plasTiSiPi

Transposable Elements (TEs) account for 20% of the Drosophila genome (50% in human). Their mobilization in germ cells has dramatic consequences since it induces mutations and chromosomal rearrangements transmitted to the next generation. The activity of TE is thus strongly silenced in these cells by piRNAs, a class of 23-28nt long PIWI-associated small RNAs. However, to create genomic diversity the germ cell DNA information transmitted to the next generation must also be partially reshuffled. It was suggested that a supervised activity of TEs which could be achieved through a partial release of TE repression during oogenesis, might help generating this diversity. We have found that repression of P-element and Idefix TEs is strongly weakened at the most anterior part of the ovariole, in a structure called germarium that contains the dividing cysts. In addition, we found that Piwi is down-regulated in this part of the germarium accordingly called the “Piwiless pocket” (pilp). In contrast, in later oogenesis, a clear random on/off bimodal distribution of repression is observed. This variegation suggests that no transition between on and off states occurs after germarium stages. Thus, TE repression can show both plasticity in early oogenesis and cellular memory in late oogenesis. Our 1st objective is to study how modulation of the piRNA silencing pathway may regulate TE repression along germ cell differentiation, allowing an appropriate balance between genome protection and genome plasticity. To this aim, we will perform 1- in situ analyses of piRNA pathway gene expression as well as of TE expression in the pilp, 2- Micro-dissection of pilp and RNA deep-sequencing analysis, 3- Ectopic expression of Piwi in the pilp and analysis of its impact on oogenesis, gene expression and TE activity (including telomeric TE), 4- Investigation of egg chamber silencing stability using GFP-based sensors. 5- Transplantation of single germarium GSC and live monitoring of silencing capacity in later oogenesis. The biogenesis of piRNAs is still poorly understood. In a screen for novel genes involved in germ cells differentiation, we isolated a mutation of the rpp30 gene that disrupts piRNAs biogenesis. The Rpp30 protein, is a subunit of the RNase P complexe, involved in the endonucleolytic maturation of pre-tRNAs. Our 2nd objective is to understand the role of Rpp30 in piRNA biogenesis. We will test 2 hypotheses. First, RNase P could be directly required to cleave long piRNA precursors. Secondly, tRNA fragments (tRFs) generated by Rpp30 could directly silence TEs, or participate in piRNA amplification. tRFs are a novel class of small RNAs conserved in vertebrates, but their function is unknown. Analyses will involve 1- characterization of Rpp30 sub-cellular localization and of its expression during oogenesis, 2- analysis of proteins and of cellular RNAs associated to Rpp30 by mass spectrometry and deep-sequencing, 3- profiling of tRFs in WT and piRNA mutants, 4- investigation of tRFs roles in TE repression (injection of antisense tRFs in embryos pole cells). The third objective is transverse to Objectives 1 and 2. It is aimed at 1- managing, 2- mining all the sequencing datasets generated by the project to profile piRNAs, tRFs and mRNAs, 3- providing new visualization tools, 4- developing a new algorithm to analyze the piRNA signatures at genome-wide level. Thus, this project will contribute to better understand the plasticity of piRNA-mediated TE repression during oogenesis and to decipher the functional interactions between piRNAs and the new class of tRFs. In addition it provides the opportunity to develop new bioinformatic tools for small RNA deep sequencing analyses and to as well as a pilot structuration for collaborative projects that require the mining of large biological datasets.