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Requirements for multivalent Yb body assembly in transposon silencing in Drosophila

ABSTRACT: Female sterile (1) Yb (Yb) is a primary component of Yb bodies, perinuclear foci considered to be the site of PIWI-interacting RNA (piRNA) biogenesis in Drosophila ovarian somatic cells (OSCs). Yb consists of three domains: Helicase C-terminal (Hel-C), RNA helicase, and extended Tudor (eTud) domains. We previously showed that the RNA helicase domain is necessary for Yb-RNA interaction, Yb body formation, and piRNA biogenesis. Here, we investigate the functions of Hel-C and eTud and reveal that Hel-C is dedicated to Yb-Yb homotypic interaction, while eTud is necessary for Yb-RNA association, as is the RNA helicase domain. All of these domains are indispensable for Yb body formation and transposon-repressing piRNA production. Strikingly, however, genic piRNAs unrelated to transposon silencing are produced in OSCs where Yb bodies are disassembled. We also reveal that Yb bodies are liquid-like multivalent condensates whose assembly depends on Yb-Yb homotypic interaction and Yb binding particularly with flamenco RNA transcripts, the source of transposon-repressing piRNAs. New insights into Yb body assembly and biological relevance of Yb bodies in transposon silencing have emerged.

SUBMITTER: Shigeki Hirakata

PROVIDER: S-SCDT-EMBOR-2019-47708V1 | biostudies-other |

REPOSITORIES: biostudies-other

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Requirements for multivalent Yb body assembly in transposon silencing in Drosophila.

Project description:Female sterile (1) Yb (Yb) is a primary component of Yb bodies, perinuclear foci considered to be the site of PIWI-interacting RNA (piRNA) biogenesis in Drosophila ovarian somatic cells (OSCs). Yb consists of three domains: Helicase C-terminal (Hel-C), RNA helicase, and extended Tudor (eTud) domains. We previously showed that the RNA helicase domain is necessary for Yb-RNA interaction, Yb body formation, and piRNA biogenesis. Here, we investigate the functions of Hel-C and eTud and reveal that Hel-C is dedicated to Yb-Yb homotypic interaction, while eTud is necessary for Yb-RNA association, as is the RNA helicase domain. All of these domains are indispensable for Yb body formation and transposon-repressing piRNA production. Strikingly, however, genic piRNAs unrelated to transposon silencing are produced in OSCs where Yb bodies are disassembled. We also reveal that Yb bodies are liquid-like multivalent condensates whose assembly depends on Yb-Yb homotypic interaction and Yb binding particularly with flamenco RNA transcripts, the source of transposon-repressing piRNAs. New insights into Yb body assembly and biological relevance of Yb bodies in transposon silencing have emerged.

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Requirements for multivalent Yb body assembly in transposon silencing in Drosophila

Project description:Female sterile (1) Yb (Yb) is a primary component of Yb bodies, perinuclear foci known as the site of PIWI-interacting RNA (piRNA) biogenesis in Drosophila ovarian somatic cells. Yb consists of Helicase C-terminal (Hel-C), RNA helicase and extended Tudor (eTud) domains. We previously showed that the RNA helicase domain is necessary for Yb−RNA interaction, Yb body formation and piRNA biogenesis. Here, we investigated the functions of the two other domains and found that Hel-C and eTud are necessary for Yb to self-associate and to interact with RNAs and other Yb body components, respectively. Both domains were essential for Yb body formation and transposon silencing. Without eTud, piRNA production was completely impaired. Loss of Hel-C severely reduced the levels of transposon-targeting piRNAs, although genic piRNAs unrelated to transposon silencing were still produced. Similar phenotypes were observed when mutations in flamenco, the primary source of transposon-targeting piRNAs, led to the expression of attenuated RNAs. Yb bodies are liquid-like multivalent condensates whose assembly depends on Yb self-association and widespread Yb−flamenco RNA binding.

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Requirements for multivalent Yb body assembly in transposon silencing in Drosophila

Project description:Requirements for multivalent Yb body assembly in transposon silencing in Drosophila

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Export of piRNA precursors by EJC triggers assembly of cytoplasmic Yb-body in Drosophila.

Project description:PIWI-interacting RNAs (piRNAs) are effectors of transposable element (TE) silencing in the reproductive apparatus. In Drosophila ovarian somatic cells, piRNAs arise from longer single-stranded RNA precursors that are processed in the cytoplasm presumably within the Yb-bodies. piRNA precursors encoded by the flamenco (flam) piRNA cluster accumulate in a single focus away from their sites of transcription. In this study, we identify the exportin complex containing Nxf1 and Nxt1 as required for flam precursor nuclear export. Together with components of the exon junction complex (EJC), it is necessary for the efficient transfer of flam precursors away from their site of transcription. Indeed, depletion of these components greatly affects flam intra-nuclear transit. Moreover, we show that Yb-body assembly is dependent on the nucleo-cytoplasmic export of flam transcripts. These results suggest that somatic piRNA precursors are thus required for the assembly of the cytoplasmic transposon silencing machinery.

