Role of small nucleolar RNAs in alternative splicing of the doublesex gene in the silkworm, Bombyx mori.

More and more evidence shows that small noncoding RNAs (ncRNAs) play diverse roles in development, stress response and other cellular processes, but functional study of intermediate-size ncRNAs is still rare. Here, the expression profile of 16 intermediate-size ncRNAs in ovary and testis of silkworm Bombyx mori were analyzed. Twelve ncRNAs, including 5 small nucleolar RNAs (snoRNAs) and 7 unclassified ncRNAs, accumulated more in the testis than in the ovary of silkworm, especially Bm-163, Bm-51 and Bm-68. Four ncRNAs (including three orphan snoRNAs and one unclassified ncRNA) had higher expression level in the ovary than in the testis, especially Bm-86. Overexpression of the testis-enriched snoRNA Bm-68 in the female led to the accumulation of male-specific isoform of doublesex (BmdsxM) and increased the expression ratio of BmdsxM: BmdsxF. While overexpression of ovary-enriched snoRNA Bm-86 in the male decreased the expression ratio of BmdsxM: BmdsxF, indicating the roles of the two snoRNAs played in the alternative splicing of Bmdsx of silkworm, which will provide new clues for the functional study of snoRNAs in insects.

Long Noncoding RNA lncR17454 Regulates Metamorphosis of Silkworm Through let-7 miRNA Cluster.

A number of long noncoding RNAs (lncRNAs) have been identified in silkworm, but little is known about their functions. Recent study showed that the let-7 miRNA cluster (contains let-7, miR-2795, and miR-100) was transcribed from the last exon of lncRNA lncR17454 in silkworm. To investigate the functional role of lncR17454, dsRNAs of lncR17454 were injected into the hemolymph of 1-d-old third-instar larvae of Bombyx mori, repression of lncR17454 led to molting arrestment during the larval-larval and larval-pupal transition of silkworm, which was consistent to the result as let-7 knockdown in other studies. The expression level of mature let-7, miR-100, and miR-2795 decreased 40%, 36%, and 40%, respectively, while the mRNA level of two predicted target genes of let-7, the Broad Complex isoform 2 (BR-C-Z2) and the BTB-Zinc finger transcription repression factor gene Abrupt (Ab), increased significantly after lncR17454 knockdown. In contrast, when adding the 20-Hydroxyecdysone (20E) to silkworm BmN4 cell lines, the expression level of lncR17454 and let-7 cluster all increased significantly, but the expression of Abrupt, the predicted target gene of let-7, was repressed. Dual-luciferase reporter assays confirmed Abrupt was the real target of let-7. Here we found that the lncRNA lncR17454 can play regulator roles in the metamorphosis of silkworm through let-7 miRNA cluster and the ecdysone signaling pathway, which will provide new clues for lepidopteran pest control.

Transcriptome analysis reveals potential function of long non-coding RNAs in 20-hydroxyecdysone regulated autophagy in Bombyx mori.

BACKGROUND: 20-hydroxyecdysone (20E) plays important roles in insect molting and metamorphosis. 20E-induced autophagy has been detected during the larval-pupal transition in different insects. In Bombyx mori, autophagy is induced by 20E in the larval fat body. Long non-coding RNAs (lncRNAs) function in various biological processes in many organisms, including insects. Many lncRNAs have been reported to be potential for autophagy occurrence in mammals, but it has not been investigated in insects. RESULTS: RNA libraries from the fat body of B. mori dissected at 2 and 6 h post-injection with 20E were constructed and sequenced, and comprehensive analysis of lncRNAs and mRNAs was performed. A total of 1035 lncRNAs were identified, including 905 lincRNAs and 130 antisense lncRNAs. Compared with mRNAs, lncRNAs had longer transcript length and fewer exons. 132 lncRNAs were found differentially expressed at 2 h post injection, compared with 64 lncRNAs at 6 h post injection. Thirty differentially expressed lncRNAs were common at 2 and 6 h post-injection, and were hypothesized to be associated with the 20E response. Target gene analysis predicted 6493 lncRNA-mRNA cis pairs and 42,797 lncRNA-mRNA trans pairs. The expression profiles of LNC_000560 were highly consistent with its potential target genes, Atg4B, and RNAi of LNC_000560 significantly decreased the expression of LNC_000560 and Atg4B. These results indicated that LNC_000560 was potentially involved in the 20E-induced autophagy of the fat body by regulating Atg4B. CONCLUSIONS: This study provides the genome-wide identification and functional characterization of lncRNAs associated with 20E-induced autophagy in the fat body of B. mori. LNC_000560 and its potential target gene were identified to be related to 20-regulated autophagy in B. mori. These results will be helpful for further studying the regulatory mechanisms of lncRNAs in autophagy and other biological processes in this insect model.

Potato virus X-mediated constitutive expression of Plutella xylostella PxSDF2L1 gene in Nicotiana benthamiana confers resistance to Phytophthora parasitica var. nicotianae.

BACKGROUND: The Plutella xylostella PxSDF2L1 gene was previously reported to enhance insect resistance to pathogen at high basal transcription rate. PxSDF2L1 shows similitude with the stromal cell-derived factor 2 (SDF2), an ER stress-induced chaperon protein that is highly conserved throughout animals and plants. The precise biological function of SDF2 is not clear, but its expression is required for innate immunity in plants. Here, we investigate whether a continuous expression of PxSDF2L1 in Nicotiana benthamiana can similarly confer resistance to plant pathogen, particularly, the black shank Phytophthora parasitica var. nicotianae. RESULTS: The N. benthamiana plants were inoculated with agrobacteria transformed with a PVX-based binary vector carrying the PxSDF2L1 gene; similar agroinoculation experiments with a PVX vector carrying the GFP gene were used for controls. In pot trials, agroinfected N. benthamiana plants constitutively expressing PxSDF2L1 showed a significant reduction of stem disease symptoms caused by the inoculation with P. parasitica, compared with controls. CONCLUSIONS: We confirm a role of PxSDF2L1 in resistance to black shank, with a potential application to engineering active resistance against this oomycete in the commercial N. tabacum species and propose its evaluation in other crop families and plant pathogens.

Small nucleolar RNA of silkworm can translocate from the nucleolus to the cytoplasm under abiotic stress.

Small nucleolar RNAs (snoRNAs) are thought to be exclusively nuclear and guide nucleotide modifications of ribosomal RNAs. