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Siwi levels reversibly regulate secondary piRISC biogenesis by affecting Ago3 body morphology in Bombyx mori

ABSTRACT: Silkworm ovarian germ cells produce the Siwi-piRNA-induced silencing complex (piRISC) through two consecutive mechanisms, the primary pathway and the secondary ping-pong cycle. Primary Siwi-piRISC production occurs on the outer mitochondrial membrane in an Ago3-independent manner, where Tudor domain-containing Papi binds unloaded Siwi via its symmetrical dimethylarginines (sDMAs). Here, we now show that secondary Siwi-piRISC production occurs at the Ago3-positive nuage Ago3 bodies, in an Ago3-dependent manner, where Vreteno (Vret), another Tudor protein, interconnects unloaded Siwi and Ago3-piRISC through their sDMAs. Upon Siwi depletion, Ago3 is phosphorylated and insolubilized in its piRISC form with cleaved RNAs and Vret, suggesting that the complex is stalled in the intermediate state. The Ago3 bodies are also enlarged. The aberrant morphology is restored upon Siwi re-expression without Ago3-piRISC supply. Thus, Siwi depletion aggregates the Ago3 bodies to protect the piRNA intermediates from degradation until the normal cellular environment returns to re-initiate the ping-pong cycle. Overall, these findings reveal a unique regulatory mechanism controlling piRNA biogenesis.

SUBMITTER: Dr. Kazumichi, M Nishida

PROVIDER: S-SCDT-EMBOJ-2020-105130 | biostudies-other |

REPOSITORIES: biostudies-other

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Siwi levels reversibly regulate secondary piRISC biogenesis by affecting Ago3 body morphology in Bombyx mori.

Project description:Silkworm ovarian germ cells produce the Siwi-piRNA-induced silencing complex (piRISC) through two consecutive mechanisms, the primary pathway and the secondary ping-pong cycle. Primary Siwi-piRISC production occurs on the outer mitochondrial membrane in an Ago3-independent manner, where Tudor domain-containing Papi binds unloaded Siwi via its symmetrical dimethylarginines (sDMAs). Here, we now show that secondary Siwi-piRISC production occurs at the Ago3-positive nuage Ago3 bodies, in an Ago3-dependent manner, where Vreteno (Vret), another Tudor protein, interconnects unloaded Siwi and Ago3-piRISC through their sDMAs. Upon Siwi depletion, Ago3 is phosphorylated and insolubilized in its piRISC form with cleaved RNAs and Vret, suggesting that the complex is stalled in the intermediate state. The Ago3 bodies are also enlarged. The aberrant morphology is restored upon Siwi re-expression without Ago3-piRISC supply. Thus, Siwi depletion aggregates the Ago3 bodies to protect the piRNA intermediates from degradation until the normal cellular environment returns to re-initiate the ping-pong cycle. Overall, these findings reveal a unique regulatory mechanism controlling piRNA biogenesis.

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Bombyx mori Vps13d is a key gene affecting silk yield.

Project description:Bombyx mori is an important economic insect, its economic value mainly reflected in the silk yield. The major functional genes affecting the silk yield of B. mori have not been determined yet. Bombyx mori vacuolar protein sorting-associated protein 13d (BmVps13d) has been identified, but its function is not reported. In this study, BmVps13d protein shared 30.84% and 34.35% identity with that of in Drosophila melanogaster and Homo. sapiens, respectively. The expressions of BmVps13d were significantly higher in the midgut and silk gland of JS (high silk yield) than in that of L10 (low silk yield). An insertion of 9 bp nucleotides and two deficiencies of adenine ribonucleotides in the putative promoter region of BmVps13d gene in L10 resulted in the decline of promoter activity was confirmed using dual luciferase assay. Finally, the functions of BmVps13d in B. mori were studied using the CRISPR/Cas9 system, and the mutation of BmVps13d resulted in a 24.7% decline in weight of larvae, as well as a 27.1% (female) decline and a 11.8% (male) decline in the silk yield. This study provides a foundation for studying the molecular mechanism of silk yield and breeding the silkworm with high silk yield.

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Two Geminin homologs regulate DNA replication in silkworm, Bombyx mori.

