Low depths of terminal cells improve the robustness of the <i>C. elegans</i> lineage to necrosis and program failure.

<p>(A) Frequency distribution of the maximum cell depth in 10,000 lineages (grey bars), which are generated by random coalescence of the terminal cells of the <i>C. elegans</i> lineage. The arrow indicates the observed maximum cell depth in the <i>C. elegans</i> lineage. <i>P</i>-value is the probability that the maximum depth of a random lineage is smaller than that of <i>C. elegans</i>. <i>Z</i>-score is the number of standard deviations by which the observation deviates from the mean of the random lineages. (B–C) Violin plot for the robustness of randomly generated lineages with defined maximum depths in the presence of (B) necrosis or (C) program failure. Each violin is essentially a horizontal histogram showing the relative probability densities of different robustness of random lineages with the indicated maximum depth. The horizontal line in each violin plot shows the mean value. The real lineage is shown by a triangle. <i>P</i>-value is the probability that the robustness of a random lineage (with the same maximum depth as that of <i>C. elegans</i>) is higher than that of <i>C. elegans</i>. <i>Z</i>-score is the number of standard deviations by which the observation deviates from the mean of the random lineages. (D) Frequency distribution of the mean terminal cell depth in 5,000 lineages (grey bars), which are generated by random coalescence of the terminal cells of the <i>C. elegans</i> lineage with the requirement that the maximum depth is the same as in <i>C. elegans</i>. The arrow indicates the observed mean depth in the <i>C. elegans</i> lineage. <i>P</i>-value is the probability that the mean depth is smaller in a random lineage than in <i>C. elegans</i> when their maximum depths are the same. (E–F) Violin plot for the robustness of randomly generated lineages with the maximum depth equal to that of <i>C. elegans</i> and defined mean depths, in the presence of (E) necrosis or (F) program failure. The real lineage is indicated by a triangle. <i>P</i>-value is the probability that the robustness is higher in a random lineage (with the same maximum depth and similar mean depth as those of <i>C. elegans</i>) than in <i>C. elegans</i>. <i>Z</i>-score is the number of standard deviations by which the observation deviates from the mean of the random lineages.</p

Animal developmental cell lineages are robust to necrosis and program failure.

<p>(A) A hypothetical cell lineage. Internal cells are prefixed with “I” and terminal cells are prefixed with “T”. Terminal cells belonging to the same cell type have the same name. Internal cells are colored according to their cell division programs. (B) The same cell lineage showing division programs for internal cells. Internal cells having the same division programs share the same color and program name (prefixed by “P”). (C) An example showing robustness calculation upon a necrotic cell depth. The internal cell I3 dies, which causes the loss of I3 as well as all of its direct and indirect descendant cells. Robustness is calculated by the product of the fraction of live terminal cells of each cell type. (D) An example showing robustness calculation upon a program failure. The failure of program P3 results in the loss of all descendant cells of internal cells that use P3. Robustness is calculated by the product of the fraction of live terminal cells of each cell type. (E–F) The <i>Caenorhabditis elegans</i> developmental cell lineage is more robust than the corresponding random lineages in the presence of (E) necrosis or (F) program failure. The grey bars show the frequency distribution of the robustness of 10,000 random lineages, whereas the arrow indicates the robustness of the <i>C. elegans</i> cell lineage. The random lineages are generated by randomly coalescing the terminal cells of the <i>C. elegans</i> lineage. <i>P</i>-value indicates the probability that a randomly generated lineage is more robust than the real lineage. <i>Z</i>-score is the number of standard deviations (of the random lineages) by which the observation deviates from the mean of the random lineages. (G–H) The <i>Pellioditis marina</i> cell lineage is more robust than the corresponding random lineages in the presence of (G) necrosis or (H) program failure. (I–J) The <i>Halocynthia roretzi</i> cell lineage is more robust than the corresponding random lineages in the presence of (I) necrosis or (J) program failure.</p

Among residues encoded by the same codon within the same gene, those with higher demands for translational accuracy have lower elongation speeds. A total of 1,843 genes are used.

<p>(A) Synonymous codon usage does not predict elongation speed. For each amino acid, <i>OR</i>₂ is calculated for each gene and then combined across genes by the MH procedure. <i>OR</i>₂>1 indicates that preferred codons are translated faster than unpreferred codons, and vice versa. The combined <i>OR</i>₂ from all amino acids is not significantly different from 1 (<i>p</i>>0.2). (B) Among residues encoded by the same codon in the same gene, those that are more conserved are translated more slowly. For each codon, <i>OR</i>₃ is calculated for each gene and then combined across genes by the MH procedure. <i>OR</i>₃>1 indicates that conserved residues encoded by a codon are translated more slowly than unconserved ones encoded by the same codon, and vice versa. The combined <i>OR</i>₃ from all codons is significantly greater than 1 (<i>p</i><10⁻⁵). For both panels, error bar indicates one standard error, estimated by bootstrapping the genes 1,000 times. The standard error of <i>OR</i>₃ for CGA cannot be estimated because CGA with relevant information occurred in only one gene. Nominal <i>p</i> values from the MH test are indicated by asterisks. * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001.</p

Selection for simplicity cannot explain the robustness of the <i>C. elegans</i> cell lineage.

