Mutator Foci Are Regulated by Developmental Stage, RNA, and the Germline Cell Cycle in Caenorhabditis elegans.

RNA interference is a crucial gene regulatory mechanism in Caenorhabditis elegans Phase-separated perinuclear germline compartments called Mutator foci are a key element of RNAi, ensuring robust gene silencing and transgenerational epigenetic inheritance. Despite their importance, Mutator foci regulation is not well understood, and observations of Mutator foci have been largely limited to adult hermaphrodite germlines. Here we reveal that punctate Mutator foci arise in the progenitor germ cells of early embryos and persist throughout all larval stages. They are additionally present throughout the male germline and in the cytoplasm of post-meiotic spermatids, suggestive of a role in paternal epigenetic inheritance. In the adult germline, transcriptional inhibition results in a pachytene-specific loss of Mutator foci, indicating that Mutator foci are partially reliant on RNA for their stability. Finally, we demonstrate that Mutator foci intensity is modulated by the stage of the germline cell cycle and specifically, that Mutator foci are brightest and most robust in the mitotic cells, transition zone, and late pachytene of adult germlines. Thus, our data defines several new factors that modulate Mutator foci morphology which may ultimately have implications for efficacy of RNAi in certain cell stages or environments.

A tudor domain protein, SIMR-1, promotes siRNA production at piRNA-targeted mRNAs in C. elegans.

piRNAs play a critical role in the regulation of transposons and other germline genes. In Caenorhabditis elegans, regulation of piRNA target genes is mediated by the mutator complex, which synthesizes high levels of siRNAs through the activity of an RNA-dependent RNA polymerase. However, the steps between mRNA recognition by the piRNA pathway and siRNA amplification by the mutator complex are unknown. Here, we identify the Tudor domain protein, SIMR-1, as acting downstream of piRNA production and upstream of mutator complex-dependent siRNA biogenesis. Interestingly, SIMR-1 also localizes to distinct subcellular foci adjacent to P granules and Mutator foci, two phase-separated condensates that are the sites of piRNA-dependent mRNA recognition and mutator complex-dependent siRNA amplification, respectively. Thus, our data suggests a role for multiple perinuclear condensates in organizing the piRNA pathway and promoting mRNA regulation by the mutator complex.

In the biological world, a process known as RNA interference helps cells to switch genes on and off and to defend themselves against harmful genetic material. This mechanism works by deactivating RNA sequences, the molecular templates cells can use to create proteins. Overall, RNA interference relies on the cell creating small RNA molecules that can target and inhibit the harmful RNA sequences that need to be silenced. More precisely, in round worms such as Caenorhabditis elegans, RNA interference happens in two steps. First, primary small RNAs identify the target sequences, which are then combatted by newly synthetised, secondary small RNAs. A number of proteins are also involved in both steps of the process. RNA interference is particularly important to preserve fertility, guarding sex cells against 'rogue' segments of genetic information that could be passed on to the next generation. In future sex cells, the proteins involved in RNA interference cluster together, forming a structure called a germ granule. Yet, little is known about the roles and identity of these proteins. To fill this knowledge gap, Manage et al. focused on the second stage of the RNA interference pathway in the germ granules of C. elegans, examining the molecules that physically interact with a key protein. This work revealed a new protein called SIMR-1. Looking into the role of SIMR-1 showed that the protein is required to amplify secondary small RNAs, but not to identify target sequences. However, it only promotes the creation of secondary small RNAs if a specific subtype of primary small RNAs have recognized the target RNAs for silencing. Further experiments also showed that within the germ granule, SIMR-1 is present in a separate substructure different from any compartment previously identified. This suggests that each substep of the RNA interference process takes place at a different location in the granule. In both C. elegans and humans, disruptions in the RNA interference pathway can lead to conditions such as cancer or infertility. Dissecting the roles of the proteins involved in this process in roundworms may help to better grasp how this process unfolds in mammals, and how it could be corrected in the case of disease.

Visualization and Quantification of Transposon Activity in Caenorhabditis elegans RNAi Pathway Mutants.

RNA silencing pathways play critical roles in maintaining quiescence of transposons in germ cells to promote genome integrity. However the precise mechanism by which different types of transposons are recognized by these pathways is not fully understood. Furthermore, the location in the germline where this transposition occurs after disruption of transposon silencing was previously unknown. Here we utilize the spatial and temporal organization of the Caenorhabditis elegans germline to demonstrate that transposition of DNA transposons in RNA silencing pathway mutants occur in all stages of adult germ cells. We further demonstrate that the double-strand breaks generated by transposons can restore homologous recombination in a mutant defective for the generation of meiosis-specific double-strand breaks. Finally, we detected clear differences in transposase expression and transposon excision between distinct branches of the RNA silencing pathway, emphasizing that there are multiple mechanisms by which transposons can be recognized and routed for small-RNA-mediated silencing.