Hierarchical roles of mitochondrial Papi and Zucchini in Bombyx germline piRNA biogenesis.

PIWI-interacting RNAs (piRNAs) are small regulatory RNAs that bind to PIWI proteins to control transposons and maintain genome integrity in animal germ lines. piRNA 3' end formation in the silkworm Bombyx mori has been shown to be mediated by the 3'-to-5' exonuclease Trimmer (Trim; known as PNLDC1 in mammals), and piRNA intermediates are bound with PIWI anchored onto mitochondrial Tudor domain protein Papi. However, it remains unclear whether the Zucchini (Zuc) endonuclease and Nibbler (Nbr) 3'-to-5' exonuclease, both of which have pivotal roles in piRNA biogenesis in Drosophila, are required for piRNA processing in other species. Here we show that the loss of Zuc in Bombyx had no effect on the levels of Trim and Nbr, but resulted in the aberrant accumulation of piRNA intermediates within the Papi complex, and that these were processed to form mature piRNAs by recombinant Zuc. Papi exerted its RNA-binding activity only when bound with PIWI and phosphorylated, suggesting that complex assembly involves a hierarchical process. Both the 5' and 3' ends of piRNA intermediates within the Papi complex showed hallmarks of PIWI 'slicer' activity, yet no phasing pattern was observed in mature piRNAs. The loss of Zuc did not affect the 5'- and 3'-end formation of the intermediates, strongly supporting the idea that the 5' end of Bombyx piRNA is formed by PIWI slicer activity, but independently of Zuc, whereas the 3' end is formed by the Zuc endonuclease. The Bombyx piRNA biogenesis machinery is simpler than that of Drosophila, because Bombyx has no transcriptional silencing machinery that relies on phased piRNAs.

Toll9 from Bombyx mori functions as a pattern recognition receptor that shares features with Toll-like receptor 4 from mammals.

Toll/Toll-like receptors (TLRs) are key regulators of the innate immune system in both invertebrates and vertebrates. However, while mammalian TLRs directly recognize pathogen-associated molecular patterns, the insect Toll pathway is thought to be primarily activated by binding Spätzle cytokines that are processed from inactive precursors in response to microbial infection. Phylogenetic and structural data generated in this study supported earlier results showing that Toll9 members differ from other insect Tolls by clustering with the mammalian TLR4 group, which recognizes lipopolysaccharide (LPS) through interaction with myeloid differentiation-2 (MD-2)-like proteins. Functional experiments showed that BmToll9 from the silkmoth Bombyx mori also recognized LPS through interaction with two MD-2-like proteins, previously named BmEsr16 and BmPP, that we refer to in this study as BmMD-2A and BmMD-2B, respectively. A chimeric BmToll9-TLR4 receptor consisting of the BmToll9 ectodomain and mouse TLR4 transmembrane and Toll/interleukin-1 (TIR) domains also activated LPS-induced release of inflammatory factors in murine cells but only in the presence of BmMD-2A or BmMD-2B. Overall, our results indicate that BmToll9 is a pattern recognition receptor for LPS that shares conserved features with the mammalian TLR4-MD-2-LPS pathway.

Role of autophagy in midgut stem cells of silkworm Bombyx mori, during larval-pupal metamorphosis.

Autophagy is a critical mechanism for the self-renewal, proliferation, and differentiation of stem cells. Bombyx mori midgut has stem cells that play a role in the regeneration of the larval epithelium in larval stages and the formation of the pupal midgut epithelium during larval-pupal metamorphosis. In this study, the role of the autophagy mechanism in midgut stem cells during the formation of the pupal midgut was investigated. For this purpose, two different doses of autophagy inhibitor chloroquine were administered to B. mori larvae on days 7 and 8 of the fifth larval stage. Morphological changes during the formation process of the pupal epithelium, expression levels of autophagy-related genes Atg8 and Atg12 in stem cells, and the amounts of lysosomal enzyme acid phosphatase were determined after the application. The obtained findings were evaluated in comparison with the control groups. Abnormalities in the formation of the pupal midgut after inhibition of autophagy showed the significance of the autophagy mechanism during this period.

Identification of LARK as a novel and conserved G-quadruplex binding protein in invertebrates and vertebrates.

