Characterization of the meiotic regulation of Superoxide dismutase 1 in Saccharomyces cerevisiae

Meiosis is the conserved cell division process used by sexually reproducing organisms to produce gametes, which are the cells that give rise to the next generation. In humans, meiosis generates eggs and sperm, and in the model budding yeast Saccharomyces cerevisiae, meiosis generates spores. Despite considerable evolutionary divergence, the general mechanics of meiotic chromosome division are largely conserved from yeast to humans. This conservation has made yeast an invaluable tool for understanding meiosis. In addition to important discoveries related to chromosome division, yeast have shaped our knowledge of other meiotic processes, including gene regulatory changes, organelle dynamics and inheritance, protein quality control, and cellular rejuvenation.Changes in expression impact virtually every gene in the yeast genome during meiosis and spore development. In addition to increases and decreases in the transcription of classically defined genes and mRNAs, meiotic gene expression also involves the transcription of extended transcripts called Long Undecoded Transcript Isoforms (LUTIs), which have 5' extended sequences relative to their canonical counterparts. LUTIs are repressive, acting to transcriptionally interfere with canonical mRNA expression and translationally block protein synthesis from their main ORF via competitive translation of upstream open reading frames (uORFs). LUTIs are also pervasive and used to drive protein levels for approximately 8% of all yeast genes during meiosis. The majority of LUTI mRNAs have yet to be characterized in detail, and many of the proteins impacted by their expression are reported to exhibit stable expression in mitosis.Superoxide dismutase 1 (Sod1) is a highly abundant and primarily cytosolic antioxidant that detoxifies superoxide radicals (O2•–), a type of reactive oxygen species (ROS), from cells. We became interested in studying Sod1 after analysis of mRNA-sequencing and ribosome profiling data that suggested a LUTI mRNA is expressed from the SOD1 locus during meiosis. During mitotic growth, yeast express a canonical SOD1 mRNA (SOD1canon.) of approximately 600 nucleotides. During the meiotic divisions, cells initiate transcription from an extremely distal start site, resulting in the production of a putative LUTI mRNA that is nearly four times the length of SOD1canon.. We confirmed that this extended isoform (SOD1LUTI) is in fact produced as a continuous, ~2.2 kb transcript, and that LUTI expression functions to repress canonical mRNA levels. Although naturally produced during meiosis, SOD1LUTI is also made by mitotic cells experiencing ER stress, raising the possibility that the meiotic unfolded protein response (UPRER) is responsible for turning on the LUTI.In addition to highly dynamic changes in the expression of SOD1canon. and SOD1LUTI, we found that Sod1 protein levels and localization change during meiosis. During the transition from nutrient-rich media to starvation conditions, Sod1 levels decrease considerably and discrete foci of Sod1 appear. As cells go through meiosis, the brightness and number of these foci decreases, suggesting their degradation. The formation of these foci is independent of entry into meiosis, but their disappearance from cells relies on meiotic progression. During the meiotic divisions, due to the combination of LUTI expression and the degradation of preexisting protein, Sod1 levels reach their lowest levels. Once the chromosome divisions have completed, cells begin to express SOD1canon. once more, and new Sod1 protein is produced. Altogether, these data support a model in which old Sod1 protein is systematically cleared from cells to facilitate the generation of new protein. We propose that the regulation of this important antioxidant outlined in this dissertation may play a role the lifespan resetting that takes place during gametogenesis. Many important questions remain unanswered regarding the meiotic regulation of Sod1. What is the significance of this transient dip in expression during meiosis? What is the nature of the relationship between SOD1LUTI and the UPRER? Are the foci of Sod1 we observe true protein aggregates? Answering these and other questions will improve our understanding of the biology of this important antioxidant

Meiotic resetting of the cellular Sod1 pool is driven by protein aggregation, degradation, and transient LUTI-mediated repression

