[
International Worm Meeting,
2019]
RNA silencing is a critical process that can be initiated by many exogenous and endogenous sources of RNA such as viral RNA, transposons and aberrant RNA transcripts. In C. elegans, the initial response by primary siRNAs to silence these deleterious RNAs is insufficient to effectively silence and requires an amplification step that occurs at Mutator foci. The scaffolding protein MUT-16 nucleates these perinuclear, germline foci and is required for recruitment of additional known Mutator proteins involved in secondary siRNA production. To identify novel components of the Mutator foci, we performed an immunoprecipitation of MUT-16, followed by mass spectrometry. We identified all known components of the complex, some small RNA factors not previously known to interact with the Mutator complex, and several completely uncharacterized proteins. To determine whether any of the uncharacterized proteins are components of the small RNA pathway, we used CRISPR to tag each with a fluorescent reporter and, in parallel, generated mutants of each for high throughput small RNA sequencing. The most promising candidate, C06A5.6, localizes to perinuclear germline foci, much like MUT-16. These foci are partially overlapping with Mutator foci but do not depend on any components of the Mutator complex for formation. Furthermore, C06A5.6 mutants exhibit a substantial loss of some Mutator complex-dependent small RNAs, indicative of a role in this pathway. This previously uncharacterized protein contains a putative Tudor domain, which is known to bind the methylated arginine and lysine residues of their substrates and promote protein-protein interactions. Mutants for this protein exhibit a transgenerational sterility phenotype at 25 deg C associated with increased germline apoptosis and a loss of oocyte viability. We are currently assessing whether C06A5.6 plays a role in transgenerational RNAi inheritance and whether it has a dosage-dependent RNAi defective phenotype, the results of which may help determine where C06A5.6 best fits into the RNA silencing pathway. Additionally, we are continuing to investigate whether transgenerational sterility at 25 deg C is oocyte specific or if sperm viability contributes to loss of germline immortality. Understanding these novel components of the C. elegans silencing pathways will have implications in understanding their potential roles in infertility, genetic disease and cancer.