Sensor-mediated fine-tuning of siRNA levels is required for spermatogenic piRNA pathway function

Small RNA pathways provide a robust and dynamic regulatory network that enables spatiotemporal regulation of the germline genome in response to environmental cues. The flexibility of RNA interference (RNAi)-mediated gene regulation and network architecture of these pathways requires molecular mechanisms that can fine-tune their regulatory potential and function to ensure proper execution of physiological processes, such as fertility. In C. elegans, we previously discovered a set of small RNA sensors that modulate the production of one class of small RNAs to adjust amplification resources based on cellular needs. These sensors maintain homeostatic levels of 22G-RNAs for the distinct RNAi branches that compete for resources in the mutator complex. Here we show this molecular feedback is essential for restricting expression of spermatogenic transcripts to an appropriate threshold during development and preventing spermiogenesis defects. Furthermore, we demonstrate 22G-RNA homeostasis is critical for proper meiotic progression in the germline and piRNA pathway function within pachytene germ cells. Together, our work reveals that RNAi homeostasis is critical for developmental and physiological processes, such as sperm-based fertility. Further, our findings show that small RNA pathway function is more than the sum of its parts and disrupting the ability to maintain homeostasis within the regulatory pathway itself leads to deleterious physiological consequences.