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Bombyx Vasa sequesters transposon mRNAs in nuage via phase separation requiring RNA binding and self-association [RIP-Seq]

ABSTRACT: Bombyx Vasa (BmVasa) assembles non-membranous organelle, nuage or Vasa bodies, in germ cells, known as the center for Siwi-dependent transposon silencing and concomitant Ago3-piRISC biogenesis. However, details of the body assembly remain unclear. Here, we show that the N-terminal intrinsically disordered region (N-IDR) and RNA helicase domain of BmVasa are responsible for self-association and RNA binding, respectively, but N-IDR is also required for full RNA-binding activity. Both domains are essential for Vasa body assembly in vivo and droplet formation in vitro via phase separation. FAST-iCLIP reveals that BmVasa preferentially binds transposon mRNAs. Loss of Siwi function derepresses transposons but has marginal effects on BmVasa-RNA binding. This study shows that BmVasa assembles nuage by phase separation via its ability to self-associate and bind newly exported transposon mRNAs. This unique property of BmVasa allows transposon mRNAs to be sequestered and enriched in nuage, resulting in effective Siwi-dependent transposon repression and Ago3-piRISC biogenesis.

ORGANISM(S): Bombyx mori

PROVIDER: GSE213914 | GEO | 2023/03/06

REPOSITORIES: GEO

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Bombyx Vasa sequesters transposon mRNAs in nuage via phase separation requiring RNA binding and self-association [RNA-Seq]

Project description:Bombyx Vasa (BmVasa) assembles non-membranous organelle, nuage or Vasa bodies, in germ cells, known as the center for Siwi-dependent transposon silencing and concomitant Ago3-piRISC biogenesis. However, details of the body assembly remain unclear. Here, we show that the N-terminal intrinsically disordered region (N-IDR) and RNA helicase domain of BmVasa are responsible for self-association and RNA binding, respectively, but N-IDR is also required for full RNA-binding activity. Both domains are essential for Vasa body assembly in vivo and droplet formation in vitro via phase separation. FAST-iCLIP reveals that BmVasa preferentially binds transposon mRNAs. Loss of Siwi function derepresses transposons but has marginal effects on BmVasa-RNA binding. This study shows that BmVasa assembles nuage by phase separation via its ability to self-associate and bind newly exported transposon mRNAs. This unique property of BmVasa allows transposon mRNAs to be sequestered and enriched in nuage, resulting in effective Siwi-dependent transposon repression and Ago3-piRISC biogenesis.

2023-03-06 | GSE213915 | GEO

Maelstrom functions in the production of Siwi-piRISC capable of regulating transposons in Bombyx germ cells

Project description:PIWI-interacting RNAs (piRNAs) bind to PIWI proteins to assemble the piRISC, which represses germline transposons. Maelstrom (Mael) is necessary for piRISC biogenesis in germ cells, but its function remains unclear. Here, we show that Mael interconnects Spindle-E (Spn-E), a key piRISC biogenesis factor, with unloaded Siwi, one of two silkworm PIWI members. Mael also assembles a subset of nuage, a non-membranous organelle involved in piRISC biogenesis. Loss of Mael abrogated the Spn-E–Siwi interaction and Ago3-piRISC biogenesis, but Siwi-piRISC was produced. Bioinformatic analysis showed that Siwi-bound piRNAs in Mael-lacking cells were rich in transposon-targeting piRNAs as in normal cells but were biased toward transposons that are marginally controlled by Siwi-piRISC. This explains the impairment in Ago3-piRISC production because transposon mRNAs cleaved by Siwi are the origin of Ago3-loaded piRNAs. We argue that Mael plays a role in the production of primary Siwi-piRISC capable of regulating transposon expression in germ cells.

2022-03-02 | GSE180191 | GEO

UAP56 couples piRNA clusters to the perinuclear transposon silencing machinery [tiling array]

Project description:The transposon silencing piRNAs are produced from precursors that are encoded by heterochromatic clusters and processed in the perinuclear nuage. We show that the Drosophila nuclear DEAD box protein UAP56, previously implicated in mRNA splicing and nuclear export, co-localizes with the cluster-associated HP1 homologue Rhino. Prominent nuclear foci containing Rhi and UAP56 localize directly across the nuclear envelope from Vasa, a conserved DEAD box protein and core nuage component that is required for piRNA production, and piRNA precursors immunoprecipitate with both UAP56 and Vasa. A uap56 point mutation that prevents UAP56 protein co-localization with Rhino also disrupts nuage organization, transposon silencing, and expression of dual strand piRNA clusters. By contrast, this allele significantly increases ectopic piRNAs from protein coding genes. We therefore propose that UAP56 and Vasa organize a piRNA-processing compartment that spans the nuclear envelope, increasing the efficiency and specificity of piRNA biogenesis. 3 replicates of each sample (uap56, vasa), total RNA samples hybridized to tiling array.

