Final Report Summary - DROSOPHILA PIRNAS (Molecular mechanisms and biological role of the somatic piRNA / Piwi pathway - an RNA-based genome immune system)
Every genome sequenced to date including the human one contains major portions of selfish genetic elements, typically called transposable elements (TEs). These TEs multiply their numbers in the genome through mobilisation to novel locations in the host genome. This poses a massive threat to the integrity of the host genome, as novel TE insertions cause mutations, genomic instability and ultimately sterility, due to their preferential activity during gonad development. The Piwi-interacting ribonucleic acid (piRNA) pathway is a conserved, small RNA-based mechanism that suppresses expression of TEs during animal gonad development. piRNAs, the small RNAs complexed by Piwi proteins, serve as sequence specific guides determining the piRNA pathway targets via base pairing. Our conceptual understanding of the piRNA pathway derives mainly from the analysis of deep-sequenced piRNAs. In contrast to piRNAs, however, we understand very little about the long precursor transcripts that serve as source for primary piRNA biogenesis. They originate from the transcription of large graveyards of old TE insertions, called piRNA clusters. Likewise we know little about the global functional relationship between sequence specific Piwi / piRNA complexes and their cognate TE targets. For example, for many sense TE transcripts there are plenty of antisense piRNAs, yet we still do not know if these TE transcripts are actually silenced by piRNA pathway.
I proposed to study the piRNA pathway in the fruitfly Drosophila melanogaster to gain insights into its mechanistic architecture of the piRNA pathway (aim 1) and analyse its biological function by systematically profiling the RNA expression in the Drosophila ovaries under wild type and piRNA pathway mutant conditions (aim 2).
In aim 1, I proposed to perform a genome wide RNA interference (RNAi) screen to identify novel components of the piRNA pathway active in the somatic follicle cells of fly ovaries. While I contributed intellectually and by supervision of a diploma student to its set up, the screen itself was performed in the recent two years mainly by technicians under the supervision of a Doctor of Philosophy (PhD) student in the host lab. This screen now completed and provides a rich resource to study mechanistic questions using the tools I established in aim 2.
In aim 2, I outlined multiple approaches to profile the biological effects of the piRNA pathway in Drosophila ovaries using deep sequencing technology. The main incentive of this approach was to systematically profile the precursors transcripts of piRNAs and the targets of the piRNA pathway at multiple functional levels. The experimental paradigm is to compare wild type controls and specific piRNA pathway mutants to extract the relevant changes.
While my proposal was focused on the analysis of the somatic part of the ovary, I extended my systematic approaches not only to the germline of the ovary but also to the next generation by analysing the early embryo samples. Firstly, these experimental extensions allow me to contrast the situation in somatic cells with the one in the germline. Secondly, it enables me to delineate the protection of the piRNA pathway to earliest time point of the next generation.
The focus of aim 2 was to develop tools and to profile of RNA at multiple stages of its metabolism, e.g. (a) transcription in the nucleus, (b) steady state cellular RNA levels, and finally (c) its translation. While I initially established the experimental tool kit to study transcription and translation in fly ovaries, it soon became clear that steady state RNA levels are an important prerequisite to understand any regulation at the transcriptional or translational levels. This made this subproject the primary milestone to reach in aim 2. As different modular forms of the piRNA pathway are active in the somatic follicle cell and the germline, I depleted three different piRNA pathway components in the soma and the germline each by tissue specific RNAi and generated RNA sequencing (RNAseq) libraries from RNA isolated from total ovaries or early embryos.
The genome wide analysis of those RNAseq profiles allowed a number of interesting findings. First, three distinct piRNA pathway components (one novel component in the soma and two known components in the germline) did impact abundance of piRNA cluster transcript levels in the respective tissues. This clearly highlights the underlying mechanisms for piRNA cluster transcription and transcript turnover. Secondly, the tissue specific piRNA pathway depletions in the germline upregulated 51 TEs in the ovary and 39 of them gain specific access to early embryos indicating that they specifically were transported and accumulated in the future oocyte. Thirdly, piRNA pathway depletion in the soma led to very coherent expression of roughly 20 gypsy type retro-transposons in the somatic tissue. At least four of these retro-elements specifically gained access to the early embryo showing that they traffic from the somatic cells to the oocyte. All these four elements encode an envelope protein (like those of retroviruses) and likely infect the oocyte as retroviral particles. In summary, in the germline many types of TEs are deregulated and contributed to the next generation, however in the soma only a small proportion of LTR-retrotransposons are de-silenced and few of them gain access to the next generation. On the one hand this shows the different TE mobility strategies and the other hand it underscores the tissue-specific adaption of the piRNA pathway to contain the elements in the two different tissues in order to prevent TEs to access the genome of the next generation. Furthermore this dataset allows the design of directed experimentation to study specific mechanistic such as the soma-to-germline trafficking of LTR-elements.
To what degree the TEs damage the host genome when freely mobilising without piRNA pathway control? Using quantitative polymerase chain reaction (qPCR) on genomic deoxyribonucleic acid (DNA) isolated from mutant ovaries and early embryos, I find increased TE copy number in mutant samples. This suggests that global DNA sequencing (DNAseq) may be a suitable and direct approach to study TE transposition.