Double-stranded DNAs are usually present in the form of linear B-form double-helix with the base pairs of adenine (A) and thymine (T) or cytosine (C) and guanine (G), but G-rich DNA can form four-stranded G-quadruplex (G4) structures, which plays important roles in transcription, replication, translation and protection of telomeres. In this study, a RNA recognition motif (RRM)-containing protein, BmLARK, was identified and demonstrated to bind G4 structures in the promoters of a transcription factor BmPOUM2 and other three unidentified genes of Bombyx mori, as well as three well-defined G4 structures in the human genes. Homologous LARKs from Bombyx mori, Drosophila melanogaster, Mus musculus and Homo sapiens bound G4 structures in BmPOUM2 and other genes in B. mori and H. sapiens. Upon binding, LARK facilitated the formation and stability of the G4 structure, enhancing the transcription of target genes. The G4 structure was visualized in vivo in cells and testis from invertebrate B. mori and vertebrate Chinese hamster ovary (CHO) cells. The results of this study strongly suggest that LARK is a novel and conserved G4-binding protein and that the G4 structure may have developed into an elaborate epigenetic mechanism of gene transcription regulation during evolution.

Bombyx mori nucleopolyhedrovirus F-like protein Bm14 is a type I integral membrane protein that facilitates ODV attachment to the midgut epithelial cells.

Our previous study showed that the Bombyx mori nucleopolyhedrovirus (BmNPV) F-like protein Bm14 is intrinsically related to the production of occlusion bodies, occlusion-derived virus (ODV) embedding and virulence in infected larvae. However, the exact mechanism by which Bm14 affects primary infection remains unknown. In this report, we characterized the detailed distribution and topology of Bm14 in occlusion bodies (OBs) and ODVs, and then further investigated the functional role of Bm14 in primary infection. A combination of Western blot and immunoelectron microscopy showed that Bm14 is mainly present on the surface of ODVs within OBs, but rarely in the OB matrix. Further phase separation and topology analysis of Bm14 by selective permeabilization revealed that Bm14 is a type I integral membrane protein with an N-terminus hidden in the endoplasmic reticulum (ER) lumen and a C-terminus exposed to the cytosol. In vivo assays demonstrated that the disruption of bm14 impaired the interactions of ODV with midgut epithelia, resulting in delayed spread in larval tissues. As the essential trigger of primary infection, some per os infectivity factors (PIFs) were verified to interact with Bm14 via a series of coimmunoprecipitation analyses. Further partially denaturing SDS-PAGE and BN-PAGE assays clearly showed that the deletion of bm14 did not affect the formation and presence of the PIF complex. In conclusion, Bm14 functions as a type I integral membrane protein to regulate ODV attachment to the midgut epithelial cells.

Efficient production of recombinant SARS-CoV-2 spike protein using the baculovirus-silkworm system.

In the case of a new viral disease outbreak, an immediate development of virus detection kits and vaccines is required. For COVID-19, we established a rapid production procedure for SARS-CoV-2 spike protein (S protein) by using the baculovirus-silkworm expression system. The baculovirus vector-derived S proteins were successfully secreted to silkworm serum, whereas those formed insoluble structure in the larval fat body and the pupal cells. The ectodomain of S protein with the native sequence was cleaved by the host furin-protease, resulting in less recombinant protein production. The S protein modified in furin protease-target site was efficiently secreted to silkworm serum and was purified as oligomers, which showed immunoreactivity for anti-SARS-CoV-2 S2 antibody. By using the direct transfection of recombinant bacmid to silkworms, we achieved the efficient production of SARS-CoV-2 S protein as fetal bovine serum (FBS)-free system. The resultant purified S protein would be useful tools for the development of immunodetection kits, antigen for immunization for immunoglobulin production, and vaccines.

The PIWI-Interacting RNA Molecular Pathway: Insights From Cultured Silkworm Germline Cells.

The PIWI-interacting RNA (piRNA) pathway, one of the major eukaryotic small RNA silencing pathways, is a genome surveillance system that silences selfish genes in animal gonads. piRNAs guide PIWI protein to target genes through Watson-Crick RNA-RNA base-parings. Loss of piRNA function causes genome instability, inducing failure in gametogenesis and infertility. Studies using fruit flies and mice as key experimental models have resulted in tremendous progress in understanding the mechanism underlying the piRNA pathway. Recent work using cultured silkworm germline cells has also expanded our knowledge of piRNA biogenesis in particular, since these silkworm cells are the only cells of germline origin that can be cultured. In this review, we describe elucidation of the piRNA pathway using cultured silkworm cells as an experimental model by focusing on recent work in biochemistry and structural biology. Earlier studies that made important contributions to the field are also described.

