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Survival from Hypoxia in C. elegans by Inactivation of Aminoacyl-tRNA Synthetases

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Science  30 Jan 2009:
Vol. 323, Issue 5914, pp. 630-633
DOI: 10.1126/science.1166175

Abstract

Hypoxia is important in a wide range of biological processes, such as animal hibernation and cell survival, and is particularly relevant in many diseases. The sensitivity of cells and organisms to hypoxic injury varies widely, but the molecular basis for this variation is incompletely understood. Using forward genetic screens in Caenorhabditis elegans, we isolated a hypoxia-resistant reduction-of-function mutant of rrt-1 that encodes an arginyl–transfer RNA (tRNA) synthetase, an enzyme essential for protein translation. Knockdown of rrt-1, and of most other genes encoding aminoacyl-tRNA synthetases, rescued animals from hypoxia-induced death, and the level of hypoxia resistance was inversely correlated with translation rate. The unfolded protein response was induced by hypoxia and was required for the hypoxia resistance of the reduction-of-function mutant of rrt-1. Thus, translational suppression produces hypoxia resistance, in part by reducing unfolded protein toxicity.

Oxygen requirements of cells and organisms have wide-ranging implications in behavior and disease. Forward genetic screens offer the possibility of discovering genes not previously known to control hypoxic sensitivity. Such genes are likely to play an important role in emergent organismal traits such as habitat range and ability to hibernate. Additionally, these genes may lead to the development of novel therapies for conditions where cellular hypoxic sensitivity is a pathological determinant, such as stroke, myocardial infarction, and cancer. Wild-type C. elegans when placed in a severe hypoxic environment (oxygen concentration <0.3 volume percent) become immobile but fully recover when returned to normoxia within 4 hours (1). After 4 hours, permanent behavioral deficits and cellular death ensue, and after a 22-hour hypoxic incubation, >99% of wild-type animals are dead. To identify genes that control hypoxic sensitivity, we screened for ethylmethane sulfonate (EMS)–derived mutants that survived a 22-hour hypoxic incubation. In a screen of 3884 F1 mutant worm genomes, we recovered 14 mutants that had a hypoxia-resistant phenotype (table S1). These mutants fell into 13 complementation groups. We selected gc47, one of the strongest hypoxia-resistant mutants, for further characterization and mapping.

After outcrossing to the wild-type strain N2, the hypoxia resistance of gc47 was quantified. Immediately after removal from a 20-hour hypoxic incubation, both N2 and gc47 were paralyzed, but the gc47 worms recovered the ability to move completely over the next 1 to 2 hours. After a 24-hour recovery, essentially all of the gc47 animals were alive, whereas almost all wild-type worms failed to survive (Fig. 1, A and B); gc47 prolonged the hypoxic incubation time required for complete killing by a factor of >3 (Fig. 1C). The hypoxia-resistant phenotype was fully recessive and segregated as a single locus in a Mendelian fashion (Fig. 1D) (2). gc47 was mapped to a 106-kb interval on the left arm of chromosome III (Fig. 2A) (2). Double-stranded RNA interference (RNAi) of 29 of the 32 genes in the interval identified only one gene, rrt-1, whose knockdown produced high-level hypoxia resistance (Fig. 2B). Simultaneously, five fosmids that together spanned the entire interval were individually injected to attempt transformation rescue of gc47. Only one fosmid restored normal hypoxia sensitivity to gc47 (Fig. 2C); the rescuing fosmid contained the rrt-1 gene implicated by RNAi. Sequencing rrt-1 in gc47 found a single G → A transition, resulting in a change of amino acid residue 271 from an aspartate to an asparagine in gc47 (D271N, Fig. 2A). Thus, gc47 is an allele of rrt-1 and behaves like a reduction-of-function allele.

Fig. 1.

gc47 is a potent regulator of hypoxic cell death in C. elegans. (A and B) Time-lapse images (magnification 4×) of gc47 (A) and N2 (B) adult worms after a 24-hour recovery from a 20-hour hypoxic insult. (C) Percent dead animals for N2 (squares) and gc47 (circles) after a 24-hour recovery as a function of length of hypoxic insult. (D) Percent death among homozygous gc47 (open bar, 13 trials), heterozygous gc47/+ (hatched bar, 9 trials), and N2 (solid bar, 16 trials) animals; the results show that gc47 is recessive. Data are means ± SEM with >30 animals per trial; *P < 0.01 (two-tailed t test).