Lastly, I developed advanced systematic tools that pinpoint distinct features of the life of an RNA. The first tissue specific method uses fluorescence-activated cell sorting (FACS)-based nuclear purification coupled to RNAseq. Furthermore, I developed two approaches that target either transcription or translation utilising tissue-specific RNA polymerase II chromatin-IP-seq (ChIPseq) or Ribosome profiling.
The combination of the completed RNAi screen and the developed tools now serve as an extremely competitive basis to mechanistically dissect selected novel piRNA pathway components and profile their effects in a systematic manner.
I proposed to study the piRNA pathway in the fruitfly Drosophila melanogaster to gain insights into its mechanistic architecture of the piRNA pathway (aim 1) and analyse its biological function by systematically profiling the RNA expression in the Drosophila ovaries under wild type and piRNA pathway mutant conditions (aim 2).
In aim 1, I proposed to perform a genome wide RNA interference (RNAi) screen to identify novel components of the piRNA pathway active in the somatic follicle cells of fly ovaries. While I contributed intellectually and by supervision of a diploma student to its set up, the screen itself was performed in the recent two years mainly by technicians under the supervision of a Doctor of Philosophy (PhD) student in the host lab. This screen now completed and provides a rich resource to study mechanistic questions using the tools I established in aim 2.
In aim 2, I outlined multiple approaches to profile the biological effects of the piRNA pathway in Drosophila ovaries using deep sequencing technology. The main incentive of this approach was to systematically profile the precursors transcripts of piRNAs and the targets of the piRNA pathway at multiple functional levels. The experimental paradigm is to compare wild type controls and specific piRNA pathway mutants to extract the relevant changes.
While my proposal was focused on the analysis of the somatic part of the ovary, I extended my systematic approaches not only to the germline of the ovary but also to the next generation by analysing the early embryo samples. Firstly, these experimental extensions allow me to contrast the situation in somatic cells with the one in the germline. Secondly, it enables me to delineate the protection of the piRNA pathway to earliest time point of the next generation.
The focus of aim 2 was to develop tools and to profile of RNA at multiple stages of its metabolism, e.g. (a) transcription in the nucleus, (b) steady state cellular RNA levels, and finally (c) its translation. While I initially established the experimental tool kit to study transcription and translation in fly ovaries, it soon became clear that steady state RNA levels are an important prerequisite to understand any regulation at the transcriptional or translational levels. This made this subproject the primary milestone to reach in aim 2. As different modular forms of the piRNA pathway are active in the somatic follicle cell and the germline, I depleted three different piRNA pathway components in the soma and the germline each by tissue specific RNAi and generated RNA sequencing (RNAseq) libraries from RNA isolated from total ovaries or early embryos.
The genome wide analysis of those RNAseq profiles allowed a number of interesting findings. First, three distinct piRNA pathway components (one novel component in the soma and two known components in the germline) did impact abundance of piRNA cluster transcript levels in the respective tissues. This clearly highlights the underlying mechanisms for piRNA cluster transcription and transcript turnover. Secondly, the tissue specific piRNA pathway depletions in the germline upregulated 51 TEs in the ovary and 39 of them gain specific access to early embryos indicating that they specifically were transported and accumulated in the future oocyte. Thirdly, piRNA pathway depletion in the soma led to very coherent expression of roughly 20 gypsy type retro-transposons in the somatic tissue. At least four of these retro-elements specifically gained access to the early embryo showing that they traffic from the somatic cells to the oocyte. All these four elements encode an envelope protein (like those of retroviruses) and likely infect the oocyte as retroviral particles. In summary, in the germline many types of TEs are deregulated and contributed to the next generation, however in the soma only a small proportion of LTR-retrotransposons are de-silenced and few of them gain access to the next generation. On the one hand this shows the different TE mobility strategies and the other hand it underscores the tissue-specific adaption of the piRNA pathway to contain the elements in the two different tissues in order to prevent TEs to access the genome of the next generation. Furthermore this dataset allows the design of directed experimentation to study specific mechanistic such as the soma-to-germline trafficking of LTR-elements.
To what degree the TEs damage the host genome when freely mobilising without piRNA pathway control? Using quantitative polymerase chain reaction (qPCR) on genomic deoxyribonucleic acid (DNA) isolated from mutant ovaries and early embryos, I find increased TE copy number in mutant samples. This suggests that global DNA sequencing (DNAseq) may be a suitable and direct approach to study TE transposition.
Lastly, I developed advanced systematic tools that pinpoint distinct features of the life of an RNA. The first tissue specific method uses fluorescence-activated cell sorting (FACS)-based nuclear purification coupled to RNAseq. Furthermore, I developed two approaches that target either transcription or translation utilising tissue-specific RNA polymerase II chromatin-IP-seq (ChIPseq) or Ribosome profiling.
The combination of the completed RNAi screen and the developed tools now serve as an extremely competitive basis to mechanistically dissect selected novel piRNA pathway components and profile their effects in a systematic manner.