The near-universal conservation of small RNAs (sRNAs) as a way of regulating gene expression and the demonstrated importance of this for germline maintenance underscores the need for understanding the mechanisms by which these agents control their mRNA targets. Prior work studying the localization of proteins required for this process in different developmental models has shown that many components of the sRNA machinery localize to perinuclear structures within the germline, known generally as nuage. In C. elegans, factors such as Argonaute proteins required for sRNA targeting and RNA-dependent RNA polymerases (RdRPs) needed for amplifying sRNA signals have been observed to be enriched within these perinuclear organelles. The significance of these structures and how they facilitate sRNA related processes, however, remains unknown and has become a topic of intense interest. While the cell biology of sRNA protein factors has been previously characterized, a well-documented study of how RNAs are localized when targeted by this process remains to be conducted. In an effort to address this gap in the field, we have for the first time visualized RNAs in the process of being knocked down by sRNAs in a developmental context. By initiating the sRNA-mediated knockdown of transcripts though RNAi interference (RNAi), I have found that upon RNAi treatment, targeted RNAs become specifically enriched within the nuage, which houses the very factors required for sRNA-mediated silencing, such as RdRPs. This exciting observation now opens the doors into studying the sRNA pathway through a previously uncharted cell biological lens. I hypothesize that recruitment of sRNA-targeted RNAs into nuage aids in RdRP-mediated amplification of sRNAs against the transcript. To study the basis for this newly characterized cell biological phenomenon, I will first explore the molecular players required for this sRNA-induced RNA recruitment as described in my first aim. Using known RNAi mutants, I will systematically assess each mutant's ability to accumulate RNAs within nuage on a cell biological level upon induction with RNAi. Furthermore, I will complement this analysis with an unbiased RNA-centric proteomics approach to identify factors that may be responsible for this RNAi-induced recruitment. In my second aim, I will assay the effects that concentrating RNAs within condensates could have on RdRP activity through an in vitro analysis of condensate residency on RdRP kinetics. Altogether, this proposal seeks to understand a newly identified aspect of sRNA targeting that could have profound implications in human health and fertility given the conservation of nuage and small RNA pathways in the human germline.
It is now increasingly understood that the conserved germline structures known as nuage help maintain germ cell homeostasis via sRNA-mediated gene regulation; the mechanism by which this occurs, however, remains unknown. With our new findings that sRNA-targeted RNAs are recruited into nuage, we now have a framework through which we can more closely interrogate how these structures are contributing to germ cell maintenance. Given the conserved presence of nuage across metazoan germlines, our findings regarding the mechanisms of nuage functioning are likely to be broadly relevant and could have potential implications in human health and fertility.