Gametogenesis requires packaging of the cellular components needed for the next generation. In budding yeast, this process includes degradation of many mitotically stable proteins, followed by their resynthesis. Here, we show that one such case-Superoxide dismutase 1 (Sod1), a protein that commonly aggregates in human ALS patients-is regulated by an integrated set of events, beginning with the formation of pre-meiotic Sod1 aggregates. This is followed by degradation of a subset of the prior Sod1 pool and clearance of Sod1 aggregates. As degradation progresses, Sod1 protein production is transiently blocked during mid-meiotic stages by transcription of an extended and poorly translated SOD1 mRNA isoform, SOD1LUTI. Expression of SOD1LUTI is induced by the Unfolded Protein Response, and it acts to repress canonical SOD1 mRNA expression. SOD1LUTI is no longer expressed following the meiotic divisions, enabling a resurgence of canonical mRNA and synthesis of new Sod1 protein such that gametes inherit a full complement of Sod1 protein. Failure to aggregate and degrade Sod1 results in reduced gamete fitness in the presence of oxidants, highlighting the importance of this regulation. Investigation of Sod1 during yeast gametogenesis, an unusual cellular context in which Sod1 levels are tightly regulated, could shed light on conserved aspects of its aggregation and degradation, with relevance to understanding Sod1's role in human disease

Tunable Transcriptional Interference at the Endogenous Alcohol Dehydrogenase Gene Locus in Drosophila melanogaster

Neighboring sequences of a gene can influence its expression. In the phenomenon known as transcriptional interference, transcription at one region in the genome can repress transcription at a nearby region in cis. Transcriptional interference occurs at a number of eukaryotic loci, including the alcohol dehydrogenase (Adh) gene in Drosophila melanogaster. Adh is regulated by two promoters, which are distinct in their developmental timing of activation. It has been shown using transgene insertion that when the promoter distal from the Adh start codon is deleted, transcription from the proximal promoter becomes de-regulated. As a result, the Adh proximal promoter, which is normally active only during the early larval stages, becomes abnormally activated in adults. Whether this type of regulation occurs in the endogenous Adh context, however, remains unclear. Here, we employed the CRISPR/Cas9 system to edit the endogenous Adh locus and found that removal of the distal promoter also resulted in the untimely expression of the proximal promoter-driven mRNA isoform in adults, albeit at lower levels than previously reported. Importantly, transcription from the distal promoter was sufficient to repress proximal transcription in larvae, and the degree of this repression was dependent on the degree of distal promoter activity. Finally, upregulation of the distal Adh transcript led to the enrichment of histone 3 lysine 36 trimethylation over the Adh proximal promoter. We conclude that the endogenous Adh locus is developmentally regulated by transcriptional interference in a tunable manner

Transaldolase inhibition impairs mitochondrial respiration and induces a starvation-like longevity response in <i>Caenorhabditis elegans</i>

<div><p>Mitochondrial dysfunction can increase oxidative stress and extend lifespan in <i>Caenorhabditis elegans</i>. Homeostatic mechanisms exist to cope with disruptions to mitochondrial function that promote cellular health and organismal longevity. Previously, we determined that decreased expression of the cytosolic pentose phosphate pathway (PPP) enzyme transaldolase activates the mitochondrial unfolded protein response (UPR^mt) and extends lifespan. Here we report that transaldolase (<i>tald-1</i>) deficiency impairs mitochondrial function <i>in vivo</i>, as evidenced by altered mitochondrial morphology, decreased respiration, and increased cellular H₂O₂ levels. Lifespan extension from knockdown of <i>tald-1</i> is associated with an oxidative stress response involving p38 and c-Jun N-terminal kinase (JNK) MAPKs and a starvation-like response regulated by the transcription factor EB (TFEB) homolog HLH-30. The latter response promotes autophagy and increases expression of the flavin-containing monooxygenase 2 (<i>fmo-2</i>). We conclude that cytosolic redox established through the PPP is a key regulator of mitochondrial function and defines a new mechanism for mitochondrial regulation of longevity.</p></div

Transaldolase deficiency causes a starvation-like response that decreases animal fat content and rewires lipid metabolism gene expression.

