Validation of tissue-specific RNAi systems in C. elegans reveals a converging role for polyubiquitin UBQ-1/UBC in vitellogenin metabolism and lifespan

ABSTRACT Numerous studies in C. elegans have taken advantage of gene silencing RNAi libraries and tissue-specific RNAi systems to temporally and spatially understand gene function. Tissue-specific RNAi strains were created via tissue-specific functional expression of a key gene in the RNAi system in a presumably RNAi insensitive correspondingly mutated background. Here, we tested the level of RNAi insensitivity across different commonly used RNAi-defective mutants ( rde-1 and sid- 1) using lifespan analyses. Remarkably, while we found similarly important lifespan shortening in wild-type animals by silencing the polyubiquitin gene ubq-1 ( UBC ) at both 20°C and 25°C, we found wide and temperature-sensitive variations in RNAi sensitivity across different rde-1 and sid-1 strains from substantial sensitivity to complete insensitivity. This riveting finding warrants a re-evaluation of the tissue-specific interpretations in numerous tissue-specific RNAi studies in C. elegans . Using validated tissue-specific RNAi systems, we determined that proteasomal burden largely differs between the germline and the soma. Notably, gonadally accumulating vitellogenin proteins substantially contributed to the total polyubiquitinated proteins observed during aging. Interestingly, we found that UBQ-1 was essential to maintain the transcription of highly expressed genes, including vitellogenins, supporting a broad and key role for polyubiquitin beyond proteostasis. Altogether, we authenticated commonly used tissue-specific RNAi systems and uncovered a new approach to improve RNAi insensitivity in RNAi-defective strains. With validated tissue-specific RNAi strains, we spatially analyzed proteasomal function and age-related polyubiquitinated protein accumulation and revealed a converging regulatory link between UBQ-1, vitellogenin metabolism and lifespan in C. elegans .