Hierarchical roles of mitochondrial PAPI and Zucchini in Bombyx germline piRNA biogenesis
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ABSTRACT:
PROVIDER: PRJNA419912 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR6324420.fastq.gz | Fastqsanger.gz | |||
| SRR6324421.fastq.gz | Fastqsanger.gz | |||
| SRR6324422.fastq.gz | Fastqsanger.gz | |||
| SRR6324423.fastq.gz | Fastqsanger.gz | |||
| SRR6324424.fastq.gz | Fastqsanger.gz |
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Project description:PIWI-interacting RNAs (piRNAs) are germline-enriched small RNAs that control transposons to maintain genome integrity1,2,3. To achieve this, piRNAs bind PIWI proteins upon being processed from piRNA precursors1,2,3. Bioinformatic studies of piRNA biogenesis in Drosophila showed that the piRNA 5′ end is formed by PIWI-Slicer or Zucchini (Zuc) endonucleolytic cleavage, while the 3′ end is formed by Zuc or Nibbler (Nbr) 3′-to-5′ exonucleolytic activity4,5,6. piRNA 3′-end formation in Bombyx was shown to be mediated by PNLDC1/Trimmer (Trim) 3′-to-5′ exonuclease7, while piRNA intermediates are bound with PIWI anchored onto mitochondrial protein PAPI8. However, the requirement for Zuc and Nbr in piRNA biogenesis in Bombyx has not been elucidated. Here, we applied biochemical approaches to understand their involvement in piRNA biogenesis and revealed that Zuc endonuclease, but not Trim and Nbr exonucleases, plays a crucial role in Bombyx piRNA 3′-end formation. Loss of Zuc had little effect on the levels of Trim and Nbr, but led to the aberrant accumulation of piRNA intermediates within the PAPI complex, which were processed to mature piRNAs by recombinant Zuc. Zuc copurified with PAPI, and PAPI exerted RNA-binding activity only when Siwi coexisted with it and PAPI was phosphorylated, suggesting that complex assembly proceeds via a hierarchical process. Bioinformatic analyses of piRNA intermediates within the PAPI complex revealed that both the 5′ and the 3′ ends showed the hallmark of PIWI-Slicer, yet no phasing pattern was observed in mature piRNAs. These findings strongly support the notion that, in Bombyx piRNA, the 5′ end is formed by PIWI-Slicer, but independently of Zuc, while the 3′ end is formed by Zuc endonuclease. The Bombyx piRNA biogenesis is simpler than that of Drosophila, which is reasonable considering that Bombyx has no transcriptional silencing machinery relying on phased piRNAs.
2018-03-01 | GSE107371 | GEO
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.
2015-06-07 | E-GEOD-64802 | biostudies-arrayexpress
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.
2015-06-07 | GSE64802 | GEO
Project description:Bombyx Papi contains two K-homology (KH) domains and one Tudor domain, and acts as a scaffold for Siwi-piRISC biogenesis on the mitochondrial surface. To initiate this process, Papi binds first to Siwi via the Tudor domain and subsequently to piRNA precursors loaded onto Siwi via the KH domains. This second action depends on phosphorylation of Papi. However, its underlying mechanism remains unknown. Here, we show that Siwi targets Par-1 kinase to mitochondrial Papi to promote its phosphorylation at Ser547 in the auxiliary domain and that this modification enhances the ability of Papi to bind Siwi-bound piRNA precursors via the KH domains. Papi S547A mutant still bound to Siwi, like wild-type (WT) Papi, although it evaded phosphorylation by Par-1: Consequently, Papi lost the ability to bind RNAs, abrogating the generation of Siwi-piRISC. Papi mutant lacking the Tudor and auxiliary domains escaped coordinated regulation by Siwi and Par-1 and lost their bias to bind piRNA precursors. Pseudo-phosphorylation mutants of Papi restored Siwi-piRISC formation in Papi-lacking cells, but their ability to bind RNAs required Siwi, similar to WT Papi. Par-1-dependent, multilayered mechanism by which Siwi regulates the role of Papi in Siwi-piRISC biogenesis was revealed.
2022-01-25 | GSE179432 | GEO
Project description:The piRNA pathway is a small RNA-based immune system that silences mobile genetic elements in animal germlines. piRNA biogenesis requires a specialised machinery that converts long single-stranded precursors into small RNAs of ~25-nucleotides in length. This process involves factors that operate in two different subcellular compartments: the nuage/Yb-body and mitochondria. How these two sites communicate to achieve accurate substrate selection and efficient processing remains unclear. Here, we investigate a previously uncharacterized piRNA biogenesis factor, Daedalus (Daed), that is located on the outer mitochondrial membrane. Daed is essential for Zucchini-mediated piRNA production and for the correct localisation of the indispensable piRNA biogenesis factor Armitage (Armi). We find that Gasz and Daed interact with each other and likely provide a mitochondrial “anchoring platform” to ensure that Armi is held in place, proximal to Zucchini, during piRNA precursor processing. Our data suggest that Armi initially identifies piRNA precursors in nuage/Yb-body in a manner that depends upon Piwi and then moves to mitochondria to present them to the biogenesis machinery. These results represent a significant step in understanding a critical aspect of transposon silencing, namely how RNAs are chosen to instruct the piRNA machinery in the nature of its silencing targets.
2019-04-17 | GSE129321 | GEO
Project description:The piRNA pathway is a small RNA-based immune system that silences mobile genetic elements in animal germlines. piRNA biogenesis requires a specialised machinery that converts long single-stranded precursors into small RNAs of ~25-nucleotides in length. This process involves factors that operate in two different subcellular compartments: the nuage/Yb-bodies and mitochondria. How these two sites communicate to achieve accurate substrate selection and efficient processing remains unclear. Here, we investigate a previously uncharacterized piRNA biogenesis factor, Daedalus (Daed), that is located on the outer mitochondrial membrane. Daed is essential for Zucchini-mediated piRNA production and for the correct localisation of the indispensable piRNA biogenesis factor, Armitage (Armi). We find that Gasz and Daed interact with each other and likely provide a mitochondrial “anchoring platform” to ensure that Armi is held in place, proximal to Zucchini, during piRNA processing. Our data suggest that Armi initially identifies piRNA precursors in nuage/Yb-bodies in a manner that depends upon Piwi and then moves to mitochondria to present precursors to the mitochondrial biogenesis machinery. These results represent a significant step in understanding a critical aspect of transposon silencing, namely how RNAs are chosen to instruct the piRNA machinery in the nature of its silencing targets.
2019-04-18 | PXD013403 | Pride
Project description:The piRNA pathway is a small RNA-based immune system that silences mobile genetic elements in animal germlines. piRNA biogenesis requires a specialised machinery that converts long single-stranded precursors into small RNAs of ~25-nucleotides in length. This process involves factors that operate in two different subcellular compartments: the nuage/Yb-body and mitochondria. How these two sites communicate to achieve accurate substrate selection and efficient processing remains unclear. Here, we investigate a previously uncharacterized piRNA biogenesis factor, Daedalus (Daed), that is located on the outer mitochondrial membrane. Daed is essential for Zucchini-mediated piRNA production and for the correct localisation of the indispensable piRNA biogenesis factor, Armitage (Armi). We find that Gasz and Daed interact with each other and likely provide a mitochondrial “anchoring platform” to ensure that Armi is held in place, proximal to Zucchini, during piRNA processing. Our data suggest that Armi initially identifies piRNA precursors in nuage/Yb-body in a manner that depends upon Piwi and then moves to mitochondria to present precursors to the mitochondrial biogenesis machinery. These results represent a significant step in understanding a critical aspect of transposon silencing, namely how RNAs are chosen to instruct the piRNA machinery in the nature of its silencing targets.
