Witmer, M. | bioRxiv Science

bioRxiv2026

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 C. elegans and plants, our molecular understanding of systemic RNAi remains very limited. We here investigated the highly-effective systemic RNAi of the planarian S. mediterranea, 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 C. elegans, 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.

molecular biology↗

bioRxiv2026

Two tiers of piRNA clusters balance diversification of piRNAs with limitation of off-target effects

PIWI-interacting RNAs (piRNAs) are important factors in the protection of the genome against nucleic acid invaders such as transposons. piRNAs are encoded in the genome in regions known as piRNA clusters, but major questions remain surrounding their regulation. Two such questions are how the piRNA clusters are recognized as piRNA sources, and how the piRNA response can expand from these regions to facilitate the recognition of novel invading sequences without risking the targeting of essential cellular mRNAs. Here, we investigated the piRNA clusters of the planarian S, mediterranea, and found that the clusters differ regarding the PIWI proteins their piRNAs bind to, as well as their chromatin features. We uncovered a subset of piRNA clusters that had unique chromatin features and atypical transcription. piRNAs from these clusters were depleted of genic content, stabilized by 3 methylation and were not dependent on the ping-pong mechanism. We therefore named these clusters Seed clusters. We identified a second set of piRNA clusters that we called Spread clusters, which had features reminiscent of pseudogenes or aberrant genic transcripts. piRNA generation from these clusters relied on ping-pong, and piRNAs largely remained unmethylated. Further, we found that many more regions in the genome generated single ping-pong events, suggesting that ping-pong is used as a means to diversify the collection of piRNA-generating transcripts beyond the Seed clusters. We propose that this two-tiered organization of the piRNA clusters allows the stable targeting of known genomic threats by the piRNAs generated from the Seed clusters, while the flexible generation of additional diverse piRNAs from Spread clusters as well as other aberrant transcripts increases the sequence space probed by the piRNA system. The absence of methylation on these additional piRNAs decreases their lifespan and limits the chances of a run-away response. This two-tiered organization thus solves a core challenge of the piRNA system and may be a widespread feature of piRNA systems across animals.

molecular biology↗