Organisms rely on a stable genome in their germ cells to successfully establish cellularly competent offspring. However, the mobilization of many transposons and retroviruses called retrotransposons can interrupt important or essential genes in the host’s genome, rendering the effected germ cells mutagenized. Such attacks are so common that retrotransposon-derived sequences make up nearly 40% of most mammalian genomes. To combat the possibly deleterious effects of these mobile elements, metazoans have evolved a robust genome defense system housed in their germ cells. This defense system is composed of thousands of endogenously encoded small non-coding RNAs called piRNAs. A PIWI Argonaute protein traffics piRNAs to mRNA targets that share sequence complementarity and helps regulate their expression. In the first chapter of this dissertation, I review the current literature on small non-coding RNA pathways in general, and how those pathways function in the C. elegans germline in particular. I then discuss the connection between germline small RNA pathways and phase separated condensates enriched in animal germ cells called germ granules. Finally, I outline what is currently known about how particular mRNAs become targeted and silenced by small RNAs. In the second chapter, I demonstrate that a reporter assay can successfully uncover novel components involved in piRNA mediated gene silencing. I discuss where in the pathway each factor likely functions, and further characterize how each factor likely plays a role in gene silencing. This chapter demonstrates that highly conserved and ancient cellular machinery that is essential for snRNA biogenesis, pre-mRNA splicing, nuclear protein import, and nuclear pore formation has evolved to also contribute to small RNA mediated germline defense. In the third chapter, I discuss the role that the hallmark of animal germ cells, the germ granule, plays in the piRNA pathway. I show that, surprisingly, these highly conserved, phase separated structures are largely dispensable for small RNA-mediated gene silencing. However, without germ granules, competing small RNA pathways lose robustness, allowing a subset of typically silenced genes to become expressed and a subset of typically expressed genes to become silenced. This mis-regulation likely contributes to the deleterious fertility phenotypes associated with germ granule loss described here and in previous work. In the fourth chapter, I characterize signals that determine whether particular germline transcripts will become targeted and silenced by the piRNA pathway. The coding region of germline mRNAs rather than the untranslated regions are uniquely vulnerable to piRNA targeting. This finding suggests that the piRNA pathway has evolved to target regions of mRNAs which are less likely to escape detection due to genetic drift. In the fifth chapter, I present a collaborative effort to make the suite of tools used to perform the complex analyses discussed in this dissertation available to the scientific community. Many analyses involved in parsing next generation sequencing data require advanced domain knowledge to replicate. We have made these tools available in a convenient graphical format for the community to use without specific knowledge of the underlying code required to use them. I hope that the availability of such tools will become a standard in academia to allow for greater transparency and to open barriers closed to many biologists who do not have the bioinformatic expertise necessary to analyze the data already available to the community. In the final chapter, I discuss several important implications of this work for the field of small RNA biology. I address how the findings presented here influence our understanding of piRNA-mediated transcriptome regulation, and I suggest future work that could further clarify our understanding of how self and non-self is determined for germline mRNAs.