Membrane-associated cytoplasmic granules carrying the Argonaute protein WAGO-3 enable paternal epigenetic inheritance in Caenorhabditis elegans.

Epigenetic inheritance describes the transmission of gene regulatory information across generations without altering DNA sequences, enabling offspring to adapt to environmental conditions. Small RNAs have been implicated in this, through both the oocyte and the sperm. However, as much of the cellular content is extruded during spermatogenesis, it is unclear whether cytoplasmic small RNAs can contribute to epigenetic inheritance through sperm. Here we identify a sperm-specific germ granule, termed the paternal epigenetic inheritance (PEI) granule, that mediates paternal epigenetic inheritance by retaining the cytoplasmic Argonaute protein WAGO-3 during spermatogenesis in Caenorhabditis elegans. We identify the PEI granule proteins PEI-1 and PEI-2, which have distinct functions in this process: granule formation, Argonaute selectivity and subcellular localization. We show that PEI granule segregation is coupled to the transport of sperm-specific secretory vesicles through PEI-2 in an S-palmitoylation-dependent manner. PEI-like proteins are found in humans, suggesting that the identified mechanism may be conserved.

Arginine methylation promotes siRNA-binding specificity for a spermatogenesis-specific isoform of the Argonaute protein CSR-1.

CSR-1 is an essential Argonaute protein that binds to a subclass of 22G-RNAs targeting most germline-expressed genes. Here we show that the two isoforms of CSR-1 have distinct expression patterns; CSR-1B is ubiquitously expressed throughout the germline and during all stages of development while CSR-1A expression is restricted to germ cells undergoing spermatogenesis. Furthermore, CSR-1A associates preferentially with 22G-RNAs mapping to spermatogenesis-specific genes whereas CSR-1B-bound small RNAs map predominantly to oogenesis-specific genes. Interestingly, the exon unique to CSR-1A contains multiple dimethylarginine modifications, which are necessary for the preferential binding of CSR-1A to spermatogenesis-specific 22G-RNAs. Thus, we have discovered a regulatory mechanism for C. elegans Argonaute proteins that allows for specificity of small RNA binding between similar Argonaute proteins with overlapping temporal and spatial localization.

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.

henn-1/HEN1 Promotes Germline Immortality in Caenorhabditis elegans.

The germline contains an immortal cell lineage that ensures the faithful transmission of genetic and, in some instances, epigenetic information from one generation to the next. Here, we show that in Caenorhabditis elegans, the small RNA 3'-2'-O-methyltransferase henn-1/HEN1 is required for sustained fertility across generations. In the absence of henn-1, animals become progressively less fertile, becoming sterile after ∼30 generations at 25°C. Sterility in henn-1 mutants is accompanied by severe defects in germline proliferation and maintenance. The requirement for henn-1 in transgenerational fertility is likely due to its role in methylating and, thereby, stabilizing Piwi-interacting RNAs (piRNAs). However, despite being essential for piRNA stability in embryos, henn-1 is not required for piRNA stability in adults. Thus, we propose that methylation is important for the role of piRNAs in establishing proper gene silencing during early stages of development but is dispensable for their role in the proliferated germline.