0.01; Mantel-Cox log rank test) with corresponding experiments in parentheses (the p-value is stated, if not significant). All the life span assays were repeated at least three times. Data shown is a sum of all experiments.</p

Tissue specific RNAi against <i>vang-1</i>.

<p><b>(A) </b><b><i>vang-1</i></b><b> function interferes with life span extension in </b><b><i>C. elegans</i></b><b>. </b><i>vang-1(RNAi)</i> (orange) animals showed a significantly extended mean life span (<b>14.9±0.2 d</b>, n = 576*) in comparison to control: RNAi HT115 bacteria (blue; <b>12.8±0.1 d</b>, n = 936*, p<0.001**). <b>(B) Germline-specific RNAi against </b><b><i>vang-1</i></b><b> effects </b><b><i>C. elegans</i></b><b> life span.</b> After <i>vang-1(RNAi)</i> in germline-specific RNAi strain NL2098 a significant increase (13%) of mean life span (<b>14.6±0.3 d</b>, red spotted line, n = 274*) in comparison to the control (NL2098 kept on RNAi HT115 bacteria carrying the empty “feeding”-vector) can be observed (<b>12.9±0.3 d</b>, blue spotted line, n = 309*, p<0.01**). <b>(C–D) Neuron-specific RNAi against </b><b><i>vang-1</i></b><b> does not effect </b><b><i>C. elegans</i></b><b> life span.</b> After depletion of VANG-1 in the enhanced-neuronal RNAi strain TU3311 <i>([unc-119p::YFP+unc-119p::sid-1])</i>, the mean life span is <b>21.1±0.4 d</b> (orange solid line, n = 280*) compared to <b>19.5±0.5 d</b> (green solid line, n = 92*, p = 0.02**) in the control (TU3311 kept on RNAi HT115 bacteria carrying the empty “feeding”-vector). The same is true in the neuron-specific RNAi strain TU3401 <i>(sid-1(pk3321) V; [pCFJ90(myo-2p::mCherry)+unc-119p::sid-1])</i>, which only has SID-1 in neurons. Depletion of VANG-1 in this strain leads to a mean life span of <b>17.7±0.3 d</b> (red spotted line, n = 284*) and <b>17±0.3 d</b> (blue spotted line, n = 380*, no significant difference**) in the control (TU3401 kept on RNAi HT115 bacteria carrying the empty “feeding”-vector). <b>(E) Intestine-specific RNAi against </b><b><i>vang-1</i></b><b> does not effect </b><b><i>C. elegans</i></b><b> life span.</b> After depletion of VANG-1 in the intestine-specific RNAi strain OLB11 {<i>rde-1(ne219);[pOLB11(elt-2p::rde-1)+pRF4(rol-6(su1006))]</i>}, the mean life span is <b>14.4±0.3 d</b> (red solid line, n = 195*) compared to <b>14.0±0.3 d</b> (blue solid line, n = 250*, no significant difference**) in the control (OLB11 kept on RNAi HT115 bacteria carrying the empty “feeding”-vector). (*three or more independent trials, **Mantel-Cox log rank test).</p

<i>vang-1</i> shows reproduction- and aging-related defects.

<p><b>(A) </b><b><i>vang-1(tm1422)</i></b><b> populations have a reduced brood size.</b> The average brood size at 25°C in <i>vang-1(tm1422)</i> (red, <b>111±41</b> progeny; n = 28*) is significantly reduced (p<0.0001**) in comparison to WT (blue, <b>194±50</b> progeny; n = 56*). Results are shown as mean±standard deviation. <b>(B–C) </b><b><i>ok1142</i></b><b> and </b><b><i>tm1422</i></b><b> show decreased lipofuscin accumulation five and ten days after hatching.</b> (B) Five days after hatching, <i>ok1142</i> (green, <b>RFU = 792.35±25</b>, n = 31, p<0.001**) and <i>tm1422</i> (red, <b>RFU = 543.1±18</b>, n = 37, p<0.001**) accumulate significantly less lipofuscin in comparison to WT (blue, <b>RFU = 900.4±17.27</b>, n = 45). (C) Ten days after hatching, <i>ok1142</i> (green, <b>RFU = 1083±32</b>, n = 33, p<0.05**) and <i>tm1422</i> (red, <b>RFU = 940.9±27</b>, n = 29, p<0.01**) still accumulate significantly less lipofuscin in comparison to WT (blue, <b>RFU = 1196±37</b>, n = 27). Results are shown as mean±SEM of relative fluorescence units (RFU: OD_individual−OD_background/mm²). <b>(D) In </b><b><i>tm1422</i></b><b> the ovulation rate is reduced in comparison to WT. </b><i>tm1422</i> has an ovulation rate of <b>0.7±0.1</b> (n = 25***) and the WT shows <b>2.3±0.7</b> (n = 15***) what is significantly more (p<0.05**). Ovulations were counted per gonad arm per hour at 20°C for synchronous WT and mutant populations. <b>(E) </b><b><i>vang-1</i></b><b> populations have a prolonged reproductive span.</b> The reproductive span in <i>ok1142</i> (green, <b>6.6</b> d; n = 20***) and <i>tm1422</i> (red, <b>6.9</b> d; n = 20***) is significantly prolonged (p<0.05^##) in comparison to WT (blue, <b>5.7</b> d; n = 20***). (*three independent trials, **unpaired t-test, ***two independent trials; animals grown on OP50 bacteria, ^##Mantel-Cox log rank test).</p