<p><b>(A)</b> Intestinal fat staining decreases from RNAi knockdown of <i>tald-1</i> or <i>cco-1</i>. Oil Red O (ORO) staining was performed on day 3 from hatching animals propagated at 20°C. Scale bar, 50 μm. <b>(B)</b> Quantification of ORO staining within anterior intestine (N = 2 independent experiments, pooled individual worm values, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(C)</b> RNAi knockdown of <i>tald-1</i> causes an increase in adipose triglyceride lipase ATGL-1 protein levels. Scale bar, 200 μm. <b>(D)</b> Mean relative fluorescence of ATGL-1::GFP signal in animals grown on <i>tald-1(RNAi)</i> or <i>cco-1(RNAi)</i>. Fluorescence is calculated relative to <i>EV(RNAi)</i> controls (N = 4 independent experiments, pooled individual worm values, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(E)</b> RNAi knockdown of <i>tald-1</i> or <i>cco-1</i> causes a decrease in stearoyl-CoA desaturase <i>fat-7p</i>::<i>gfp</i> reporter expression. Scale bar, 200 μm. <b>(F)</b> Mean relative fluorescence of <i>fat-7p</i>::<i>gfp</i> reporter animals grown on <i>tald-1(RNAi)</i> or <i>cco-1(RNAi)</i>. Fluorescence is calculated relative to <i>EV(RNAi)</i> controls (N = 3 independent experiments, pooled individual worm values, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(G)</b> Gene expression of starvation-responsive lipid metabolism genes is altered in <i>tald-1(RNAi)</i> animals. Log2 fold change calculated to emphasize the increases and decreases in gene expression levels from RNAi treatments (N = 6–8 independent experiments, error bars indicate s.e.m., paired student’s t-tests with Bonferroni’s correction). <b>(H)</b> RNAi knockdown of <i>tald-1</i> does not robustly extend lifespan of BD animals. N2 fed <i>EV(RNAi)</i> (mean 18.2±0.2 days, n = 161), N2 fed <i>tald-1(RNAi)</i> (mean 20.4±0.2 days, n = 151), BD animals developed on <i>EV(RNAi)</i> (mean 20.2±0.2 days, n = 123), BD animals developed on <i>tald-1(RNAi)</i> (mean 21.5±0.3 days, n = 150). Lifespans were performed at 25°C, with one experiment shown. <b>(I)</b> RNAi knockdown of <i>cco-1</i> extends lifespan dissimilar from BD. N2 fed <i>EV(RNAi)</i> (mean 18.2±0.2 days, n = 161), N2 fed <i>cco-1(RNAi)</i> (mean 24±0.3 days, n = 156), BD animals developed on <i>EV(RNAi)</i> (mean 20.2±0.2 days, n = 123), BD animals developed on <i>cco-1(RNAi)</i> (mean 27.1±0.3 days, n = 148). Lifespans were performed at 25°C, with one experiment shown. <b>(J)</b> RNAi knockdown of <i>tald-1</i> does not require NHR-49 for lifespan extension. N2 fed <i>EV(RNAi)</i> (mean 17.5±0.1 days, n = 366), N2 fed <i>tald-1(RNAi)</i> (mean 20.1±0.1 days, n = 397), <i>nhr-49(nr2041)</i> fed <i>EV(RNAi)</i> (mean 11.5±0.1 days, n = 310), <i>nhr-49(nr2041)</i> fed <i>tald-1(RNAi)</i> (mean 12.9±0.1 days, n = 333). Lifespans were performed at 25°C, with pooled data from three independent experiments shown. <b>(K)</b> RNAi knockdown of <i>cco-1</i> does not require NHR-49 for lifespan extension. N2 fed <i>EV(RNAi)</i> (mean 17±0.1 days, n = 532), N2 fed <i>cco-1(RNAi)</i> (mean 22.6±0.2 days, n = 344), <i>nhr-49(nr2041)</i> fed <i>EV(RNAi)</i> (mean 11.1±0.1 days, n = 495), <i>nhr-49(nr2041)</i> fed <i>cco-1(RNAi)</i> (mean 15±0.1 days, n = 489). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. Lifespans in this figure are indicated as mean±s.e.m. and statistical analysis is provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006695#pgen.1006695.s011" target="_blank">S1 Table</a>. In this figure, statistics are displayed as: * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001.</p

The flavin-containing monooxygenase FMO-2 is upregulated in a HLH-30 and PMK-1 dependent fashion and regulates the lifespan extension from <i>tald-1(RNAi)</i>.

<p><b>(A)</b><i>fmo-2p</i>::<i>mCherry</i> reporter expression is increased by <i>tald-1(RNAi)</i> or BD in a HLH-30 and PMK-1 dependent fashion. BD animals were starved for 24 hours on FUDR plates prior to imaging. Scale bar, 200 μm. <b>(B)</b> Mean relative fluorescence of <i>fmo-2p</i>::<i>mCherry</i> reporter animals in the context of the <i>hlh-30(tm1978)</i> mutation. Fluorescence is calculated relative to N2 <i>EV(RNAi)</i> controls (N = 3 independent experiments, pooled individual worm values, error bars indicate s.e.m., ANOVA with Bonferroni’s post-hoc). <b>(C)</b> Mean relative fluorescence of <i>fmo-2p</i>::<i>mCherry</i> reporter animals in the context of the <i>pmk-1(km25)</i> mutation. Fluorescence is calculated relative to N2 <i>EV(RNAi)</i> controls (N = 5 independent experiments, pooled individual worm values, error bars indicate s.e.m., ANOVA with Bonferroni’s post-hoc). <b>(D)</b> Gene expression of <i>fmo-2</i> is upregulated by <i>tald-1(RNAi)</i> or <i>cco-1(RNAi)</i> (N = 11 biological replicates, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(E)</b> Gene expression of <i>fmo-2</i> is upregulated by <i>tald-1(RNAi)</i> in a HLH-30 and PMK-1 dependent fashion (N = 3–6 biological replicates, error bars indicate s.e.m., ANOVA with Bonferroni’s post-hoc). <b>(F)</b> Percent of animals displaying HLH-30 nuclear localization. BD animals were starved for 8 hours on FUDR plates prior to imaging (N = 5 independent experiments, error bars indicate s.e.m., ANOVA with Bonferroni’s post-hoc). <b>(G)</b> FMO-2 is required for the lifespan extension from <i>tald-1(RNAi)</i>. N2 fed <i>EV(RNAi)</i> (mean 15.3±0.1 days, n = 341), N2 fed <i>tald-1(RNAi)</i> (mean 17.8±0.1 days, n = 353), <i>fmo-2(ok2147)</i> fed <i>EV(RNAi)</i> (mean 18±0.2 days, n = 314), <i>fmo-2(ok2147)</i> fed <i>tald-1(RNAi)</i> (mean 17.4±0.2 days, n = 382). Lifespans were performed at 25°C, with pooled data from three independent experiments shown. <b>(H)</b> FMO-2 is partially required for the lifespan extension from <i>cco-1(RNAi)</i>. N2 fed <i>EV(RNAi)</i> (mean 15.7±0.1 days, n = 562), N2 fed <i>cco-1(RNAi)</i> (mean 23.3±0.2 days, n = 616), <i>fmo-2(ok2147)</i> fed <i>EV(RNAi)</i> (mean 18.3±0.1 days, n = 474), <i>fmo-2(ok2147)</i> fed <i>cco-1(RNAi)</i> (mean 20.5±0.2 days, n = 473). Lifespans were performed at 25°C, with pooled data from five independent experiments shown. <b>(I)</b> Lifespan extension from <i>fmo-2</i> overexpression is not additive with <i>tald-1(RNAi)</i>. N2 fed <i>EV(RNAi)</i> (mean 16.5±0.1 days, n = 453), N2 fed <i>tald-1(RNAi)</i> (mean 20.6±0.1 days, n = 421), <i>eft-3p</i>::<i>fmo-2</i> fed <i>EV(RNAi)</i> (mean 18.2±0.1 days, n = 439), <i>eft-3p</i>::<i>fmo-2</i> fed <i>tald-1(RNAi)</i> (mean 19.1±0.1 days, n = 435). Lifespans were performed at 25°C, with pooled data from three independent experiments shown. <b>(J)</b> Lifespan extension from <i>fmo-2</i> overexpression is additive with <i>cco-1(RNAi)</i>. N2 fed <i>EV(RNAi)</i> (mean 16.5±0.1 days, n = 453), N2 fed <i>cco-1(RNAi)</i> (mean 23.3±0.2 days, n = 259), <i>eft-3p</i>::<i>fmo-2</i> fed <i>EV(RNAi)</i> (mean 18.2±0.1 days, n = 439), <i>eft-3p</i>::<i>fmo-2</i> fed <i>cco-1(RNAi)</i> (mean 25.5±0.2 days, n = 352). Lifespans were performed at 25°C, with pooled data from three independent experiments shown. Lifespans in this figure are indicated as mean±s.e.m. and statistical analysis is provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006695#pgen.1006695.s011" target="_blank">S1 Table</a>. In this figure, statistics are displayed as: * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001.</p

