Project description:Organisms require mechanisms to distinguish self and non-self RNA. This distinction is crucial to initiate the biogenesis of piRNAs. In Drosophila ovaries, PIWI-guided slicing and the recognition of piRNA precursor transcripts by the DEAD-box RNA helicase Yb are the two known mechanisms to initiate biogenesis in the germline and the soma, respectively. Both, the PIWI proteins and Yb are highly conserved across most Drosophila species and are thought to be essential to the piRNA pathway and for silencing TEs. However, we find that species closely related to D. melanogaster have lost the yb gene, as well as the PIWI gene Ago3. We show that neither mechanism is required to efficiently produce TE antisense piRNAs in Drosophila. Thus, there are more possible routes through which the piRNA pathway can achieve specificity than previously suggested.
Project description:Organisms require mechanisms to distinguish self and non-self RNA. This distinction is crucial to initiate the biogenesis of piRNAs. In Drosophila ovaries, PIWI-guided slicing and the recognition of piRNA precursor transcripts by the DEAD-box RNA helicase Yb are the two known mechanisms to initiate biogenesis in the germline and the soma, respectively. Both, the PIWI proteins and Yb are highly conserved across most Drosophila species and are thought to be essential to the piRNA pathway and for silencing TEs. However, we find that species closely related to D. melanogaster have lost the yb gene, as well as the PIWI gene Ago3. We show that neither mechanism is required to efficiently produce TE antisense piRNAs in Drosophila. Thus, there are more possible routes through which the piRNA pathway can achieve specificity than previously suggested.
Project description:Organisms require mechanisms to distinguish self and non-self RNA. This distinction is crucial to initiate the biogenesis of piRNAs. In Drosophila ovaries, PIWI-guided slicing and the recognition of piRNA precursor transcripts by the DEAD-box RNA helicase Yb are the two known mechanisms to initiate biogenesis in the germline and the soma, respectively. Both, the PIWI proteins and Yb are highly conserved across most Drosophila species and are thought to be essential to the piRNA pathway and for silencing TEs. However, we find that species closely related to D. melanogaster have lost the yb gene, as well as the PIWI gene Ago3. We show that neither mechanism is required to efficiently produce TE antisense piRNAs in Drosophila. Thus, there are more possible routes through which the piRNA pathway can achieve specificity than previously suggested.
Project description:In animal gonads, 23-30nt long PIWI interacting RNAs (piRNAs) guarantee genome integrity by guiding the sequence specific silencing of selfish genetic elements such as transposons. Two major branches of piRNA biogenesis, namely primary processing and ping-pong amplification, feed into the PIWI clade of Argonaute proteins. Despite our conceptual understanding of piRNA biogenesis, major gaps exist in the mechanistic understanding of the underlying molecular processes as well as in the knowledge of the involved players. Here, we demonstrate an essential role for the female sterility gene shutdown in the piRNA pathway. Shutdown, an evolutionarily conserved co-chaperone of the immunophilin class is the first piRNA biogenesis factor that is essential for all primary and secondary piRNA populations in Drosophila. Based on these findings, we define distinct groups of piRNA biogenesis factors and reveal the core concept of how PIWI family proteins are hard-wired into piRNA biogenesis processes. small-RNA libraries from 2 control samples and 7 knock-down samples of D. mel. ovaries and 2 small-RNA profiles from Piwi IP and Aub IP from OSCs.
Project description:In animal gonads, 23-30nt long PIWI interacting RNAs (piRNAs) guarantee genome integrity by guiding the sequence specific silencing of selfish genetic elements such as transposons. Two major branches of piRNA biogenesis, namely primary processing and ping-pong amplification, feed into the PIWI clade of Argonaute proteins. Despite our conceptual understanding of piRNA biogenesis, major gaps exist in the mechanistic understanding of the underlying molecular processes as well as in the knowledge of the involved players. Here, we demonstrate an essential role for the female sterility gene shutdown in the piRNA pathway. Shutdown, an evolutionarily conserved co-chaperone of the immunophilin class is the first piRNA biogenesis factor that is essential for all primary and secondary piRNA populations in Drosophila. Based on these findings, we define distinct groups of piRNA biogenesis factors and reveal the core concept of how PIWI family proteins are hard-wired into piRNA biogenesis processes.
Project description:Piwi interacting (pi)RNAs repress diverse transposable elements in the germ cells of metazoans and are essential for fertility in both invertebrates and vertebrates. The precursors of piRNAs are transcribed from distinct genomic regions, the so-called piRNA clusters; however, how piRNA clusters are differentiated from the rest of the genome is not known. To address this question, we studied piRNA biogenesis in two Drosophila virilis strains that show differential ability to generate piRNAs from several genomic regions. We found that active piRNA biogenesis correlates with high levels of histone 3 lysine 9 trimethylation (H3K9me3) over genomic regions that give rise to piRNAs. Furthermore, piRNA biogenesis in the progeny requires the trans-generational inheritance of an epigenetic signal, presumably in form of homologous piRNAs that are generated in the maternal germline and deposited into the oocyte. The inherited piRNAs enhance piRNA biogenesis by installment of H3K9me3 mark on piRNA clusters and by promoting ping-pong processing of homologous transcripts into mature piRNAs. We submitted the resequencing data together with the functional genomic datasets because it was generated with the sole purpose of supporting those. The SRA accession numbers are SRR1536176 and SRR1536175. ChIP-seq against H3K9me3 and Pol2, Total RNA-seq, in Drosophila virilis Strain9 and Strain160 as well as crosses between them
Project description:PIWI-clade Argonaute proteins repress transposable elements in animal gonads. Their sequence specificity is conferred via bound ~23-30nt long piRNAs, which are processed from single stranded precursor RNAs. How transcripts are specified as precursors and processed into stereotypical piRNA populations are central unresolved questions. Here we show that piRNA-guided RNA cleavage in Drosophila results not only in generation of a ping-pong partner piRNA but further triggers efficient 3′ directed and phased primary piRNA biogenesis. Phasing is a feature of primary piRNAs in somatic and germline cells and a consequence of consecutive endo-nucleolytic cleavage events catalyzed by Zucchini. Formation of 3′ and 5′ ends of flanking piRNAs is therefore tightly coupled. Zucchini also participates in 3′ end formation of secondary piRNAs but its function can be bypassed by additional downstream piRNA-guided cleavages and subsequent precursor trimming. Hallmarks of Zucchini-dependent phased piRNA biogenesis are also evident in mouse testes, pointing to an evolutionarily conserved mechanism of piRNA biogenesis. This study aims at understanding how piRNA biogenesis is intiated in the Drosophila germline and understanding the role of the nuclease Zucchini/MitoPLD in piRNA biogenesis in Drosophila/Mouse by analysing small RNA sequencing data of various genotypes and sensor constructs.