Bom-miR-2805 upregulates the expression of Bombyx mori fibroin light chain gene in vivo.

MicroRNAs (miRs) are inner regulatory RNAs mainly by regulating expression of genes at the posttranscriptional level. To investigate the regulatory function of Bombyx mori (B. mori) fibroin protein genes, the mRNA 3'-untranslated region (3'-UTR) of fibroin light chain gene (BmFib-L) was used as the target and one miRNA, miR-2805 was predicted by using the Software. miR-2805 expression plasmid pcDNA3.0[ie1-egfp-pre-miR-2805-SV40] and BmFib-L 3'-UTR plasmid pGL3.0[A3-luc-Fib-L-3'-UTR-SV40] were constructed, respectively. The mentioned plasmids were cotransfected in BmN cells, and the regulatory function of miR-2805 on BmFib-L was detected by assay of dual luciferase activities, as well as synthesized mimic and inhibitor of miR-2805. The results revealed that miR-2805 significantly downregulated the expression of BmFib-L in BmN cells. To validate the function of miR-2805 in vivo, cultured silk glands or larvae were injected with solution containing pcDNA3.0[ie1-egfp-SV40], pcDNA3.0[ie1-egfp-pre-miR-2805-SV40], mimic, inhibitor respectively. BmFib-L expression was analyzed by quantitative reverse transcription polymerase chain reaction using total RNAs extracted from silk glands. The results showed that miR-2805 significantly upregulated the expression of BmFib-L in both cultured tissues and individuals. To find out how miR-2805 differentially regulates BmFib-L expression in cells and tissues or individuals, we analyzed the expression level of transcription factors (TFs) involved in expression of silk protein genes. The results showed that miR-2805 upregulated the expression of TFs BmAwh and Bmdimm. These results suggest that miR-2805 may up-regulate the expression of BmFib-L interaction with BmAwh and/or Bmdimm in vivo. These findings are beneficial to clarify the molecular mechanism of miRNAs in regulating B. mori silk protein biosynthesis.

Development of SpyTag/SpyCatcher-Bacmid Expression Vector System (SpyBEVS) for Protein Bioconjugations Inside of Silkworms.

Protein conjugations at post-translational levels are known to be essential to protein stability and function. Recently, it has been proven that the split protein CnaB2 (SpyTag/SpyCatcher, ST/SC) from Streptococcus pyogenes can induce covalent conjugation rapidly and efficiently under various conditions. The protein of interest fused with the split protein SC/ST could be assembled spontaneously. In light of this finding, we introduced the ST/SC protein coupling concept into the silkworm-bacmid protein expression system (SpyBEVS). As a proof of concept, we first examined and confirmed that a competent ligation occurred between ST/SC-fused protein partners in vitro in cultured silkworm cells and in vivo in silkworm larvae by co-infection of several recombinant baculoviruses. The protein conjugation could be also achieved sufficiently by a simple one-step mixture of purified ST/SC-tagged peptide-protein pairs in vitro. Given the flexibility and robustness of silkworm-BEVS, our results on SpyBEVS show an alternative method for enabling the production of protein decorations in vitro and inside of silkworms.

Bombyx mori nucleopolyhedrovirus ORF40 is essential for budded virus production and occlusion-derived virus envelopment.

ORF40 (bm40) of the Bombyx mori nucleopolyhedrovirus (BmNPV) encodes a homologue of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) AC51 and is a highly conserved gene in sequenced alphabaculoviruses. To investigate the role of bm40 in the baculovirus infection cycle, a bm40 knockout BmNPV bacmid was constructed via homologous recombination in Escherichia coli. Western blotting analysis revealed that bm40 is a late gene during virus infection. Compared with wild-type and repair viruses, the knockout virus exhibited a single-cell infection phenotype. Titration assays confirmed that no infectious budded viruses (BVs) were produced due to the bm40 deletion, while there was no effect on viral DNA replication. Electron microscopy revealed that Bm40 is required for nucleocapsid egress from the nucleus to the cytoplasm, nucleocapsid envelopment to form occlusion-derived viruses (ODVs) and subsequent embedding of ODVs into polyhedra. Confocal microscopy showed that Bm40 was predominantly localized in the nuclei from 48 h post-infection and subsequently condensed on the nuclear membrane and polyhedra at the late phase of infection. Taken together, these results demonstrate that Bm40 plays an essential role in BV production and ODV envelopment in the BmNPV infection cycle.

