Organisms have evolved a robust set of gene-silencing pathways that provide protection against invasive genetic elements. The Piwi-interacting, piRNA pathway acts as one such surveillance mechanism, silencing transposable elements, foreign transgene DNA, and many endogenous genes in the C. elegans germline. The Piwi Argonaute PRG-1 localizes within peri-nulcear nuage, where it functions along with several other Argonautes to mediate surveillance of essentially all germline mRNAs. The family of DEAD-box helicase proteins known as germline helicases (GLH proteins) includes four distinct homologues that are required for fertility and for the maintenance of the germline nuage (Spike et al., 2008). The GLH proteins share several highly conserved domains, including a helicase domain with the Drosophila VASA protein (Orsborn et al., 2007). While our lab has identified GLH-1 and GLH-4 as factors required for cold-sensitive RNAi and for the Piwi-mediated silencing pathway (Dai et al., 2017), little is known regarding the function and regulation of GLH-2 and GLH-3. To explore the role of GLH-3 in the piRNA pathway, we created
glh-3 null mutations using CRISPR and introduced them into a sensitive piRNA reporter strain (Seth et al., 2018). We found that three independent
glh-3 null alleles caused partial de-silencing of the reporter. A similar study is ongoing with
glh-2. Further experiments are planned to examine double and multiple mutant strains and to explore the effects of these mutations on small-RNA pathways. The transcriptome-wide surveillance that occurs within germline nuage involves millions of Argonaute guide complexes. We hope these studies will advance our understanding of how GLH-3, and the multitude of DEAD-box co-factors that reside and function within germline nuage, promote and facilitate Argonuate mRNA interactions on such a remarkable scale. Literature Cited Dai, SY, Shen EZ, Mello CC. (2017) International Worm Meeting. "The germline DEAD-box protein, GLH-1, interacts with PRG-1 and a JNK-related kinase, KGB-1, to promote epigenetic gene silencing, and to preserve an RNA interference response that is robust to low temperatures." Orsborn AM, Li W, McEwen TJ, Mizuno T, Kuzmin E, et al. (2007) GLH-1, the C. elegans P granule protein, is controlled by the JNK KGB-1 and by the COP9 subunit CSN-5. Development 134: 3383-3392. Seth M, Shirayama M, Tang W, et al. The Coding Regions of Germline mRNAs Confer Sensitivity to Argonaute Regulation in C. elegans. Cell Rep. 2018;22(9):2254-2264. Spike C, Meyer N, Racen E, Orsborn A, Kirchner J, Kuznicki K, Yee C, Bennett K, & Strome S (2008). Genetic analysis of the Caenorhabditis elegans GLH family of P-granule proteins. Genetics, 178, 1973-87.