Project description:DNA replication is rigorously controlled in cells to ensure that the genome duplicates exactly once per cell cycle. Geminin is a small nucleoprotein, which prevents DNA rereplication by directly binding to and inhibiting the DNA replication licensing factor, Cdt1. In this study, we have identified 2 Geminin genes, BmGeminin1 and BmGeminn2, in silkworm, Bombyx mori. These genes contain the Geminin conserved coiled-coil domain and are periodically localized in the nucleus during the S-G2 phase but are degraded at anaphase in mitosis. Both BmGeminin1 and BmGeminin2 are able to homodimerize and interact with BmCdt1 in cells. In addition, BmGeminin1 and BmGeminin2 can interact with each other. Overexpression of BmGeminin1 affects cell cycle progression: cell cycle is arrested in S phase, and RNA interference of BmGeminin1 leads to rereplication. In contrast, overexpression or knockdown of BmGeminin2 with RNAi did not significantly affect cell cycle, while more rereplication occurred when BmGeminin1 and BmGeminin2 together were knocked down in cells than when only BmGeminin1 was knocked down. These data suggest that both BmGeminin1 and BmGeminin2 are involved in the regulation of DNA replication. These findings provide insight into the function of Geminin and contribute to our understanding of the regulation mechanism of cell cycle in silkworm.

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Enteroendocrine peptides regulate feeding behavior via controlling intestinal contraction of the silkworm Bombyx mori.

Project description:Our previous study demonstrated that predominant feeding inhibitory effects were found in the crude extracts of foregut and midgut of the silkworm Bombyx mori larvae. To address the entero-intestinal control crucial for the regulation of insect feeding behavior, the present study identified and functionally characterized feeding inhibitory peptides from the midgut of B. mori larvae. Purification and structural analyses revealed that the predominant inhibitory factors in the crude extracts were allatotropin (AT) and GSRYamide after its C-terminal sequence. In situ hybridization revealed that AT and GSRYamide were expressed in enteroendocrine cells in the posterior and anterior midgut, respectively. Receptor screening using Ca2+-imaging technique showed that the B. mori neuropeptide G protein-coupled receptor (BNGR)-A19 and -A22 acted as GSRYamide receptors and BNGR-A5 acted as an additional AT receptor. Expression analyses of these receptors and the results of the peristaltic motion assay indicated that these peptides participated in the regulation of intestinal contraction. Exposure of pharynx and ileum to AT and GSRYamide inhibited spontaneous contraction in ad libitum-fed larvae, while exposure of pharynx to GSRYamide did not inhibit contraction in non-fed larvae, indicating that the feeding state changed their sensitivity to inhibitory peptides. These different responses corresponded to different expression levels of their receptors in the pharynx. In addition, injection of AT and GSRYamide decreased esophageal contraction frequencies in the melamine-treated transparent larvae. These findings strongly suggest that these peptides exert feeding inhibitory effects by modulating intestinal contraction in response to their feeding state transition, eventually causing feeding termination.

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MiR-2 family targets awd and fng to regulate wing morphogenesis in Bombyx mori.

Project description:MicroRNAs (miRNAs) are post-transcriptional regulators that target specific mRNAs for repression and thus play key roles in many biological processes, including insect wing morphogenesis. miR-2 is an invertebrate-specific miRNA family that has been predicted in the fruit fly, Drosophila melanogaster, to be involved in regulating the Notch signaling pathway. We show here that miR-2 plays a critical role in wing morphogenesis in the silkworm, Bombyx mori, a lepidopteran model insect. Transgenic over-expression of a miR-2 cluster using a Gal4/UAS system results in deformed adult wings, supporting the conclusion that miR-2 regulates functions essential for normal wing morphogenesis. Two genes, abnormal wing disc (awd) and fringe (fng), which are positive regulators in Notch signaling, are identified as miR-2 targets and validated by a dual-luciferase reporter assay. The relative abundance of both awd and fng expression products was reduced significantly in transgenic animals, implicating them in the abnormal wing phenotype. Furthermore, somatic mutagenesis analysis of awd and fng using the CRISPR/Cas9 system and knock-out mutants also resulted in deformed wings similar to those observed in the miR-2 overexpression transgenic animals. The critical role of miR-2 in Bombyx wing morphogenesis may provide a potential target in future lepidopteran pest control.

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Circadian Clock Genes Regulate Temperature-Dependent Diapause Induction in Silkworm <i>Bombyx mori</i>.

Project description:The bivoltine strain of the domestic silkworm, Bombyx mori, exhibits a facultative diapause phenotype that is determined by maternal environmental conditions during embryonic and larval development. Although a recent study implicated a circadian clock gene period (per) in circadian rhythms and photoperiod-induced diapause, the roles of other core feedback loop genes, including timeless (tim), Clock (Clk), cycle (cyc), and cryptochrome2 (cry2), have to be clarified yet. Therefore, the aim of this study was to elucidate the roles of circadian clock genes in temperature-dependent diapause induction. To achieve this, per, tim, Clk, cyc, and cry2 knockout (KO) mutants were generated, and the percentages of diapause and non-diapause eggs were determined. The results show that per, tim, Clk, cyc, and cry2 regulated temperature-induced diapause by acting upstream of cerebral γ-aminobutyric acid (GABA)ergic and diapause hormone signaling pathways. Moreover, the temporal expression of the clock genes in wild-type (wt) silkworms was significantly different from that of thermosensitive transient receptor potential ankyrin 1 (TRPA1) KO mutants during embryonic development. Overall, the findings of this study provide target genes for regulating temperature-dependent diapause induction in silkworms.