Secreted glycoprotein BmApoD1 plays a critical role in anti-oxidation and anti-apoptosis in Bombyx mori.

Recent studies highlighted that apolipoprotein D (ApoD) and its homologs exert neuroprotective and antioxidant functions in mammals and Drosophila. Unlike mammals and Drosophila, lepidopteran insects possess three distinct ApoD homologs. However, few information on their functions in lepidopteran insects are available. In this study, we investigated the protective potential of a novel ApoD homolog, BmApoD1, in Bombyx mori. Quantitative PCR analyses demonstrated that BmApoD1 is extensively expressed at low levels during the larval stage but abundantly expressed in the testis during the pupal and adult stages. Tryptophan fluorescence titration demonstrated that recombinant BmApoD1 protein can bind retinoic acid and ergosterol. In addition, we provided evidence that N-linked glycans of BmApoD1 are essential to BmApoD1 secretion, and three residues, namely, Asp69, Asp104, and Asp196, are the glycosylation sites of BmApoD1. Furthermore, we showed that BmApoD1 is significantly up-regulated in the larvae after oxidant or starvation treatment. The recombinant BmApoD1 protein can protect cells from oxidative stress induced by H2O2 and reduce actinomycin D-induced cell apoptosis. These observations, together with the transcriptional up-regulation of BmApoD1 in several tissues upon oxidative insult, identify BmApoD1 as a potent antioxidant. Our results demonstrate that BmApoD1 is critical for metabolic adaptation of B. mori to environmental challenges.

Vitellogenin receptor selectively endocytoses female-specific and highly-expressed hemolymph proteins in the silkworm, Bombyx mori.

VgR, a member of the LDLR family, functions to transport vitellogenin into the ovaries to protome ovarian growth and embryonic development. In insects, the only widely accepted ligand of VgR is Vg. Recently, BmVgR has been shown to interact with BmSP1 in vitro. Therefore, in this study, we evaluated whether BmVgR could transport BmSP1 into certain cells. Although BmVgR could combine with BmVg and BmSP1, BmVgR did not affect the amount of BmSP1 taken up by Sf9 cells. Parallel immunofluorescence showed that most BmVg and BmVgR were localized in the inner oocyte membrane, showing tissue localization similar to that of BmVg labeled with pHrodo Red absorbed by the ovaries on day 2 of pupation. Although BmSP1 showed localization similar to BmVgR during the same phase, little BmSP1 was present in the ovary. Additionally, BmSP1 did not exist in ovaries when the ovaries contained BmVgR on day 5 of pupation, suggesting that BmSP1 in the ovaries was not endocytosed by BmVgR. In summary, BmVgR could facilitate uptake of BmVg by developing oocytes, but did not modulate in the transport of BmSP1.

Characterization of Armitage and Yb containing granules and their relationship to nuage in ovary-derived cultured silkworm cell.

PIWI-interacting RNAs (piRNAs) are a class of endogenous small non-coding RNAs, which are mostly 24-32 nucleotides in length and interact specifically with PIWI subfamily of argonaute proteins. Despite the significant research progress in germ line piRNA pathway, its role in somatic cell is not well known. In Drosophila ovarian somatic cell, maturation of primary piRNA and its loading onto Piwi occurs at perinuclear Yb body. The Armitage (Armi) and Yb proteins are the major components of Yb body and specially expressed in ovarian somatic cell. Based on the reports, here we studied the BmArmi and BmYb in Bombyx mori ovary-derived BmN4 cells expressing BmVasa. In this study, we show that BmArmi and BmYb co-localized with BmVasa at nuage. The helicase domains of BmArmi and BmYb are important for nuage localization. Moreover, RNAi of piRNA components reveal that BmArmi depend on BmAgo3 for nuage localization, and BmArmi and BmYb form cytoplasmic granules independently in the absence of BmVasa. Our results provide evidence that the BmArmi and BmYb coexist with BmVasa and play an important role in perinuclear nuage granules formation in ovary-derived BmN4 cell.

Molecular characterization of mitochondrial Zucchini and its relation to nuage-piRNA pathway components in Bombyx mori ovary-derived BmN4 cells.