Fig. 2.

gc47 is an allele of rrt-1. (A) Genetic mapping of gc47. Three-factor mapping with the indicated visible markers (black text) and single-nucleotide polymorphisms (gray text) placed gc47 in a 106-kb interval on chromosome III between CE3-141 and snpLA3. Fosmids injected for transformation rescue are shown. Amino acid alignment is with rrt-1 orthologs; the gc47 mutation is boxed. Amino acid abbreviations: A, Ala; C, Cys; D, Asp; E, Glu; G, Gly; H, His; I, Ile; L, Leu; M, Met; N, Asn; R, Arg; S, Ser; T, Thr; V, Val. (B) RNAi of genes within the 106,000–base pair mapping interval. Death was induced by hypoxia in animals treated with 29 of 32 predicted genes within the mapping interval. rrt-1 RNAi conferred highly significant hypoxia resistance relative to empty vector (*P < 0.01; two-tailed t test, n = 30 animals per data point). (C) Transformation rescue of gc47. Hypoxia-induced animal death was scored for N2, gc47 [full genotype: rrt-1(gc47) dpy-17(e164)], and gc47 transformed with the transformation marker pPHGFP alone or in addition to the rescuing fosmid WRM0615bG07. Data are means ± SEM of at least two trials, >20 animals per trial; *P < 0.01 (two-tailed t test).

rrt-1 encodes an arginyl-tRNA synthetase, one of the aminoacyl-tRNA synthetases (AARSs). AARSs catalyze the adenosine triphosphate (ATP)–dependent acylation of their cognate tRNA(s) with a specific amino acid (3). AARSs fall into two distinct structural classes. RRT-1 is a class I enzyme, characterized by HIGH and MRSK domains (4, 5). In higher eukaryotes, the RRT-1 ortholog has been isolated in a cytoplasmic complex with six other AARSs and three accessory subunits (6, 7). Some AARSs are specific for mitochondrial DNA, and besides their role in translation, a subset has been implicated in non–translation-related functions (8, 9). To test whether control of hypoxic sensitivity is a general function of AARSs or is restricted to a particular subset, we used feeding RNAi constructs against 23 of the 33 predicted AARSs in the C. elegans genome. All but one of the 23 RNAi constructs conferred significant hypoxia resistance (table S2). Thus, most, if not all, AARSs control hypoxic sensitivity. Knockdown of some AARSs produced stronger hypoxia-resistant phenotypes than others, which was not explained by the class of AARSs or whether the tRNA substrate was cytoplasmic or mitochondrial. Although the degree of RNAi knockdown was variable (table S2), RNAi efficacy did not explain all of the phenotypic variance. To examine whether the variable levels of hypoxia resistance could be explained by differences in translational suppression, we measured [35S]methionine incorporation in animals treated with select AARS RNAi constructs as well as in rrt-1(gc47) (fig. S1). The level of hypoxia resistance had a strong inverse correlation with the relative translation rate. However, the absolute amount of translational suppression was relatively modest even in the strongest hypoxia-resistant animals, where the [35S]methionine incorporation was about half that of vector controls. Thus, hypoxic sensitivity appears to be exquisitely sensitive to even small changes in translation rate. Consistent with translational suppression as the proximate mechanism of hypoxia resistance, treatment with cycloheximide also conferred hypoxia resistance (fig. S2).

Reduced translation has been shown to lengthen life span in C. elegans and other organisms (1012) and a known, long-lived mutant was also found to be highly hypoxia-resistant (1). Thus, we hypothesized that rrt-1(gc47) would be long-lived. Indeed, rrt-1(gc47) had a small but significantly increased life span (fig. S3). Although this further links hypoxia resistance and long life span, the difference in the strength of the two phenotypes suggests that their mechanisms downstream of RRT-1 are distinct or that hypoxic sensitivity is much more responsive than life span to alterations in the translation machinery. Other phenotypes seen in gc47 were a modest decrease in fecundity, a small but significant level of embryonic lethality, a reduction in speed of movement to about half that of wild-type animals, and a slight developmental delay (table S3). Thus, as expected for a gene with such an essential function, rrt-1(gc47) has a pleiotropic phenotype but the strongest observed phenotype was hypoxia resistance.

