Piwi proteins and Piwi-interacting RNAs (piRNAs) are essential for animal fertility because theyrepress transposable elements (TE) and regulate germ cell-specific genes. Although vertebrate genomesencode hundreds of piRNA cluster loci, we know little about the impact and gene regulation roles ofindividual piRNA clusters. The reason is because Piwi pathway mutants disrupt all piRNAs and causewidespread germ cell death, thereby obscuring the dissection of functions for individual piRNA Clusterloci. Major knowledge gaps include: (1) Which genes besides TEs impact fertility and are being targetedby PIWI/piRNA complexes? (2) Will a single piRNA cluster mutation affect the production of piRNAs fromother clusters? (3) How will the regulation of Piwi targets and vertebrate germ cell development beaffected 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. Wewill then create an intergenic piRNA cluster locus mutant in Xenopus tropicalis, and examine which PATsare affected in this mutant so as to gain insight into the relationship between specific groups of piRNAsand target genes regulated by Piwi/piRNA complexes. The frog is an experimentally tractable vertebratesystem for dissecting the role of individual piRNA clusters. These Xenopus studies will reveal how asingle major intergenic piRNA cluster will affect gonadogenesis and embryonic development. The futurecharacterizations of mutant phenotypes are considerations for a longer-term project after the initiation ofthis exploratory project. The focus of this exploratory project is to create a list of PATs, test for PATregulation in oocyte extracts, and then create a valuable mutant that will enable precise examination ofwhich PATs are mis-regulated. Given our foundation of expertise described in this proposal, we areconfident in achieving the goals of this exploratory project.
In animal germ cells; Piwi-interacting RNAs (piRNAs) are essential for gametogenesisand may impact embryogenesis and child reproductive health. Despite hundreds ofindividual piRNA clusters encoded in our genomes; we do not know the role thatindividual piRNA clusters play in regulating gene expression during vertebrate germ celldevelopment. To address this question; we will explore the regulation of Piwi-AssociatedTranscripts (PATs) in the model vertebrate Xenopus. We will then create a Xenopusmutant 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 PIWIproteins regulate germ cell and embryonic gene expression; and the PATs we identifywill 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|