Characterization of piRNA processing factors in C. elegans

Small RNA molecules can act as regulators in gene expression pathways. The precise recognition of targets is based on base pairing, and the sRNA thus acts as specificity factors, and consequently, their processing needs to be tightly controlled. One of the well-conserved small RNA silencing pathways is the so-called Piwi pathway. This pathway is one of the main surveillance systems in germ cells to keep transposable elements under control and is steered by small RNAs known as piRNAs. Given the wide diversity and varying nature of the transposon sequences, the piRNA sequence repertoire needs to be well controlled to avoid the recognition of host sequences. This happens by selecting specific transcripts as piRNA precursors, followed by distinct processing steps. Neither the selection process nor the processing steps are in general well understood. In the nematode Caenorhabditis elegans (C. elegans), the piRNAs are derived from specific, short RNA polymerase II transcripts. Following this, they are bound by a dedicated protein complex named PETISCO, which allows the processing of their 5` end by an as-yet- unidentified nuclease. Whether this step is a compilation of different activities, such as de- capping, followed by exoribonucleolytic trimming, or is driven by a distinct endoribonucleolytic enzyme is unknown. We present preliminary data that implicate that the endoribonuclease is constituted by a heterodimeric protein complex that specifically acts on PETISCO-bound piRNA precursors in C. elegans. The proposed work plans are aimed to solidify this hypothesis and to understand this novel enzymatic activity. We want to learn about its 3-dimensional structure and how it is connected to PETISCO. In addition, we identified two additional factors that play a role in piRNA biogenesis and resemble proteins constituting dimeric endonuclease on the domain level. Our data suggest a potential modular heterodimeric assembly of potentially other types of nucleases, based on one of the two subunits of the piRNA processing nuclease. We aim to understand the function(s) of those complexes both at the biochemical and organismic levels.

How cells protect and process unusual RNA molecules In every one of our cells, genes are constantly copied into RNA molecules. Many of these RNAs are short-lived and must be actively protected so that they can do their job before they are broken down again. This is especially important in the germ cells, from which egg and sperm cells arise and through which genetic information is passed on to the next generation. Our genetic material is under constant threat from so-called jumping genes. These are small parasitic stretches of DNA that multiply within the genome and can damage it. Cells defend themselves with tiny RNA molecules called piRNAs, which silence these intruders. Before a piRNA can form, a precursor RNA first has to be trimmed at exactly the right position. Which tool carries out this precise cut was long unknown. In the first funding period, our two research groups found this tool. We discovered an enzyme, which we named PUCH, that cuts the precursor RNA at the decisive position. Surprisingly, PUCH sits on the power plants of the cell, the mitochondria. A completely different enzyme in insects and mammals, which performs the same task, works in exactly the same place. Nature has therefore chosen the same location for this process twice, independently, a sign of how important it is. Just as important is protecting the fragile precursor RNA before it is cut. This is where a protein complex called PETISCO comes in. It attaches to the precursor RNA and shields it from premature breakdown, so that enough material is available for the production of piRNAs. PETISCO has a second, quite different task as well. It protects another short-lived RNA that the mother deposits in her egg cells and that is needed for the very first steps of embryonic development. Without this protection, the embryos die. One and the same complex therefore takes on two distinct protective jobs. Taken together, our results show how cells recognize, protect, and process unusual RNA molecules. Because disturbances in the piRNA system are linked to infertility in humans, these fundamental insights will, in the long term, also contribute to our understanding of human reproduction.