Dissection of piRNA precursor biogenesis and the epigenetic mechanisms of the piRNA pathway

RNA interference (RNAi) is a cellular mechanism where small RNAs (20 to 35 nucleotides in length) pair with proteins to build effector complexes guiding them to RNA targets through homologous base pairing. The piRNA pathway is a germ line specific RNAi pathway that protects the gonads of animals from harmful genetic sequences, termed transposable elements (TEs). TEs are viral DNA remnants that can autonomously mobilize within genomes and cause damage through insertion into new genomic loci and double-strand breaks in DNA. TE activity in germline cells ultimately leads to sterility and its suppression through the piRNA pathway is therefore essential for propagation. While the piRNA pathway shares common characteristics with the two major RNAi pathways (miRNAs and siRNAs), it additionally exhibits several unique features, particularly during biogenesis. piRNAs originate from large non-genic regions, so called piRNA clusters, or from mRNAs of transposable elements. Neither of these RNA transcripts contain the requisite structural features capable of triggering small RNA production, seen in both siRNAs and miRNAs. Consequently, piRNAs are produced in a Dicer independent manner. Therefore, it is not yet understood, how target transcript are recognized by the piRNA pathway. Furthermore, the extent of piRNA functionality in vivo are not well defined, as they seem to act on multiple levels to protect the germline genome from the propagation of transposable elements. piRNAs lead to direct cleavage of TE mRNAs, but also seem to change the chromatin landscape of regions expressing RNA targets. Additionally, the effect of piRNAs reaches into the next generation through a transgenerational epigenetic mechanism. This study focuses on the characterization of the early stages in piRNA biogenesis, starting at their transcription through the first processing steps. The data presented suggest that the two different piRNA cluster types, uni-stranded and double-stranded clusters, differ greatly in the properties of their transcription. Uni-stranded clusters exhibit a defined transcription start site, giving rise to 5' capped piRNA precursor molecules that resemble canonical mRNAs. Whereas, double-stranded clusters do not show a distinct start site and can be transcribed through internal initiation or read through transcription from neighboring genes. The work furthermore reveals the existence of piRNA intermediates: they are over 200 nucleotides long, which contain a 5' monophosphorylated terminus and already exhibit the nucleotide biases similar to mature piRNAs. After processing of piRNA precursors into mature piRNAs, their effects can reach into subsequent generations. To investigate the trans-generational aspect of piRNAs, I utilized transgenic fly models. The results reveal that piRNAs are epigenetic factors influencing a plethora of mechanisms in the piRNA pathway: ensuring a strong piRNA production in the next generation through maintenance of both piRNA production pathways (primary and secondary piRNA production). Furthermore, they maintain the characteristic structural chromatin features of piRNA clusters in the next generation. However, most strikingly piRNAs are capable of transforming regions expressing targeted transcripts into loci producing primary piRNAs. Therefore, the latter result informs a revision to the current definition of piRNA sources and their subsequent targets. piRNA sources and targets might not be strictly distinct, but any piRNA target acquires piRNA cluster signatures and starts producing piRNAs. The discovery that inherited piRNAs transform a piRNA target into a source, allowed the investigation of the molecular changes at loci targeted by the piRNA pathway. These newly established piRNA clusters acquire a specific chromatin configuration consisting of the histone mark H3K9me3 and two piRNA protein factors Rhino and Cutoff. Together, these factors correlate with increased transcription of their targeted loci. Cutoff appears to be the key factor in piRNA production, through both promotion of read-through transcription beyond stop signals and protection of RNA termini against exonucleolytic degradation. Overall, the presented data contribute to an understanding of the molecular mechanisms in piRNA biogenesis, from the initial processing steps through the trans-generational effect of piRNAs. It suggests that the piRNA system is organized as a giant feed-forward loop, where piRNAs themselves reach into the next generation to ensures reliable propagation of the system and effective silencing of deleterious TE sequences.