Gene silencing by the RNA interference (RNAi) machinery is an evolutionary conserved process that is critical for control of genes expression in organisms from yeast to human. Targets of RNAi are recognized through complementary interactions with small RNAs that act as guides in the silencing process. Several discrete classes, encompassing thousands of small RNAs have been identified in recent years. For example, microRNAs interact with members of the ubiquitously expressed Argonaute protein family and together these regulate gene expression networks that impact diverse biological processes. Members ofthe other branch of the Argonaute family, the Piwi proteins, are expressed specifically in germ cells with mutant animals showing severe defects in gametogenesis that lead to sterility. Until recently, the small RNA partners of Piwi proteins were unknown, and the molecular functions of Piwis in gametogenesis remain so. We comprehensively characterized Piwi interacting RNA (piRNA) expression in germ cells of mouse and Drosophila. We have also identified the protein partners of Piwi proteins and additional components ofthe cytoplasmic granules that represent the main sites of piRNA pathway operation. In this proposal I present a focused strategy for analyzing piRNA biogenesis and function in the germline.
In aim 1, 1 will probe the mechanism of piRNA biogenesis in vivo.
In aim 2, 1 will determine the mechanism of piRNA-Piwi mediated silencing.

Public Health Relevance

These studies will produce fundamental insights into the functions of Piwi proteins and piRNAs in germline development, which can be applied for the discovery of both diagnostic markers and therapeutic targets of human infertility. Furthermore, if successful, the proposed research has the potential to expand the application of RNAi methods by providing tools to manipulate the epigenetic structure of specific loci in mammalian germ cells.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Research Transition Award (R00)
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Special Emphasis Panel (NSS)
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Taymans, Susan
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California Institute of Technology
Schools of Arts and Sciences
United States
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Liu, Yiwei; Esyunina, Daria; Olovnikov, Ivan et al. (2018) Accommodation of Helical Imperfections in Rhodobacter sphaeroides Argonaute Ternary Complexes with Guide RNA and Target DNA. Cell Rep 24:453-462
Manakov, Sergei A; Pezic, Dubravka; Marinov, Georgi K et al. (2015) MIWI2 and MILI Have Differential Effects on piRNA Biogenesis and DNA Methylation. Cell Rep 12:1234-43
Le Thomas, Adrien; Stuwe, Evelyn; Li, Sisi et al. (2014) Transgenerationally inherited piRNAs trigger piRNA biogenesis by changing the chromatin of piRNA clusters and inducing precursor processing. Genes Dev 28:1667-80
Olovnikov, Ivan; Le Thomas, Adrien; Aravin, Alexei A (2014) A framework for piRNA cluster manipulation. Methods Mol Biol 1093:47-58
Hur, Junho K; Olovnikov, Ivan; Aravin, Alexei A (2014) Prokaryotic Argonautes defend genomes against invasive DNA. Trends Biochem Sci 39:257-9
Le Thomas, Adrien; Tóth, Katalin Fejes; Aravin, Alexei A (2014) To be or not to be a piRNA: genomic origin and processing of piRNAs. Genome Biol 15:204
Pezic, Dubravka; Manakov, Sergei A; Sachidanandam, Ravi et al. (2014) piRNA pathway targets active LINE1 elements to establish the repressive H3K9me3 mark in germ cells. Genes Dev 28:1410-28
Stuwe, Evelyn; Tóth, Katalin Fejes; Aravin, Alexei A (2014) Small but sturdy: small RNAs in cellular memory and epigenetics. Genes Dev 28:423-31
Olovnikov, Ivan; Chan, Ken; Sachidanandam, Ravi et al. (2013) Bacterial argonaute samples the transcriptome to identify foreign DNA. Mol Cell 51:594-605
Le Thomas, Adrien; Rogers, Alicia K; Webster, Alexandre et al. (2013) Piwi induces piRNA-guided transcriptional silencing and establishment of a repressive chromatin state. Genes Dev 27:390-9

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