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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Transition Award (R00)
Project #
5R00HD057233-04
Application #
8230632
Study Section
Special Emphasis Panel (NSS)
Program Officer
Taymans, Susan
Project Start
2010-03-15
Project End
2014-02-28
Budget Start
2012-03-01
Budget End
2014-02-28
Support Year
4
Fiscal Year
2012
Total Cost
$242,811
Indirect Cost
$75,466
Name
California Institute of Technology
Department
None
Type
Schools of Arts and Sciences
DUNS #
009584210
City
Pasadena
State
CA
Country
United States
Zip Code
91125
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
Stuwe, Evelyn; Tóth, Katalin Fejes; Aravin, Alexei A (2014) Small but sturdy: small RNAs in cellular memory and epigenetics. Genes Dev 28:423-31
Hur, Junho K; Olovnikov, Ivan; Aravin, Alexei A (2014) Prokaryotic Argonautes defend genomes against invasive DNA. Trends Biochem Sci 39:257-9
Olovnikov, Ivan; Le Thomas, Adrien; Aravin, Alexei A (2014) A framework for piRNA cluster manipulation. Methods Mol Biol 1093:47-58
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
Olovnikov, Ivan; Aravin, Alexei A; Fejes Toth, Katalin (2012) Small RNA in the nucleus: the RNA-chromatin ping-pong. Curr Opin Genet Dev 22:164-71
Muerdter, Felix; Olovnikov, Ivan; Molaro, Antoine et al. (2012) Production of artificial piRNAs in flies and mice. RNA 18:42-52