Piwi-interacting RNAs (piRNAs) are a class of small non-coding RNAs that associate with PIWI subfamily proteins, which play an important role in transposon silencing in animal germ cell. The piRNAs biogenesis is divided into two major pathways: primary and secondary, and both pathways are independent of double-stranded RNA-processing enzyme Dicer, which processes the single-stranded RNA transcripts in microRNA (miRNA) and siRNA (small interfering RNA) pathway. Primary piRNAs are processed from long non-coding RNA precursors transcribed from piRNA clusters. Zucchini (Zuc), a mitochondrial phospholipase D (PLD) superfamily protein is conserved among the animals and involved in piRNA biogenesis. Recent studies showed that the Zucchini is an endoribonuclease essential for primary piRNA maturation and production of phased piRNA in secondary piRNA biogenesis of drosophila germ cell. Based on these reports, here we identified and studied the silkworm Zucchini (BmZuc) at subcellular level in ovary-derived BmN4 cell. The silkworm Zuc specifically expressed in germ-related tissues and localized on mitochondria and partially co-localized with perinuclear nuage-piRNA pathway components and nuage marker protein BmVasa. Molecular dissection analyses revealed that the conserved mitochondrial localization sequence, RGV motif, PLDc 2 domain and HKD motif are important for the BmZuc mitochondrial localization. Moreover, the knockdown analyses showed that the piRNA pathway components are independent on BmZuc for their nuage localization, whereas BmZuc depend on piRNA pathway components for the proper localization. Our data provides vital information on mitochondrial BmZuc and its relationship to "nuage" in ovary-derived BmN4 cell.

Proteasome inhibitor MG132 impairs autophagic flux through compromising formation of autophagosomes in Bombyx cells.

MG132 has been used as a proteasome inhibitor on Bombyx cells, but its physiological effects on autophagy still have not been elucidated. In this study, we find that the lipidated BmAtg8, BmAtg8-PE as an autophagosomal marker protein, is only localized to membranes. Then we established systems to monitor autophagic flux in Bombyx cells: Induction of autophagy reduces exogenous BmAtg8 and exogenous BmAtg8-PE, facilitates formation of autophagosomes indicated by green EGFP-BmAtg8 puncta after cotreatment by Rapamycin and Bafilomycin A1, and causes accumulation of free EGFP from EGFP-BmAtg8 cleavage in autolysosomes. Using these established systems, we find that exposure of MG132 inhibits both basal and Rapamycin-induced autophagy when polyubiquitinated proteins are accumulated markedly in Bombyx cells. Interestingly, we reveal that attenuation of autophagy in these cells is ascribed as distinct suppression of formation of autophagosomes after MG132 treatment.

Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture.

A somatic transformation vector, pDP9, was constructed that provides a simplified means of producing permanently transformed cultured insect cells that support high levels of protein expression of foreign genes. The pDP9 plasmid vector incorporates DNA sequences from the Junonia coenia densovirus that are involved in integration of the densovirus in insect cell chromosomes and a promoter/enhancer system that results in high levels of expression. The plasmid also contains two markers that permit selection of transformed insect cells by antibiotic resistance or by cell-sorting for fluorescent protein expression. Transformation of Bombyx mori Bm5 or Spodoptera frugiperda Sf9 cultured cells with the pDP9 vectors results in the integration of the pDP9 plasmid into genomic DNA of Bm5 and Sf9 cells. pDP9 contains a multiple cloning site (MCS) 3' of the densoviral P9 promoter and insertion of a protein coding sequence within the MCS results in high level expression by pDP9 transformed cells. P9 driven transcription in the pDP9 transformed Sf9 cells produced foreign gene transcript levels that were 30 fold higher than actin 3 driven transgenes and equivalent to hr5IE1 driven transgenes. The pDP9 vector transformation results in the efficient selection of clones for assessment of promoter activity.

Two types of local interneurons are distinguished by morphology, intrinsic membrane properties, and functional connectivity in the moth antennal lobe.

Neurons in the silkmoth antennal lobe (AL) are well characterized in terms of their morphology and odor-evoked firing activity. However, their intrinsic electrical properties including voltage-gated ionic currents and synaptic connectivity remain unclear. To address this, whole cell current- and voltage-clamp recordings were made from second-order projection neurons (PNs) and two morphological types of local interneurons (LNs) in the silkmoth AL. The two morphological types of LNs exhibited distinct physiological properties. One morphological type of LN showed a spiking response with a voltage-gated sodium channel gene expression, whereas the other type of LN was nonspiking without a voltage-gated sodium channel gene expression. Voltage-clamp experiments also revealed that both of two types of LNs as well as PNs possessed two types of voltage-gated potassium channels and calcium channels. In dual whole cell recordings of spiking LNs and PNs, activation of the PN elicited depolarization responses in the paired spiking LN, whereas activation of the spiking LN induced no substantial responses in the paired PN. However, simultaneous recording of a nonspiking LN and a PN showed that activation of the nonspiking LN induced hyperpolarization responses in the PN. We also observed bidirectional synaptic transmission via both chemical and electrical coupling in the pairs of spiking LNs. Thus our results indicate that there were two distinct types of LNs in the silkmoth AL, and their functional connectivity to PNs was substantially different. We propose distinct functional roles for these two different types of LNs in shaping odor-evoked firing activity in PNs.

