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.