<p>(A–B) Complex relationships between lineage complexity and robustness to (A) necrosis or (B) program failure among three types of random lineages. Each dot represents a random lineage, whereas the triangle shows the real lineage of <i>C. elegans</i>. <i>P</i>₁ is the probability that the complexity is equal between the dots of two colors compared (Mann-Whitney <i>U</i> test), whereas <i>P</i>₂ is the probability that the robustness (<i>f</i>_n or <i>f</i>_p) is equal between the dots of two colors compared (Mann-Whitney <i>U</i> test). All <i>P</i> values are calculated based on 10,000 dots of each color. For clarity, however, only 100 dots of each color are shown here. (C–D) Lineage complexity and robustness to (C) necrosis or (D) program failure of lineages generated in the macroevolution. Each evolutionary simulation is conducted 100 times, shown by 100 dots of the same color. The quantity being selected for is defined in the symbol legend, where <i>S</i> and <i>R</i> represent simplicity (i.e., 1/complexity) and robustness against necrosis (<i>f</i>_n), respectively. The fitness functions used in various simulations (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004501#s4" target="_blank">Materials and Methods</a>) are shown in the dash-lined box. The actual <i>C. elegans</i> lineage is indicated by a triangle.</p

Messenger RNA folding serves as elongation brakes to modulate the speed and accuracy of protein translation in yeast.

<p>(A) Schematic diagram of a translating ribosome. The codon being decoded is at the ribosome A site. (B) Rank correlation (black dots) between the elongation speed (measured at the ribosome A site) and mRNA folding strength at different offsets. The correlations are calculated among codons within each gene; the 1,232 within-gene correlations are then averaged. Error bar indicates 95% confidence interval of the mean correlation, estimated from bootstrapping the genes 100 times. The <i>p</i> values (red line) are based on a binomial test of the null hypothesis that equal numbers of genes have positive and negative correlations. (C) Rank correlation between the elongation <i>s</i>peed (measured at the ribosome A site) and experimentally determined <i>m</i>RNA folding strength (ρ_m-s) at different offsets for individual genes. Genes are ordered according to their protein sequence conservation among orthologs. Rank correlation between ρ_m-s at offset +12 and the evolutionary conservation of the protein is −0.158 (<i>p</i><10⁻⁴). In (B) and (C), only those genes that have ρ_m-s values for all offsets are shown. (D) Similar to (C), except that genes are ordered according to their expression levels in a rich medium. Rank correlation between ρ_m-s at offset +12 and the gene expression level is −0.320 (<i>p</i><10⁻⁴⁷). (E) Rank correlation (black dots) between the amino acid conservation of the codon being decoded and the mRNA folding strength at various offsets. Correlations are calculated for each gene and then averaged across 2,214 genes. Error bar indicates 95% confidence interval of mean correlation, estimated from bootstrapping the genes 100 times. The <i>p</i> values (red line) are based on a binomial test of the null hypothesis that equal numbers of genes have positive and negative correlations.</p

The tendency for rare cells to have low depths improves the robustness of the <i>C. elegans</i> lineage.

<p>(A) Positive correlation between the depth of a terminal cell and its cell type size. Spearman's rank correlation (ρ) for the original unbinned data and the associated <i>P</i>-value are presented. Error bars show one standard deviation of the depth within a cell type. Bla, blast; Epi, epithelial; Ger, germ; Gla, gland; Int, intestinal; Mus, muscle; Str, neural structural; Neu, neuron. The rare-early correlation remains strong even when the germ cells are removed (ρ = 0.515, <i>P</i><10⁻³⁸). (B) Frequency distribution of the rare-early correlation coefficient from 10,000 random lineages that have the same topology as that of <i>C. elegans</i> but have their terminal cells randomly relabeled. The arrow indicates the correlation coefficient for the <i>C. elegans</i> lineage. <i>P</i>-value is the probability that a random lineage above generated has a higher rare-early correlation than that observed in <i>C. elegans</i>. <i>Z</i>-score is the number of standard deviations by which the observed correlation deviates from the expected correlation of the random lineages with the same topology. (C–D) The stronger the rare-early correlation (ρ_rare-early) in a random lineage, the higher the robustness of the lineage in the presence of (C) necrosis or (D) program failure. Although 10,000 random lineages are generated, for clarity, only 1000 are shown (grey dots). The dashed line is the linear least-square regression of these 1000 dots. The rank correlation between ρ_rare-early and robustness, as well as the associated <i>P</i>-value, are calculated from all 10,000 lineages. The <i>C. elegans</i> lineage is represented by a triangle.</p

Within individual yeast genes, codons with higher demands for translational accuracy have slower elongations. A total of 1,843 genes are used.