RRT-1 presumably functions in all cells to mediate translation. However, because of their high metabolic activity, germ cells might be particularly vulnerable to hypoxic injury and thereby determine the hypoxic sensitivity of the whole organism. To examine this issue, we made use of a mutation in the rrf-1 gene, which encodes an RNA-directed RNA polymerase required for the somatic but not germline actions of RNAi (13). The hypoxia resistance of rrt-1(RNAi) was greatly reduced in an rrf-1 mutant versus wild-type background (fig. S4), indicating that RRT-1 acts in both somatic and germline cells to mediate hypoxic sensitivity. Myocytes and neurons are two major somatic cell types where hypoxia produces characteristic pathological changes (1, 14). Hypoxia-induced myocyte nuclear fragmentation and neuron axonal degeneration were both abated in rrt-1(gc47) (fig. S5).

To determine when RRT-1 functions to regulate hypoxic sensitivity, we applied RNAi to wild-type animals early in development, early in adulthood before the hypoxic insult, or in adulthood after the hypoxic insult (Fig. 3A). RNAi either during early development or during early adulthood protected equally well from subsequent hypoxic death (Fig. 3B). Thus, the hypoxia-resistant phenotype of rrt-1 reduction of function is not dependent on developmental stage and can be induced after development is complete. Exposure to rrt-1(RNAi) only after the hypoxic insult also increased survival from delayed hypoxic death (Fig. 3C).

Fig. 3.

rrt-1 acutely controls hypoxic sensitivity, both during and after the insult. (A) Schematic of the experimental protocol. Wild-type animals were exposed to rrt-1 or L4440 empty vector RNAi at the developmental stages indicated, and were then treated with a 20-hour (B) or 16-hour (C) hypoxic incubation (HYP, yellow boxes); Emb, embryo. (B) Hypoxia resistance by rrt-1(RNAi) is not dependent on developmental stage [n = 40 animals per condition; *P < 0.05 versus L4440 (Fisher's exact test, two-sided)]. (C) Inhibition of rrt-1 is effective after hypoxic insult. Percent survived = [(number of animals alive at day of interest)/(number of animals alive initially after 24-hour recovery)] × 100; *P < 0.05 versus L4440 (Fisher's exact test, two-sided); n > 300 initially alive worms per RNAi over three independent trials.

The reduction-of-function mutant rrt-1(rf) reduces global translation rate and consequently should reduce oxygen consumption. Indeed, the rate of paralysis by hypoxia, which should correlate with oxygen consumption, is decreased in rrt-1(gc47) (fig. S6). Reduced oxygen consumption by translational arrest is a logical and established mechanism for reducing cellular injury during hypoxia but not after (1517). Thus, the mechanism of protection by rrt-1 knockdown, at least that functioning after the hypoxic insult, appears to be more complex than a global reduction in oxygen consumption by translational arrest.