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Yb body assembly on the flamenco piRNA precursor transcripts reduces genic piRNA production

Project description:In Drosophila ovarian somatic cells, piRNAs against transposable elements are mainly produced from the ~180-kb flamenco (flam) locus. flam transcripts are gathered into one-two foci, located close to nuclear envelope, and processed into piRNAs in the cytoplasmic Yb bodies. The mechanism of Yb body formation remains unknown. Using RNA FISH, we found that in the follicle cells of ovaries the 5’-ends of flam transcripts are usually located in close proximity to the nuclear envelope and outside of Yb bodies, while their extended downstream regions mostly overlap with Yb bodies. In flamKG mutant ovaries, flam transcripts containing the 1st and, partially, 2nd exons but lacking downstream regions are gathered into foci at the nuclear envelope, but Yb bodies are not assembled. Strikingly, piRNA production from the protein-coding gene transcripts is enhanced in flamKG ovaries. We propose that normally in ovarian follicle cells flam downstream transcript regions function not only as a substrate for generation of piRNAs but also as a scaffold for Yb body assembly, which is necessary to minimize piRNA production from protein-coding gene transcripts. By contrast, in ovarian somatic cap and escort cells Yb body assembly does not require flam transcription.

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Yb body assembly on the flamenco piRNA precursor transcripts reduces genic piRNA production.

Project description:In Drosophila ovarian somatic cells, PIWI-interacting small RNAs (piRNAs) against transposable elements are mainly produced from the ?180-kb flamenco (flam) locus. flam transcripts are gathered into foci, located close to the nuclear envelope, and processed into piRNAs in the cytoplasmic Yb bodies. The mechanism of Yb body formation remains unknown. Using RNA fluorescence in situ hybridization, we found that in the follicle cells of ovaries the 5'-ends of flam transcripts are usually located in close proximity to the nuclear envelope and outside of Yb bodies, whereas their extended downstream regions mostly overlap with Yb bodies. In flamKG mutant ovaries, flam transcripts containing the first and, partially, second exons but lacking downstream regions are gathered into foci at the nuclear envelope, but Yb bodies are not assembled. Strikingly, piRNAs from the protein-coding gene transcripts accumulate at higher levels in flamKG ovaries indicating that piRNA biogenesis may occur without Yb bodies. We propose that normally in follicle cells, flam downstream transcript regions function not only as a substrate for generation of piRNAs but also as a scaffold for Yb body assembly, which competitively decreases piRNA production from the protein-coding gene transcripts. By contrast, in ovarian somatic cap and escort cells Yb body assembly does not require flam transcription.

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Requirement of Nuclear Pore Complex subunits for transposon silencing in Drosophila

Project description:The piRNA pathway is a small RNA-based immune system that silences mobile genetic elements in animal germlines. In Drosophila ovaries, piRNAs are produced from discrete genomic loci, called piRNA clusters, which are composed of inactive transposon copies and fragments and thus constitute a genetically encoded memory of past transposon challenges. Two types of piRNA clusters exist in flies: dual-strand clusters, expressed only in the germline via a highly specialised machinery, and uni-strand cluster, which are predominantly expressed in the somatic follicle cells. Flamenco (flam) is the major uni-strand piRNA cluster in Drosophila, giving rise to the majority of somatic piRNAs. Flam resembles a canonical RNA polymerase II transcriptional unit, nonetheless it can be specifically recognised by the piRNA pathway and directed to the biogenesis machinery. Recent work has implicated the RNA helicase Yb in the licensing of somatic piRNA production, however a detailed understanding of the molecular mechanisms underlying flam export and specification is still lacking. Here, we show that flam export triggers the assembly of peri-nuclear condensates of Yb and provide evidence that piRNA production from flam specifically requires subunits of the Nuclear Pore Complex (NPC). In the absence of some NPC subunits, transposons become de-silenced and piRNA biogenesis is compromised exclusively from flam. We also show that Yb transiently associates with the NPC to promote flam export. Taken together, our data shed light on how the export of uni-strand cluster transcripts is achieved and suggest the evolution of a specialised machinery that couples transcription, nuclear export and piRNA production.

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Requirement of Nuclear Pore Complex subunits for transposon silencing in Drosophila

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