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Increasing cell density globally enhances the biogenesis of Piwi-interacting RNAs in Bombyx mori germ cells.

Project description:Piwi proteins and their bound Piwi-interacting RNAs (piRNAs) are predominantly expressed in the germline and play crucial roles in germline development by silencing transposons and other targets. Bombyx mori BmN4 cells are culturable germ cells that equip the piRNA pathway. Because of the scarcity of piRNA-expressing culturable cells, BmN4 cells are being utilized for the analyses of piRNA biogenesis. We here report that the piRNA biogenesis in BmN4 cells is regulated by cell density. As cell density increased, the abundance of Piwi proteins and piRNA biogenesis factors was commonly upregulated, resulting in an increased number of perinuclear nuage-like granules where Piwi proteins localize. Along with these phenomena, the abundance of mature piRNAs also globally increased, whereas levels of long piRNA precursor and transposons decreased, suggesting that increasing cell density promotes piRNA biogenesis pathway and that the resultant accumulation of mature piRNAs is functionally significant for transposon silencing. Our study reveals a previously uncharacterized link between cell density and piRNA biogenesis, designates cell density as a critical variable in piRNA studies using BmN4 cell system, and suggests the alteration of cell density as a useful tool to monitor piRNA biogenesis and function.

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An extended Tudor domain within Vreteno interconnects Gtsf1L and Ago3 for piRNA biogenesis in Bombyx mori.

Project description:Piwi-interacting RNAs (piRNAs) direct PIWI proteins to transposons to silence them, thereby preserving genome integrity and fertility. The piRNA population can be expanded in the ping-pong amplification loop. Within this process, piRNA-associated PIWI proteins (piRISC) enter a membraneless organelle called nuage to cleave their target RNA, which is stimulated by Gtsf proteins. The resulting cleavage product gets loaded into an empty PIWI protein to form a new piRISC complex. However, for piRNA amplification to occur, the new RNA substrates, Gtsf-piRISC, and empty PIWI proteins have to be in physical proximity. In this study, we show that in silkworm cells, the Gtsf1 homolog BmGtsf1L binds to piRNA-loaded BmAgo3 and localizes to granules positive for BmAgo3 and BmVreteno. Biochemical assays further revealed that conserved residues within the unstructured tail of BmGtsf1L directly interact with BmVreteno. Using a combination of AlphaFold modeling, atomistic molecular dynamics simulations, and in vitro assays, we identified a novel binding interface on the BmVreteno-eTudor domain, which is required for BmGtsf1L binding. Our study reveals that a single eTudor domain within BmVreteno provides two binding interfaces and thereby interconnects piRNA-loaded BmAgo3 and BmGtsf1L.

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Morphology and physiology of olfactory neurons in the lateral protocerebrum of the silkmoth Bombyx mori.

Project description:Insect olfaction is a suitable model to investigate sensory processing in the brain. Olfactory information is first processed in the antennal lobe and is then conveyed to two second-order centres-the mushroom body calyx and the lateral protocerebrum. Projection neurons processing sex pheromones and plant odours supply the delta area of the inferior lateral protocerebrum (?ILPC) and lateral horn (LH), respectively. Here, we investigated the neurons arising from these regions in the brain of the silkmoth, Bombyx mori, using mass staining and intracellular recording with a sharp glass microelectrode. The output neurons from the ?ILPC projected to the superior medial protocerebrum, whereas those from the LH projected to the superior lateral protocerebrum. The dendritic innervations of output neurons from the ?ILPC formed a subdivision in the ?ILPC. We discuss pathways for odour processing in higher order centres.

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Infectious Virions of Bombyx Mori Latent Virus Are Incorporated into Bombyx Mori Nucleopolyhedrovirus Occlusion Bodies.

Project description:The Bombyx mori latent virus (BmLV) belongs to the unassigned plant virus family Tymoviridae and contains a positive-sense, single-stranded RNA genome. BmLV has infected almost all B. mori-derived cultured cell lines through unknown routes. The source of BmLV infection and the BmLV life cycle are still unknown. Here, we examined the interaction between BmLV and the insect DNA virus Bombyx mori nucleopolyhedrovirus (BmNPV). Persistent infection with BmLV caused a slight delay in BmNPV propagation, and BmLV propagation was enhanced in B. mori larvae via co-infection with BmNPV. We also showed that BmLV infectious virions were co-occluded with BmNPV virions into BmNPV occlusion bodies. We propose a new relationship between BmLV and BmNPV.

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