Transaldolase deficiency alters mitochondrial morphology and decreases <i>in vivo</i> mitochondrial respiration.

<p><b>(A)</b> Diagram depicting the posterior intestinal cells that were visualized for mitochondrial morphology. <b>(B)</b> Intestinal mitochondrial morphology is altered by <i>tald-1(RNAi)</i> and <i>cco-1(RNAi)</i>. The top panel represents a single 0.34 μm slice imaged using confocal microscopy, with a magnified area displayed in a white dotted box to highlight morphology differences. The bottom panel consists of a max intensity projection of five z-slices to emphasize mitochondrial content in these cells. Scale bar, 10 μm. <b>(C)</b> Quantification of percent mitochondrial area per cell. (N = 2 independent experiments, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(D)</b> Mitochondrial morphology changes from <i>tald-1(RNAi)</i> are regulated by DRP-1. RNAi treatments include <i>EV(RNAi)</i>, <i>tald-1(RNAi)</i> [50:50 with <i>EV(RNAi)</i>], <i>drp-1(RNAi)</i> [50:50 with <i>EV(RNAi)</i>], and <i>tald-1(RNAi)</i> [50:50 with <i>drp-1(RNAi)</i>]. Scale bar, 10 μm. <b>(E)</b> Oxygen consumption rate decreases with <i>tald-1(RNAi)</i> and <i>cco-1(RNAi)</i>. OCR was measured using the Seahorse XF Analyzer and normalized to animal number (N = 6 independent experiments, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(F)</b> P/O ratio (the ATP produced per oxygen atom reduced), <b>(G)</b> respiratory control index (State 3:State 4 rates), <b>(H)</b> malate-driven respiration (Complex I-IV), succinate-driven respiration (Complex II-IV), and TMPD/ascorbate-driven respiration (Complex IV) were measured using the OXPHOS assay on isolated mitochondria from RNAi treated animals. Respiratory rates were measured as rate of disappearance of oxygen (nmol[O₂]) per minute per mg protein (N = 4 independent experiments, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). Also, in this figure, color coating of bars and lines reflect the legend in (C).</p

Lifespan extension from <i>tald-1(RNAi)</i> or <i>cco-1(RNAi)</i> requires stress-activated MAPKs.

