The PIWI/piRNA pathway: Genome defense guided by small RNAs
Transposons and other selfish genetic elements populate every eukaryotic genome, indicating an ancient genetic conflict. To amplify and spread within host DNA, transposons utilize the cellular gene expression machinery. Their selective silencing therefore poses a complex challenge for the host cell. In plants, fungi, and animals, small RNA silencing pathways—commonly referred to as RNA interference processes—meet this challenge. We focus on the PIWI/piRNA pathway, which is the central transposon silencing system in animal gonads. This pathway has remarkable conceptual similarities to adaptive immune systems and intersects the fascinating world of small RNAs with heterochromatin biology and epigenetic phenomena that play central roles in evolution.
RNA interference & transposon silencing
Transposons are universal components of eukaryotic genomes and contribute significantly to genome size (~50% in humans). These selfish genetic elements are central agents for genome evolution by diversifying the gene regulatory landscape or by serving as building blocks of heterochromatin. On the other hand, their mobility causes DNA double-strand breaks and insertional mutagenesis.
To safeguard genome integrity and to prevent the uncontrolled spread of transposons, host defense systems evolved early in evolution. After the discovery of RNA interference it became clear that small RNA-silencing systems are at the root of transposon control in fungi, plants, and animals. Central to all small RNA pathways are Argonaute proteins. These silencing machines are guided to their target transcripts via bound small RNAs. The success of small RNA pathways in the battle against transposons likely rests upon their conceptual set-up: By fuelling small RNA biogenesis with transcripts of the targets themselves, these pathways provide flexibility to any nucleic acid sequence and adaptability to new challenges originating from sequence drift or the horizontal invasion of new elements.
The conceptual logic of small RNA-guided genome defense pathways
Figure 1 shows the three core principles that characterize small RNA-based genome defense pathways: (1) Silencing information is stored in genomic loci. These are insertions of active transposons or large loci composed of libraries of transposon fragments. The memory loci are evolutionarily plastic and acquire or loose transposon sequences over time. Transcription of the genomic storage loci provides the small RNA precursors. (2) Dedicated RNA processing systems parse precursor transcripts into small RNAs, which are loaded into Argonaute proteins. (3) Argonaute proteins act at the center of small RNA pathways. Their bound small RNAs are sequence-specific guides that target the RNA-induced silencing complex (RISC) to complementary transcripts. RISC recruitment elicits target silencing at the transcriptional or post-transcriptional level. Remarkably, recent insights into CRISPR/Cas—bacterial small RNA-based defense systems against phage—indicate intriguing conceptual similarities between eukaryotic and prokaryotic host genome defense systems.
The PIWI/piRNA pathway
The key transposon silencing system in animal gonads is the piRNA pathway, which utilizes Argonaute proteins of the PIWI clade. In contrast to the much better understood micro-RNA and siRNA pathways, its mechanistic framework is largely unknown. We study this fascinating genome surveillance system in Drosophila melanogaster, combining our strong expertise in genetics with biochemistry, cell biology, next-generation sequencing, and computational biology. Our interest is focused on three main areas (Fig. 2):
1. piRNA biogenesis: Thousands of different ~23 to 30 nt long piRNAs are processed from single-stranded precursor RNAs in a poorly understood manner. Genetic screens have disclosed more than a dozen proteins to be required for piRNA biogenesis. Among these are RNA helicases and several uncharacterized proteins. Interestingly, several of them are transmembrane proteins of the outer mitochondrial membrane. We aim to discover the rules of piRNA biogenesis and to dissect the underlying molecular mechanisms in order to understand how the cell distinguishes piRNA precursor transcripts from other RNAs in the cell.
2. Piwi-mediated transcriptional silencing: Of the three Drosophila PIWI clade proteins, one (Piwi) localizes to the nucleus. Based on our recent work, piRNAs guide Piwi to nascent transposon transcripts to trigger the buildup of a potent silencing complex that represses target transcription and leads to local heterochromatin formation. Our aim is to understand how Piwi-RISC recruitment to a nascent RNA elicits these processes.
3. The biology of piRNA clusters: piRNA clusters are the system’s heritable repositories for transposon sequence information as they provide the piRNA precursors transcripts. These up to several hundreds of kilobase long heterochromatic regions resemble transposon graveyards and encompass a selection of selfish sequences that a population has been exposed to. Our recent findings indicate that piRNA clusters recruit a battery of germline specific factors that bypass the canonical rules of gene expression. We are interested in the transcription of piRNA clusters as well as in the specification, export and processing of their transcripts.