2012-11-28 | E-GEOD-35636 | biostudies-arrayexpress

Siwi cooperates with Par-1 kinase to regulate mitochondrial Papi scaffolding Siwi-piRISC biogenesis

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

UAP56 couples piRNA clusters to the perinuclear transposon silencing machinery [RNA-Seq and RIP-Seq]

Project description:The transposon silencing piRNAs are produced from precursors that are encoded by heterochromatic clusters and processed in the perinuclear nuage. We show that the Drosophila nuclear DEAD box protein UAP56, previously implicated in mRNA splicing and nuclear export, co-localizes with the cluster-associated HP1 homologue Rhino. Prominent nuclear foci containing Rhi and UAP56 localize directly across the nuclear envelope from Vasa, a conserved DEAD box protein and core nuage component that is required for piRNA production, and piRNA precursors immunoprecipitate with both UAP56 and Vasa. A uap56 point mutation that prevents UAP56 protein co-localization with Rhino also disrupts nuage organization, transposon silencing, and expression of dual strand piRNA clusters. By contrast, this allele significantly increases ectopic piRNAs from protein coding genes. We therefore propose that UAP56 and Vasa organize a piRNA-processing compartment that spans the nuclear envelope, increasing the efficiency and specificity of piRNA biogenesis. RNA-Seq: 3 samples examined: w1118, uap56 mutant un-oxidized, uap56 mutant oxidized RIP-Seq: 6 samples: UAP56-Venus, sz-Venus, and wild type w1 with anti-flag and input control each.

2012-11-28 | E-GEOD-35637 | biostudies-arrayexpress

UAP56 couples piRNA clusters to the perinuclear transposon silencing machinery [RNA-Seq and RIP-Seq]

2012-11-28 | GSE35637 | GEO

UAP56 couples piRNA clusters to the perinuclear transposon silencing machinery [tiling array]

2012-11-28 | GSE35636 | GEO

High throughput sequencing of Siwi and BmAGO3 associated piRNAs from Bombyx mori BmN4 cells

Project description:In germ cells, piRNAs are amplified through the Ping-Pong cycle that depends on reciprocal Slicer-mediated target RNA cleavage by two PIWI members. A germ-specific DEAD-box protein Vasa is required for the process. However, Vasa’s function is poorly understood. Here, we show that target RNAs cleaved by a Bombyx mori (silkworm) PIWI, Siwi, remain to be bound with the protein upon cleavage, but are released in the presence of Vasa in B. mori (BmVasa) and ATP. Under normal conditions, BmVasa co-purifies with Siwi, but not with second B. mori PIWI BmAgo3. However, when BmVasa loses the ATP-binding and RNA-unwinding activities, BmVasa avidly associates with Siwi and BmAgo3, which contains transposon transcripts predominantly in sense orientation, the sources of BmAgo3-piRNAs. Without BmVasa, BmAgo3 is devoid of piRNAs. Thus, BmVasa actively releases target RNAs from Siwi, upon its cleavage, to urge BmAgo3-piRNA complex formation in the Ping-Pong cycle, enabling continuous supply of piRNAs in germ cells.

2014-12-31 | GSE58221 | GEO

Krimper enforces an antisense bias on piRNA pools by binding AGO3 in the Drosophila germline

Project description:Piwi-interacting RNAs (piRNAs) suppress transposon activity in animal germ cells. In the Drosophila ovary, primary Aubergine (Aub)-bound antisense piRNAs initiate the ping-pong cycle to produce secondary AGO3-bound sense piRNAs. This increases the number of secondary Aub-bound antisense piRNAs that can act to destroy transposon mRNAs. Here we show that Krimper (Krimp), a Tudor-domain protein, directly interacts with piRNA-free AGO3 to promote symmetrical dimethylarginine (sDMA) modification, ensuring sense piRNA-loading onto sDMA-modified AGO3. In aub mutant ovaries, AGO3 associates with ping-pong signature piRNAs, suggesting AGO3’s compatibility with primary piRNA loading. Krimp sequesters ectopically expressed AGO3 within Krimp bodies in cultured ovarian somatic cells (OSCs), in which only the primary piRNA pathway operates. Upon krimp-RNAi in OSCs, AGO3 loads with piRNAs, further showing the capacity of AGO3 for primary piRNA loading. We propose that Krimp enforces an antisense bias on piRNA pools by binding AGO3 and blocking its access to primary piRNAs.

2015-05-28 | GSE69293 | GEO