<i>vang-1(tm1422)</i> life span modulation depends on Insulin/IGF-1-like signaling and leads to higher DAF-16 activity.

<p><b>(A) </b><b><i>vang-1(tm1422)</i></b><b> induced life span extension interferes with RNAi against </b><b><i>daf-2</i></b><b> and </b><b><i>daf-16</i></b><b>.</b> Depletion of DAF-2 by RNAi in <i>tm1422</i> (brown spotted line) and WT (green solid line) causes an increase of mean life span to <b>25.6±1.3 d</b> (n = 135*) and <b>25.8±1.1 d</b> (n = 126*), respectively (p<0.62**), which is in agreement with published results for <i>daf-2</i> mutants <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032183#pone.0032183-Hertweck1" target="_blank">[23]</a>. In contrast, depletion of DAF-16 by RNAi in <i>tm1422</i> (rose spotted line) and WT (purple solid line) causes a decrease of mean life span to <b>12±0.3 d</b> (n = 247*) and <b>11.9±0.2 d</b> (n = 142*), respectively (p<0.96**). Life spans of WT (blue solid line) and <i>tm1422</i> (red spotted line) fed with RNAi HT115 bacteria carrying the empty “feeding”-vector are <b>12.8±0.1 d</b> (n = 936*) and <b>15.8±0.2 d</b> (n = 480*), respectively (p<0.001**). <b>(B) </b><b><i>vang-1(tm1422)</i></b><b> populations are dauer constitutive.</b> Synchronous populations were scored after 60 h at 27°C (OP50 bacteria) for dauers and L1 in diapause. All farther grown and adult animals were pooled as “other”. WT animals developed <b>21%</b>, <b>0%</b> and <b>79%</b> dauers, L1 diapause and “other”, respectively (n = 390*). <i>daf-2(e1370)</i> animals showed <b>94.7%</b>, <b>4.6%</b> and <b>0.7%</b> dauers, L1 diapause and “other”, respectively (n = 281*). <i>daf-16(mu86)</i> animals developed <b>100%</b> “other” (n = 111*). <i>tm1422</i> showed <b>57.6%</b>, <b>18%</b> and <b>24.4%</b> dauers, L1 diapause and “other”, respectively (n = 205*, p<0.05^§). (*three or more independent trials, **Mantel-Cox log rank test, animals grown on OP50 bacteria, if not stated otherwise, ^§Data analyzed by Chi-square test).</p

MOESM5 of Differences in the genetic control of early egg development and reproduction between C. elegans and its parthenogenetic relative D. coronatus

Additional file 5: Fig. S3. Binning of different combinations of replicates. Combining all four replicates the numbers of expressed sequences appear to saturate at about 6500 transcripts (see Table 2)

MOESM3 of Differences in the genetic control of early egg development and reproduction between C. elegans and its parthenogenetic relative D. coronatus

Additional file 3: Fig. S1. GO terms enriched in the D. coronatus 1–8 cell transcriptomic proteome in comparison with the complete D. coronatus proteome. Associated functional descriptions and test statistics are given in tabular format in Additional file 4

Additional file 13: of Genome analysis of Diploscapter coronatus: insights into molecular peculiarities of a nematode with parthenogenetic reproduction

3′EST clustering program (cluster3.sh). This shell script originally written for C.elegans EST analysis was used. (TXT 5 kb