Final Report Summary - PISILENCE (Small RNA-guided complex machinery for epigenetic silencing) The ERC Starting Grant “pisilence” had the aim to shed light on how a newly-discovered class of small RNAs called Piwi-interacting RNAs (piRNAs) are produced and how they function in repressing transposable genetic elements in the animal germline. piRNAs are bound by Piwi proteins and we used this as a starting point in our investigations. We raised antibodies to all the mouse Piwi proteins, and used these reagents to immunoprecipitate endogenous Piwi complexes from mouse testes. Mass spectrometry identified a number of factors which were then put through a series of genetic and biochemical analyses. This led to the identification of a number of biogenesis factors (Tdrd12, Fkbp6, Exd1). Mice mutant for these factors affect piRNA biogenesis and lead to de-repression of endogenous transposons in the mouse genome. Such mutants display male-specific infertility due to arrested spermatogenesis, which is likely due to genome damage caused by uncontrolled transposition. Our investigations reveal that the lost piRNAs are those that normally bind a nuclear Piwi protein Miwi2. Consequently, unloaded Miwi2 fails to enter nucleus and is unable to participate in its transcriptional repression role, as transposon genomic loci show reduced DNA methylation. Using mice and flies expressing artificial piRNA precursors, we could demonstrate that endonuclease activity (slicer activity) of Piwi proteins on a target RNA is able to trigger the generation of a wave of piRNAs in a non-overlapping manner. Such piRNAs preferentially associate with the nuclear Miwi2. Finally, we could demonstrate that slicer activity of Piwi is also important for post-transcriptional silencing of transposons and mice lacking such activity are infertile. Taken together, this work reveals the biochemical framework and functional pathways that lead to production and function of germline small RNAs that are needed to ensure germline genome integrity and fertility in animals.
