RNAi Screening Implicates a SKN-1–Dependent Transcriptional Response in Stress Resistance and Longevity Deriving from Translation Inhibition

Joslin Diabetes Center, Harvard Stem Cell Institute, and Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.
PLoS Genetics (Impact Factor: 7.53). 08/2010; 6(8). DOI: 10.1371/journal.pgen.1001048
Source: PubMed


Caenorhabditis elegans SKN-1 (ortholog of mammalian Nrf1/2/3) is critical for oxidative stress resistance and promotes longevity under reduced insulin/IGF-1-like signaling (IIS), dietary restriction (DR), and normal conditions. SKN-1 inducibly activates genes involved in detoxification, protein homeostasis, and other functions in response to stress. Here we used genome-scale RNA interference (RNAi) screening to identify mechanisms that prevent inappropriate SKN-1 target gene expression under non-stressed conditions. We identified 41 genes for which knockdown leads to activation of a SKN-1 target gene (gcs-1) through skn-1-dependent or other mechanisms. These genes correspond to multiple cellular processes, including mRNA translation. Inhibition of translation is known to increase longevity and stress resistance and may be important for DR-induced lifespan extension. One model postulates that these effects derive from reduced energy needs, but various observations suggest that specific longevity pathways are involved. Here we show that translation initiation factor RNAi robustly induces SKN-1 target gene transcription and confers skn-1-dependent oxidative stress resistance. The accompanying increases in longevity are mediated largely through the activities of SKN-1 and the transcription factor DAF-16 (FOXO), which is required for longevity that derives from reduced IIS. Our results indicate that the SKN-1 detoxification gene network monitors various metabolic and regulatory processes. Interference with one of these processes, translation initiation, leads to a transcriptional response whereby SKN-1 promotes stress resistance and functions together with DAF-16 to extend lifespan. This stress response may be beneficial for coping with situations that are associated with reduced protein synthesis.