Hypoxia produces intracellular misfolded proteins and thereby induces the unfolded protein response (UPR) (18). One effect of UPR induction is phosphorylation of the translation initiation factor eIF2-α, thereby suppressing translation (19); this has been proposed as an adaptive mechanism to reduce the load of newly synthesized and unfolded proteins, particularly in the context of cancer cell biology. To determine whether the hypoxia-resistant phenotype of rrt-1(rf) may be due to a reduction in unfolded proteins, we used strains carrying a transgene, Phsp-4::GFP, consisting of a fusion between the hsp-4 promoter and GFP (green fluorescent protein). Phsp-4::GFP expression has been shown to be a reliable indicator of the level of unfolded proteins and of activation of the UPR (2022). Hypoxia induced a significant increase in expression of Phsp-4::GFP that peaked 4 hours after recovery from hypoxia and was dependent on the length of hypoxic incubation (Fig. 4, A to C). The glycosylation inhibitor tunicamycin, which increases the level of unfolded proteins, also induced Phsp-4::GFP expression. Induction of Phsp-4::GFP expression by either hypoxia or tunicamycin was blocked virtually completely by a loss-of-function mutation in ire-1 [ire-1(lf)], which encodes an endoplasmic reticulum transluminal kinase essential for the UPR (22, 23) (Fig. 4, A and B). Consistent with a reduction in unfolded protein load, rrt-1(RNAi) completely blocked hypoxic induction of Phsp-4::GFP (Fig. 4, A and B); however, it did not diminish induction by tunicamycin. Thus, the level of translational suppression by rrt-1(RNAi) does not preclude synthesis of the GFP marker under strong inducing conditions. Further supporting the hypothesis that rrt-1(rf) reduces the load of unfolded proteins, rrt-1(gc47) was highly resistant to tunicamycin-induced developmental arrest (Fig. 4D). Finally, we found that ire-1(lf) and its downstream target xbp-1(lf) (22) significantly suppressed the hypoxia resistance produced by rrt-1(RNAi), but neither ire-1(lf) nor xbp-1(lf) induced hypersensitivity in rrt-1(+) animals (Fig. 4E). Rather, the ire-1(lf) and xbp-1(lf) animals were weakly resistant. These data indicate that inhibition of translation by rrt-1(lf) and the UPR interact synergistically to reduce hypoxic sensitivity, but that in the absence of translational suppression by rrt-1(lf), an intact UPR can promote death after a severe hypoxic insult.

Fig. 4.

The unfolded protein response is induced by hypoxia and is required for high-level hypoxia resistance of rrt-1(RNAi). (A) Phsp-4::GFP expression in age-matched young adult C. elegans after incubation for 6 hours in M9 buffer in a normoxic or hypoxic environment or with tunicamycin (25 μg/ml). zcIs4[Phsp-4::GFP] animals were raised on empty vector or rrt-1(RNAi) bacteria; ire-1(zc14);zcIs4 animals were raised on empty vector. Scale bar, 200 μm. (B) Quantification of Phsp-4::GFP expression. *P < 0.01, unpaired two-sided t test; au, arbitrary units. (C) Time course of Phsp-4::GFP induction after hypoxic (H) or normoxic (N) incubations of 2, 4, or 8 hours. (D) Sensitivity to developmental arrest by tunicamycin in N2 and rrt-1(gc47) animals. Freshly laid eggs were allowed to develop on agar plates containing the indicated concentrations of tunicamycin; percent of animals reaching adulthood after 7 days of development was scored. (E) Hypoxic sensitivity of wild-type or UPR pathway mutant animals exposed to rrt-1(RNAi) (30-hour hypoxic incubation) or empty vector control (20-hour hypoxic incubation). *P < 0.05, paired t test.

Translational repression is a well-established mechanism of survival for hibernating animals in a prolonged hypoxic environment (24). Translational mechanisms are important in the tumorigenicity of cancer cells (25) and are increasingly implicated in the sensitivity of normal cell types to hypoxic and ischemic injury (15, 26). In these diverse scenarios, translational repression results in several secondary changes in the biology of the cell, including decreases in ATP consumption and protein aggregates and an alteration of the proteome. Our data show that a modest suppression of translation that allows relatively normal growth and physiology can produce a profound hypoxia resistance that requires the UPR for its full phenotypic expression. One effect of UPR activation is translational suppression, which occurs by a mechanism distinct from limiting aminoacylated tRNA levels. A logical model is that a reduction in translation rate by a decrement in AARS activity reduces the unfolded protein load to a level that is manageable by the UPR and may synergize with the translational suppression produced by the UPR itself. However, without translational inhibition, the activity of the UPR may be maladaptive in the context of hypoxic injury. This interaction between translational activity and the UPR may be exploited to regulate hypoxic cell death.

Supporting Online Material

www.sciencemag.org/cgi/content/full/323/5914/630/DC1

Materials and Methods

Figs. S1 to S6

Tables S1 to S4

References

References and Notes

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