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.

Download full-text


Available from: T Keith Blackwell, Dec 10, 2014
14 Reads
  • Source
    • "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]. "
    [Show abstract] [Hide abstract]
    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.
    PLoS ONE 09/2014; 9(9):e106085. DOI:10.1371/journal.pone.0106085 · 3.23 Impact Factor
  • Source
    • "Positive controls for the heat shock responsive reporter strains SJ4005 (hsp-4p::GFP), CL2070 (hsp-16-2p::GFP) and AM446 (hsp-70p::GFP) young adults were heated to 37 °C for 2 h and then allowed to recover for 6 h at 20 °C before being evaluated for GFP expression (Link et al., 1999; Morley and Morimoto, 2004). As a positive control for sod-3p::GFP, gcs-1p::GFP and gst-4p::GFP, the atypical protein kinase riok-1 was knocked down using RNA interference (RNAi) (Wang et al., 2010). Briefly, HT115 bacteria were induced to express riok-1 double stranded (ds)RNA by seeding on NGM plates containing 6 mM isopropyl b-D-1-thiogalactopyranoside (IPTG, Sigma) (Kamath et al., 2001). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Traditional healers in Sarawak, Malaysia, use plants such as Picria fel-terrae, Linariantha bicolor and Lansium domesticum to treat gastrointestinal infections. This study aimed to test whether their nematocidal activities could be confirmed in vitro using highly standardised Caenorhabditis elegans models. We applied eight different ethanol solubilised plant extracts and two commercial anthelmintic drugs to larval and adult stages of C. elegans in vitro. Seven C. elegans strains were evaluated, one wild type and six strains with GFP-tagged stress response pathways to help characterise and compare the pathways affected by plant extracts. Our in vitro screen confirmed that both of the commercial anthelmintic drugs and five of the eight traditionally used plant extracts had significant nematocidal activity against both larval and adult C. elegans. The most effective extracts were from P. fel-terrae. The plant extracts triggered different stress response pathways from the commercial anthelmintic drugs. This study showed that using traditional knowledge of plant medicinal properties in combination with a C. elegans in vitro screen provided a rapid and economical test with a high hit rate compared with the random screening of plants for nematocidal activities. The use of transgenic C. elegans strains may allow this approach to be refined further to investigate the mode of action of active extracts.
    International journal for parasitology 02/2014; 44(5). DOI:10.1016/j.ijpara.2014.01.008 · 3.87 Impact Factor
  • Source
    • "An identical group of worms was treated with the vehicle (Ve), and a third group was grown on daf-2 RNAi bacteria. For the qPCR reactions, we used primers toward the DAF-16 target genes sod-3 and hsp-12.6 (Murphy et al., 2003), the HSF-1 regulated genes hsp-16.1 (Link et al., 1999) and hsp-70, and the SKN-1 controlled genes gst-4 and gst-10 (Wang et al., 2010). Three independent experiments showed that NT219 (Fig. 2D–F, spotted bars) elevates the expression levels of all genes compared with the levels seen in control worms (black bars). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Aging manipulation is an emerging strategy aimed to postpone the manifestation of late-onset neurodegenerative disorders such as Alzheimer's (AD) and Huntington's diseases (HD) and to slow their progression once emerged. Reducing the activity of the insulin/IGF signaling cascade (IIS), a prominent aging-regulating pathway, protects worms from proteotoxicity of various aggregative proteins, including the AD-associated peptide, Aβ- and the HD-linked peptide, polyQ40. Similarly, IGF1 signaling reduction protects mice from AD-like disease. These discoveries suggest that IIS inhibitors can serve as new drugs for the treatment of neurodegenerative maladies including AD and HD. Here, we report that NT219, a novel IIS inhibitor, mediates a long-lasting, highly efficient inhibition of this signaling cascade by a dual mechanism; it reduces the autophosphorylation of the IGF1 receptor and directs the insulin receptor substrates 1 and 2 (IRS 1/2) for degradation. NT219 treatment promotes stress resistance and protects nematodes from AD- and HD-associated proteotoxicity without affecting lifespan. Our discoveries strengthen the theme that IIS inhibition has a therapeutic potential as a cure for neurodegenerative maladies and point at NT219 as a promising compound for the treatment of these disorders through a selective manipulation of aging.
    Aging cell 11/2013; 13(1). DOI:10.1111/acel.12171 · 6.34 Impact Factor
Show more