Deciphering the role of RG motifs in piRNA-mediated silencing and transgenerational fertility
openNICHD - Eunice Kennedy Shriver National Institute of Child Health and Human Development
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.
Up to $348K
health research