<p>(A) Cumulative frequency distributions for the observed and randomly expected within-gene rank correlations between the evolutionary conservation of the encoded amino acid of a codon and its elongation speed. KS test, Kolmogorov–Smirnov test of the equality of the two distributions. Only 1,590 genes are used here because correlation cannot be calculated for the other 254 genes due to the lack of variation (or having too few sites with necessary data) in either evolutionary conservation or elongation speed. (B) Cumulative frequency distributions for the observed and randomly expected odds ratio <i>OR</i>₁, which measures the enrichment of slow-elongation codons at evolutionary conserved residues within a gene. (C) Combined <i>OR</i>₁ for all genes examined, by the MH procedure. The combined <i>OR</i>₁ significantly exceeds 1 (<i>p</i><10⁻⁷, MH test). Error bar indicates one standard error, estimated by bootstrapping the genes 1,000 times.</p

Dual luciferase assay demonstrating the impact of the pairing status of the offset +12 nucleotide in mRNA on mistranslation rate.

<p>(A) Experimental design. Concentration-independent firefly activity (<i>f</i>) is measured by the ratio between the firefly (<i>F</i>) and Renilla (<i>R</i>) signals. The firefly lysine codon AAA at position 529 (marked in blue in the fusion gene) is replaced with AGG, UAG, and UUU in three mutants, respectively. Because only protein molecules with lysine at position 529 would display luciferase activity, the rate of mistranslation to lysine at position 529 can be estimated by <i>f</i>/<i>f</i>_wt, where <i>f</i>_wt is the <i>f</i> value for the wild-type (wt) (i.e., AAA at codon 529). Three versions of the 3′ sequence (region depicted in green in the fusion gene) are respectively used for the white, red, and blue dots in panel (B). (B) The rate of mistranslation to lysine at codon 529 (<i>f</i>/<i>f</i>_wt) is influenced by the pairing status at the +12 nucleotide. Each genotype was measured in three biological replicates, depicted by three dots. Each dot represents the mean value from four technical repeats of each biological replicate, and the error bar shows the associated standard error. The <i>p</i> values are from <i>t</i> tests based on the three biological replicates. Note that for UUU, each white dot is higher than each red dot, which has a probability of  = 0.05 under the null hypothesis of no difference between white and red dots.</p

The macroevolution simulation suggests the possibility that the robustness of the <i>C. elegans</i> lineage arose as an adaptation to necrosis but not program failure.

<p>(A–E) Frequency distributions of (A) lineage robustness in the presence of necrosis (<i>f</i>_n), (B) maximum depth, (C) mean depth, (D) rare-early correlation, and (E) lineage robustness in the presence of program failure (<i>f</i>_p) among lineages generated from the macroevolution with different intensities of selection for high <i>f</i>_n. The observed values from the <i>C. elegans</i> lineage are indicated by black arrows. Each distribution in each panel is based on 100 simulation replications. The number next to the color scheme shows the fraction of most robust lineages from which the progenitor of next evolutionary expansion of cell lineage is randomly chosen. That is, the lower the number, the stronger the selection. (F–J) Frequency distributions of (F) lineage robustness in the presence of program failure (<i>f</i>_p), (G) maximum depth, (H) mean depth, (I) rare-early correlation, and (J) lineage robustness in the presence of necrosis (<i>f</i>_n) among lineages generated from the macroevolution with different intensities of selection for high <i>f</i>_p. The observed values from the <i>C. elegans</i> lineage are indicated by black arrows.</p

Slower translational elongation of yeast genes with higher demands for translational accuracy.

<p>(A) Average elongation speed for a gene decreases as the mean evolutionary conservation of the gene at the protein sequence level increases. The 1,862 yeast genes analyzed are grouped into 30 equal-sized bins. Error bars indicate the 95% confidence interval of the mean, estimated by bootstrapping the genes 1,000 times. The plot is shown in a log scale on the x axis because evolutionary conservation is calculated by the inverse of evolutionary rate and hence can be very large for proteins with very low rates of evolution. Spearman's rank correlation of the original unbinned data is shown. Note that the rank correlation does not depend on the scale used in the plot. (B) Average elongation speed for a gene decreases as the expression level of the gene increases. The 2,237 yeast genes analyzed are grouped into 30 equal-sized bins. Error bars indicate the 95% confidence interval of the mean, estimated by bootstrapping the genes 1,000 times. The plot is shown in a log scale on the x axis because the frequency distribution of gene expression level is known to follow a power law. Spearman's rank correlation of the original unbinned data is shown. Note that the rank correlation does not depend on the scale used in the plot. (C) Gene expression rank changes due to an environmental shift from a rich medium to an amino acid starvation medium are negatively correlated with changes in the rank of translational elongation speed. The 1,653 yeast genes that have relevant information are each depicted by a dot.</p