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Available from: T Keith Blackwell, Dec 10, 2014
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    • "Our previous studies demonstrated that SKN-1 is under direct regulation by a WD40 repeat protein named WDR-23; WDR-23 directly binds to SKN-1 to restrain its nuclear accumulation under basal conditions presumably by recruiting the transcription factor to a ubiquitin ligase (Choe et al. 2009). SKN-1 also functions downstream of target of rapamycin (TOR) and insulin/IGF-1-like signaling (IIS) (Tullet et al. 2008; Wang et al. 2010; Robida-Stubbs et al. 2012) pathways that influence longevity and is required for lifespan extension by dietary restriction (Tang and Choe 2015; Bishop and Guarente 2007). Regulation of SKN-1 and the mechanisms by which it influences longevity and stress resistance are highly active areas of research (Blackwell et al. 2015; Ewald et al. 2015; Steinbaugh et al. 2015; Tang and Choe 2015; Dresen et al. 2015; Chew et al. 2015). "
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    ABSTRACT: In Caenorhabditis elegans, the transcription factor SKN-1 has emerged as a central coordinator of stress responses and longevity increasing the need for genetic tools to study its regulation and function. However, current loss of function alleles cause fully penetrant maternal effect embryonic lethality and must be maintained with genetic balancers that require careful monitoring and labor intensive strategies to obtain large populations. In this study, we identified a strong, but viable skn-1 hypomorphic allele skn-1(zj0015) from a genetic screen for suppressors of wdr-23, a direct regulator of the transcription factor. skn-1(zj0015) is a point mutation in an intron that causes mis-splicing of a fraction of mRNA and strongly reduces wildtype mRNA levels of the two long skn-1a/c variants. The skn-1(zj0015) allele reduces detoxification gene expression and stress resistance to levels comparable to skn-1 RNAi, but unlike RNAi, it is not restricted from some tissues. We also show that skn-1(zj0015) is epistatic to canonical upstream regulators demonstrating its utility for genetic analysis of skn-1 function and regulation in cases where large numbers of worms are needed, a balancer is problematic, diet is varied, or RNAi cannot be used.
    Preview · Article · Dec 2015 · G3-Genes Genomes Genetics
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    • "Therefore, researchers have turned to the use of glp-1(ts) and glp-4 (bn2ts) mutants and temperature shifts to generate populations of adult hermaphrodites that have very few germ cells. For example, glp-4(bn2ts) mutants, as well as glp-1(ts) mutants, have been used to examine the role of the germline in aging, stress resistance, pathogen resistance and fat metabolism (e.g., Wang et al. 2008; TeKippe and Aballay 2010; Greer et al. 2010; Labbadia and Morimoto 2015). Nevertheless, interpretation of findings after use of glp-4 and glp-1 mutants to assess the role of germ cell proliferation on organismal properties relies on the assumption that the mutant conditions do not also affect somatic tissues in a way that could influence aging, stress resistance, pathogen resistance and metabolism. "
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    ABSTRACT: Germline stem cell proliferation is necessary to populate the germline with sufficient numbers of cells for gametogenesis and for signaling the soma to control organismal properties such as aging. The Caenorhabditis elegans gene glp-4 was identified by the temperature sensitive allele bn2 where mutants raised at the restrictive temperature produce adults that are essentially germ cell deficient, containing only a small number of stem cells arrested in the mitotic cycle, but otherwise have a morphologically normal soma. We determined that glp-4 encodes a valyl aminoacyl tRNA Synthetase (VARS-2) and that the probable null phenotype is early larval lethality. Phenotypic analysis indicates glp-4(bn2ts) is partial loss of function in the soma. Structural modeling suggests that bn2 Gly296Asp results in partial loss of function by a novel mechanism: aspartate 296 in the editing pocket induces inappropriate deacylation of correctly charged Val-tRNA(val). Intragenic suppressor mutations are predicted to displace aspartate 296 so that it is less able to catalyze inappropriate deacylation. Thus glp-4(bn2ts) likely causes reduced protein translation due to decreased levels of Val-tRNA(val). The germline, as a reproductive preservation mechanism during unfavorable conditions, signals the soma for organismal aging, stress and pathogen resistance. glp-4(bn2ts) mutants are widely used to generate germline deficient mutants for organismal studies, under the assumption that the soma is unaffected. As reduced translation has also been demonstrated to alter organismal properties, it is unclear if changes in aging, stress resistance, etc. observed in glp-4(bn2ts) mutants are the result of germline deficiency or reduced translation.
    Preview · Article · Oct 2015 · G3-Genes Genomes Genetics
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    • "RNAi experiments clearly indicated that cct-2 and daf-19 knock down worms show enhanced susceptibility to S. flexneri infection (Figures 6.C, D and 7.D, E), suggesting that these responses form part of the nematode protective mechanism. The chaperonin CCT-2 has been predicted to interact with DAF-16, which forms part of the nematode DAF-2/DAF-16 insulin signaling pathway; RNAi knock down of cct-2 inhibits the nuclear localization of DAF-16 in intestinal cells [42], suggesting that CCT-2 is required for the nuclear translocation of DAF-16. DAF-16 is a transcription factor that has been associated with the expression of several antimicrobial genes [43]. "
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    ABSTRACT: The Gram-negative bacterium Shigella flexneri is the causative agent of shigellosis, a diarrhoeal disease also known as bacillary dysentery. S. flexneri infects the colonic and rectal epithelia of its primate host and induces a cascade of inflammatory responses that culminates in the destruction of the host intestinal lining. Molecular characterization of host-pathogen interactions in this infection has been challenging due to the host specificity of S. flexneri strains, as it strictly infects humans and non-human primates. Recent studies have shown that S. flexneri infects the soil dwelling nematode Caenorhabditis elegans, however, the interactions between S. flexneri and C. elegans at the cellular level and the cause of nematode death are unknown. Here we attempt to gain insight into the complex host-pathogen interactions between S. flexneri and C. elegans. Using transmission electron microscopy, we show that live S. flexneri cells accumulate in the nematode intestinal lumen, produce outer membrane vesicles and invade nematode intestinal cells. Using two-dimensional differential in-gel electrophoresis we identified host proteins that are differentially expressed in response to S. flexneri infection. Four of the identified genes, aco-1, cct-2, daf-19 and hsp-60, were knocked down using RNAi and ACO-1, CCT-2 and DAF-19, which were identified as up-regulated in response to S. flexneri infection, were found to be involved in the infection process. aco-1 RNAi worms were more resistant to S. flexneri infection, suggesting S. flexneri-mediated disruption of host iron homeostasis. cct-2 and daf-19 RNAi worms were more susceptible to infection, suggesting that these genes are induced as a protective mechanism by C. elegans. These observations further our understanding of the processes involved in S. flexneri infection of C. elegans, which is immensely beneficial to the routine use of this new in vivo model to study S. flexneri pathogenesis.
    Full-text · Article · Sep 2014 · PLoS ONE
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