Molecular machines for small RNA-guided transposon control in the germline – piRmachines
The germline of a wide variety of animals express a special class of small RNAs called piwi-interacting RNAs (piRNAs). Its presence in the murine germline was shown in early 2006, and since then, intense research activity is focused on understanding the role of these RNAs. Initial studies show that they fundamentally differ in their biogenesis and function from the well-known microRNAs (miRNAs) and small interfering RNAs (siRNAs). It is not clear how murine piRNAs are made and the factors involved have not been identified. One of the roles attributed to piRNAs is the maintenance of genome integrity by silencing of transposons and other repeat elements by promoting DNA methylation of these elements. We have set out to biochemically characterize Piwi-associated proteins as a starting point in understanding the mechanism by which piRNAs act in this pathway. In one such complex we identified a tudor domain containing protein called Tdrd1; a mouse mutant of tdrd1 shows loss of transposon silencing and concomitant absence of DNA methylation. This is the first such factor (apart from Piwi proteins) identified that directly links it to piRNA function. Another factor identified is an RNA helicase, the homolog of which is already known to participate in the miRNA pathway. Thus, these two findings validate our biochemical method as a very useful approach in studying the piRNA pathway. We have also generated mouse mutants that will yield insight into the in vivo roles of this small RNA pathway in the germline. As there is no cell culture system available for germline studies, we are generating a GFP-based reporter for studying piRNA function in zebrafish. This required us to set up a new fish lab in Grenoble and learn to handle fish. All these are described as preliminary data and some studies are submitted for publication. In summary, this proposal aims to use interdisciplinary approaches covering biochemistry, computational methods, and genetics (mouse and zebrafish) in achieving its goals.
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