Gene silencing by the RNA interference (RNAi) machinery is an evolutionarily conserved process that is critical for control of gene 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 of the 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. Recently we discovered the small RNA partners of Piwi proteins in mammals and comprehensively analyzed Piwi interacting RNA (piRNA) in Drosophila. In this proposal I present a focused strategy for analyzing piRNA biogenesis and function in the germline. First, I will characterize small RNAs associated with Piwi proteins during different stages of germ cell development. Second, I will probe the mechanism of piRNA biogenesis in vivo. Finally, I will determine the mechanism of piRNA-Piwi mediated silencing. These studies will produce fundamental insights into the functions of Piwi proteins and piRNAs in germline development, which can be applied for 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
Career Transition Award (K99)
Project #
1K99HD057233-01
Application #
7361947
Study Section
Pediatrics Subcommittee (CHHD)
Program Officer
Taymans, Susan
Project Start
2008-05-01
Project End
2010-04-30
Budget Start
2008-05-01
Budget End
2010-04-30
Support Year
1
Fiscal Year
2008
Total Cost
$97,200
Indirect Cost
Name
Cold Spring Harbor Laboratory
Department
Type
DUNS #
065968786
City
Cold Spring Harbor
State
NY
Country
United States
Zip Code
11724
Aravin, Alexei A; van der Heijden, Godfried W; CastaƱeda, Julio et al. (2009) Cytoplasmic compartmentalization of the fetal piRNA pathway in mice. PLoS Genet 5:e1000764
Vagin, Vasily V; Wohlschlegel, James; Qu, Jun et al. (2009) Proteomic analysis of murine Piwi proteins reveals a role for arginine methylation in specifying interaction with Tudor family members. Genes Dev 23:1749-62
Vagin, Vasily V; Hannon, Gregory J; Aravin, Alexei A (2009) Arginine methylation as a molecular signature of the Piwi small RNA pathway. Cell Cycle 8:4003-4
Aravin, Alexei A; Sachidanandam, Ravi; Bourc'his, Deborah et al. (2008) A piRNA pathway primed by individual transposons is linked to de novo DNA methylation in mice. Mol Cell 31:785-99
Aravin, A A; Hannon, G J (2008) Small RNA silencing pathways in germ and stem cells. Cold Spring Harb Symp Quant Biol 73:283-90