<p><b>(A)</b> RNAi knockdown of <i>tald-1</i> extends lifespan through the JNK MAPK JNK-1. N2 fed <i>EV(RNAi)</i> (mean 17.2±0.1 days, n = 506), N2 fed <i>tald-1(RNAi)</i> (mean 20.4±0.1 days, n = 500), <i>jnk-1(gk7)</i> fed <i>EV(RNAi)</i> (mean 17±0.1 days, n = 582), <i>jnk-1(gk7)</i> fed <i>tald-1(RNAi)</i> (mean 18.1±0.1 days, n = 488). Lifespans were performed at 25°C, with pooled data from five independent experiments shown. <b>(B)</b> RNAi knockdown of <i>cco-1</i> extends lifespan partially through the JNK MAPK JNK-1. N2 fed <i>EV(RNAi)</i> (mean 16.9±0.1 days, n = 494), N2 fed <i>cco-1(RNAi)</i> (mean 22.7±0.2 days, n = 431), <i>jnk-1(gk7)</i> fed <i>EV(RNAi)</i> (mean 16.1±0.1 days, n = 594), <i>jnk-1(gk7)</i> fed <i>cco-1(RNAi)</i> (mean 19.9±0.2 days, n = 408). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. <b>(C)</b> RNAi knockdown of <i>tald-1</i> extends lifespan through the JNK MAPK KGB-1. N2 fed <i>EV(RNAi)</i> (mean 15±0.1 days, n = 630), N2 fed <i>tald-1(RNAi)</i> (mean 18.7±0.1 days, n = 657), <i>kgb-1(um3)</i> fed <i>EV(RNAi)</i> (mean 13.1±0.1 days, n = 580), <i>kgb-1</i> fed <i>tald-1(RNAi)</i> (mean 11.9±0.1 days, n = 600). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. <b>(D)</b> RNAi knockdown of <i>cco-1</i> extends lifespan partially through the JNK MAPK KGB-1. N2 fed <i>EV(RNAi)</i> (mean 15±0.1 days, n = 630), N2 fed <i>cco-1(RNAi)</i> (mean 23.2±0.2 days, n = 511), <i>kgb-1(um3)</i> fed <i>EV(RNAi)</i> (mean 13.1±0.1 days, n = 580), <i>kgb-1</i> fed <i>cco-1(RNAi)</i> (mean 15.8±0.2 days, n = 501). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. <b>(E)</b> RNAi knockdown of <i>tald-1</i> extends lifespan through the p38 MAPK PMK-1. N2 fed <i>EV(RNAi)</i> (mean 16.8±0.1 days, n = 494), N2 fed <i>tald-1(RNAi)</i> (mean 19.3±0.1 days, n = 460), <i>pmk-1(km25)</i> fed <i>EV(RNAi)</i> (mean 14.3±0.1 days, n = 514), <i>pmk-1(km25)</i> fed <i>tald-1(RNAi)</i> (mean 14±0.1 days, n = 525). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. <b>(F)</b> RNAi knockdown of <i>cco-1</i> does not require the p38 MAPK PMK-1 for lifespan extension. N2 fed <i>EV(RNAi)</i> (mean 16±0.1 days, n = 575), N2 fed <i>cco-1(RNAi)</i> (mean 22.3±0.2 days, n = 448), <i>pmk-1(km25)</i> fed <i>EV(RNAi)</i> (mean 13.8±0.1 days, n = 609), <i>pmk-1(km25)</i> fed <i>cco-1(RNAi)</i> (mean 18.7±0.1 days, n = 535). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. <b>(G)</b> RNAi knockdown of <i>tald-1</i> extends lifespan through the MAP3K NSY-1. N2 fed <i>EV(RNAi)</i> (mean 14.6±0.1 days, n = 542), N2 fed <i>tald-1(RNAi)</i> (mean 17.2±0.1 days, n = 599), <i>nsy-1(ag3)</i> fed <i>EV(RNAi)</i> (mean 14.9±0.1 days, n = 473), <i>nsy-1(ag3)</i> fed <i>tald-1(RNAi)</i> (mean 14.4±0.1 days, n = 508). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. <b>(H)</b> RNAi knockdown of <i>cco-1</i> extends lifespan partially through the MAP3K NSY-1. N2 fed <i>EV(RNAi)</i> (mean 14.6±0.1 days, n = 542), N2 fed <i>cco-1(RNAi)</i> (mean 22.5±0.2 days, n = 454), <i>nsy-1(ag3)</i> fed <i>EV(RNAi)</i> (mean 14.9±0.1 days, n = 473), <i>nsy-1(ag3)</i> fed <i>cco-1(RNAi)</i> (mean 18.5±0.2 days, n = 458). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. Lifespans in this figure are indicated as mean±s.e.m. and statistical analysis is provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006695#pgen.1006695.s011" target="_blank">S1 Table</a>.</p