Deciphering the role of RG motifs in piRNA-mediated silencing and transgenerational fertility

About This Grant

PROJECT SUMMARY Piwi-interacting RNA (piRNA)-mediated silencing is crucial to modulate germline gene expression and promote fertility. In C. elegans, piRNAs are bound by the Piwi Argonaute, PRG-1, and interact with target RNAs through base pairing. The nuclear Argonaute protein HRDE-1 acts downstream of piRNAs to regulate target genes at the transcriptional level. Precise gene regulation by piRNAs and their downstream 22G-RNAs is critical to promote germ cell maintenance, fertility, and transgenerational inheritance. The requirement for piRNAs to maintain fertility is a conserved feature across animals, including nematodes, flies, zebrafish, and mice, and is thus of fundamental importance to the developmental and cellular biology of humans. The Argonaute proteins in the piRNA pathway, including PRG-1 and HRDE-1, are essential for piRNA-mediated silencing because they provide the link between the small RNA, which provides RNA-target specificity, and the proteins, that mediate RNA-target regulation. Argonaute proteins have several well-characterized domains (Piwi, Mid, Paz) but also have less well-characterized sequences, such as the arginine/glycine (RG) motif. RG motifs can mediate liquid-liquid phase separation, protein-protein interactions, RNA binding, and they can be post- translationally modified by arginine methylation. It is currently unknown what the function of the RG motifs are in the piRNA pathway Argonaute proteins, PRG-1 and HRDE-1. Therefore, our overall objective for this proposal is to decipher the function of the RG motif in the C. elegans piRNA pathway and to determine how this motif promotes transgenerational fertility. To support this study, we have generated preliminary data indicating that the RG motifs of both PRG-1 and HRDE-1 are methylated. Furthermore, mutating the RG motifs of PRG-1 and HRDE-1 results in fertility and small RNA-related defects similar to that of prg-1 and hrde-1 null alleles. Here we will investigate the function of Argonaute protein RG motifs in the piRNA using C. elegans, protein regions that we believe may have conserved functions in mammalian Argonaute proteins. First, we will determine the biological phenotypes associated with loss of Argonaute RG motifs in the piRNA pathway by comprehensively examining germline defects of RG-mutant piRNA pathway Argonaute proteins (Aim 1). Next, we will identify the molecular “writers” and “readers” for Argonaute protein methylation at RG motifs (Aim 2). Lastly, we will examine the molecular phenotypes, including Argonaute-small RNA interactions, mRNA expression, protein interactions, and chromatin modifications associated with disruption of PRG-1 and HRDE-1 RG motifs (Aim 3). Together, the proposed experiments will uncover the function of Argonaute protein RG motifs in the piRNA pathway. Mechanistic insights gleaned from these studies will shed light on the regulation of conserved pathways that are critical for germline development, germ cell maintenance, and fertility.