A single-copy knockin translating ribosome immunoprecipitation toolkit for tissue-specific profiling of actively translated mRNAs in C. elegans.

Here, we introduce a single-copy knockin translating ribosome immunoprecipitation (SKI TRIP) toolkit, a collection of Caenorhabditis elegans strains engineered by CRISPR in which tissue-specific expression of FLAG-tagged ribosomal subunit protein RPL-22 is driven by cassettes present in single copy from defined sites in the genome. Through in-depth characterization of the effects of the FLAG tag in animals in which endogenous RPL-22 has been tagged, we show that it incorporates into actively translating ribosomes and efficiently and cleanly pulls down cell-type-specific transcripts. Importantly, the presence of the tag does not impact overall mRNA translation, create bias in transcript use, or cause changes to fitness of the animal. We propose SKI TRIP use for the study of tissue-specific differences in translation and for investigating processes that are acutely sensitive to changes in translation like development or aging.

NHR-8 and P-glycoproteins uncouple xenobiotic resistance from longevity in chemosensory C. elegans mutants.

Longevity is often associated with stress resistance, but whether they are causally linked is incompletely understood. Here we investigate chemosensory-defective Caenorhabditis elegans mutants that are long-lived and stress resistant. We find that mutants in the intraflagellar transport protein gene osm-3 were significantly protected from tunicamycin-induced ER stress. While osm-3 lifespan extension is dependent on the key longevity factor DAF-16/FOXO, tunicamycin resistance was not. osm-3 mutants are protected from bacterial pathogens, which is pmk-1 p38 MAP kinase dependent, while TM resistance was pmk-1 independent. Expression of P-glycoprotein (PGP) xenobiotic detoxification genes was elevated in osm-3 mutants and their knockdown or inhibition with verapamil suppressed tunicamycin resistance. The nuclear hormone receptor nhr-8 was necessary to regulate a subset of PGPs. We thus identify a cell-nonautonomous regulation of xenobiotic detoxification and show that separate pathways are engaged to mediate longevity, pathogen resistance, and xenobiotic detoxification in osm-3 mutants.

Mutagenesis screen uncovers lifespan extension through integrated stress response inhibition without reduced mRNA translation.

Protein homeostasis is modulated by stress response pathways and its deficiency is a hallmark of aging. The integrated stress response (ISR) is a conserved stress-signaling pathway that tunes mRNA translation via phosphorylation of the translation initiation factor eIF2. ISR activation and translation initiation are finely balanced by eIF2 kinases and by the eIF2 guanine nucleotide exchange factor eIF2B. However, the role of the ISR during aging remains poorly understood. Using a genomic mutagenesis screen for longevity in Caenorhabditis elegans, we define a role of eIF2 modulation in aging. By inhibiting the ISR, dominant mutations in eIF2B enhance protein homeostasis and increase lifespan. Consistently, full ISR inhibition using phosphorylation-defective eIF2α or pharmacological ISR inhibition prolong lifespan. Lifespan extension through impeding the ISR occurs without a reduction in overall protein synthesis. Instead, we observe changes in the translational efficiency of a subset of mRNAs, of which the putative kinase kin-35 is required for lifespan extension. Evidently, lifespan is limited by the ISR and its inhibition may provide an intervention in aging.