2019-04-18 | PXD013405 | Pride
Project description:The piRNA pathway is a small RNA-based immune system that silences mobile genetic elements in animal germlines. piRNA biogenesis requires a specialised machinery that converts long single-stranded precursors into small RNAs of ~25-nucleotides in length. This process involves factors that operate in two different subcellular compartments: the nuage/Yb-body and mitochondria. How these two sites communicate to achieve accurate substrate selection and efficient processing remains unclear. Here, we investigate a previously uncharacterized piRNA biogenesis factor, Daedalus (Daed), that is located on the outer mitochondrial membrane. Daed is essential for Zucchini-mediated piRNA production and for the correct localisation of the indispensable piRNA biogenesis factor, Armitage (Armi). We find that Gasz and Daed interact with each other and likely provide a mitochondrial “anchoring platform” to ensure that Armi is held in place, proximal to Zucchini, during piRNA processing. Our data suggest that Armi initially identifies piRNA precursors in nuage/Yb-body in a manner that depends upon Piwi and then moves to mitochondria to present precursors to the mitochondrial biogenesis machinery. These results represent a significant step in understanding a critical aspect of transposon silencing, namely how RNAs are chosen to instruct the piRNA machinery in the nature of its silencing targets.
2019-04-18 | PXD013404 | Pride
Project description:The piRNA pathway is a small RNA-based immune system that silences mobile genetic elements in animal germlines. piRNA biogenesis requires a specialised machinery that converts long single-stranded precursors into small RNAs of ~25-nucleotides in length. This process involves factors that operate in two different subcellular compartments: the nuage/Yb-body and mitochondria. How these two sites communicate to achieve accurate substrate selection and efficient processing remains unclear. Here, we investigate a previously uncharacterized piRNA biogenesis factor, Daedalus (Daed), that is located on the outer mitochondrial membrane. Daed is essential for Zucchini-mediated piRNA production and for the correct localisation of the indispensable piRNA biogenesis factor Armitage (Armi). We find that Gasz and Daed interact with each other and likely provide a mitochondrial “anchoring platform” to ensure that Armi is held in place, proximal to Zucchini, during piRNA precursor processing. Our data suggest that Armi initially identifies piRNA precursors in nuage/Yb-body in a manner that depends upon Piwi and then moves to mitochondria to present them to the biogenesis machinery. These results represent a significant step in understanding a critical aspect of transposon silencing, namely how RNAs are chosen to instruct the piRNA machinery in the nature of its silencing targets.
2019-04-18 | PXD013417 | Pride
Project description:In Drosophila, PIWI proteins and bound PIWI interacting RNAs (piRNAs) form the core of a small RNA mediated defense system against selfish genetic elements. Within germline cells piRNAs are processed from piRNA clusters and transposons to be loaded into Piwi/Aubergine/AGO3 and a subset of piRNAs undergoes target dependent amplification. In contrast, gonadal somatic support cells express only Piwi, lack signs of piRNA amplification and exhibit primary piRNA biogenesis from piRNA clusters. Neither piRNA processing/loading nor Piwi mediated target silencing is understood at the genetic, cellular or molecular level. We developed an in vivo RNAi assay for the somatic piRNA pathway and identified the RNA helicase Armitage, the Tudor domain containing RNA helicase Yb and the putative nuclease Zucchini as essential factors for primary piRNA biogenesis. Lack of any of these proteins leads to transposon de-silencing, to a collapse in piRNA levels and to a failure in Piwi nuclear accumulation. We show that Armitage and Yb interact physically and co-localize in cytoplasmic Yb-bodies, which flank P-bodies. Loss of Zucchini leads to an accumulation of Piwi and Armitage in Yb-bodies indicating that Yb-bodies are sites of primary piRNA biogenesis. small RNA libraries were prepared from Piwi immuno-precipitates of five different genotypes
2010-10-03 | E-GEOD-23560 | biostudies-arrayexpress