Nucleus-Independent Transgenerational RNAi inheritance in C. elegans

While most scholars believed it to be impossible, for over a century, research on C. elegans nematodes demonstrated that animals can transmit parental reactions transgenerationally. In worms, ancestral RNAi responses regulate gene expression for many generations, independently of changes to the DNA. Nevertheless, it remains unclear whether the primary agent that perpetuates heritable silencing is RNA or chromatin, and whether the information is communicated to the next generation inside or outside of the nucleus. Here we use the tractability of gene-specific dsRNA-induced heritable silencing to answer these questions. We first show using ATAC-seq that RNAi condenses the chromatin around the gene target. Surprisingly, we find that chromatin condensation in the progeny does not predict heritable gene silencing. Furthermore, we demonstrate that RNAi can be inherited independently of chromatin or any other nuclear factors, from mothers that are genetically engineered to only transmit their cytoplasm but not their nuclei to the next generation. Nucleus-independent RNA inheritance depends on cytoplasmic germ granule proteins, and can be potentiated by disturbing proper germ granules segregation. Utilizing RNA sequencing, chimeras, and sequence polymorphism between different C. elegans isolates, we identify and study the function of numerous endogenous small RNAs which are inherited in a nucleus-independent manner. Last, we find that, counterintuitively, transcription, and not transcriptional inhibition, is required for long-term silencing, as transgenerationally inherited small RNAs require freshly synthesized mRNA templates for amplification. Our results shed light on the confines and independence of RNA-inherited information.