Piwi proteins and Piwi-interacting RNAs (piRNAs) are essential for animal fertility because they repress transposable elements (TE) and regulate germ cell-specific genes. Although vertebrate genomes encode hundreds of piRNA cluster loci, we know little about the impact and gene regulation roles of individual piRNA clusters. The reason is because Piwi pathway mutants disrupt all piRNAs and cause widespread germ cell death, thereby obscuring the dissection of functions for individual piRNA Cluster loci. Major knowledge gaps include: (1) Which genes besides TEs impact fertility and are being targeted by PIWI/piRNA complexes? (2) Will a single piRNA cluster mutation affect the production of piRNAs from other clusters? (3) How will the regulation of Piwi targets and vertebrate germ cell development be affected by mutating a single piRNA cluster? This exploratory project will address these questions by first generating a list of Xenopus Piwi- associated transcripts (PATs) and testing their mechanism of regulation in Xenopus oocyte extracts. We will then create an intergenic piRNA cluster locus mutant in Xenopus tropicalis, and examine which PATs are affected in this mutant so as to gain insight into the relationship between specific groups of piRNAs and target genes regulated by Piwi/piRNA complexes. The frog is an experimentally tractable vertebrate system for dissecting the role of individual piRNA clusters. These Xenopus studies will reveal how a single major intergenic piRNA cluster will affect gonadogenesis and embryonic development. The future characterizations of mutant phenotypes are considerations for a longer-term project after the initiation of this exploratory project. The focus of this exploratory project is to create a list of PATs, test for PAT regulation in oocyte extracts, and then create a valuable mutant that will enable precise examination of which PATs are mis-regulated. Given our foundation of expertise described in this proposal, we are confident in achieving the goals of this exploratory project.
In animal germ cells, Piwi-interacting RNAs (piRNAs) are essential for gametogenesis and may impact embryogenesis and child reproductive health. Despite hundreds of individual piRNA clusters encoded in our genomes, we do not know the role that individual piRNA clusters play in regulating gene expression during vertebrate germ cell development. To address this question, we will explore the regulation of Piwi-Associated Transcripts (PATs) in the model vertebrate Xenopus. We will then create a Xenopus mutant that disrupts a single piRNA cluster locus to examine which PATs are mis- regulated. This project will shed new light on the mechanisms for how piRNAs and PIWI proteins regulate germ cell and embryonic gene expression, and the PATs we identify will be useful markers for assessing the health of germ cells and embryos.
|Toombs, James A; Sytnikova, Yuliya A; Chirn, Gung-Wei et al. (2017) Xenopus Piwi proteins interact with a broad proportion of the oocyte transcriptome. RNA 23:504-520|