Systemic RNAi in planarians depends on spread of RNPs from active stem cells

RNAi is a powerful cellular defense mechanism against genomic invaders that rely on dsRNA intermediates, such as viruses and mobile elements. Due to its specificity and ease of use, RNAi is also widely used as an experimental and therapeutic strategy to reduce levels of specific RNAs. For many systems however, delivery of the silencing agents into each individual cell is a major challenge. A mechanistic understanding of the processes involved in intercellular spread in systems with effective systemic RNAi thus is key to improve applications, and to increase our understanding of this defense mechanism. Remarkably, outside of <i>C. elegans</i> and plants, our molecular understanding of systemic RNAi remains very limited. We here investigated the highly-effective systemic RNAi of the planarian <i>S. mediterranea</i> , which can be triggered by introduction of dsRNA via food or injection and rapidly spreads through the entire body. Notwithstanding its efficiency, we find no evidence of an RNA amplification mechanism or of transgenerational effects as are found in <i>C. elegans</i> , and rather find that planarian RNAi effects are limited in time. We identify the biogenesis factors involved in the planarian RNAi mechanism, and find that these are independent of the miRNA pathway, enabling the separation of the effects from these small RNA pathways. Surprisingly, we find that planarian systemic RNAi relies on active stem cells. Further, we identify Argonaute-siRNA complexes as the mobile agent that effectuates systemic spread of RNAi throughout the tissue. These findings provide new insights into the mechanisms by which small RNAs spread between cells, and by which organisms can extend protection to all their cells upon encounter of a novel invading element. Additionally, our findings may have important implications for the design of effective applications in other systems.