Identification of a transformer homolog in the acorn worm, Saccoglossus kowalevskii, and analysis of its activity in insect cells.

The transformer (tra) gene is an intermediate component of the sex determination hierarchy in many insect species. The homolog of tra is also found in two branchiopod crustacean species but is not known outside arthropods. We have isolated a tra homolog in the acorn worm, Saccoglossus kowalevskii, which is a hemichordate belonging to the deuterostome superphylum. The full-length complementary DNA (cDNA) of the S. kowalevskii tra homolog (Sktra) has a 3786-bp open reading frame that encodes a 1261-amino acid sequence including a TRA-CAM domain and an arginine/serine (RS)-rich domain, both of which are characteristic of TRA orthologs. Reverse transcription PCR (RT-PCR) analyses demonstrated that Sktra showed no differences in expression patterns between testes and ovaries, but its expression level was approximately 7.5-fold higher in the testes than in the ovaries. TRA, together with the protein product of the transformer-2 (tra-2) gene, assembles on doublesex (dsx) pre-messenger RNA (mRNA) via the cis-regulatory element, enhancing female-specific splicing of dsx in Drosophila. To understand functional conservation of the SkTRA protein as a dsx-splicing activator, we investigated whether SkTRA is capable of inducing female-specific splicing of the Drosophila dsx. Ectopic expression of Sktra cDNA in insect cultured cells did not induce the female-specific splicing of dsx. On the other hand, forced expression of Sktra-2 (a tra-2 homolog of S. kowalevskii) was able to induce the female-specific dsx splicing. These results demonstrate that the function as a dsx-splicing activator is not conserved in SkTRA even though SkTRA-2 is capable of functionally replacing the Drosophila TRA-2. We have also found a tra homolog in an echinoderm genome. This study provides the first evidence that that tra is conserved not only in arthropods but also in basal species of deuterostoms.

Remobilizing deleted piggyBac vector post-integration for transgene stability in silkworm.

Deletion of transposable elements post-genomic integration holds great promise for stability of the transgene in the host genome and has an essential role for the practical application of transgenic animals. In this study, a modified piggyBac vector that mediated deletion of the transposon sequence post-integration for transgene stability in the economically important silkworm Bombyx mori was constructed. The piggyBac vector architecture contains inversed terminal repeat sequences L1, L2 and R1, which can form L1/R1 and L2/R1 types of transposition cassettes. hsp70-PIG as the piggyBac transposase expression cassette for initial transposition, further remobilization and transgene stabilization test was transiently expressed in a helper vector or integrated into the modified vector to produce a transgenic silkworm. Shortening L2 increased the transformation frequency of L1/R1 into the silkworm genome compared to L2/R1. After the integration of L1/R1 into the genome, the remobilization of L2/R1 impaired the transposon structure and the resulting transgene linked with an impaired transposon was stable in the genome even in the presence of exogenously introduced transposase, whereas those flanked by the intact transposon were highly mobile in the genome. Our results demonstrated the feasibility of post-integration deletion of transposable elements to guarantee true transgene stabilization in silkworm. We suggest that the modified vector will be a useful resource for studies of transgenic silkworms and other piggyBac-transformed organisms.

Hsp90 facilitates accurate loading of precursor piRNAs into PIWI proteins.

PIWI-interacting RNAs (piRNAs) defend the genome against transposon activity in animal gonads. The Hsp90 chaperone machinery has been implicated in the piRNA pathway, but its exact role remains obscure. Here, we examined the effect of 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), an Hsp90-specific inhibitor, on the piRNA pathway. In the silkworm ovary-derived BmN4 cells, 17-AAG treatment reduced the level of piRNAs and PIWI proteins. In vitro, the 5'-nucleotide preference upon precursor piRNA loading was compromised by 17-AAG, whereas 3'-end trimming and 2'-O-methylation were unaffected. Our data highlight a role of Hsp90 in accurate loading of precursor piRNAs into PIWI proteins.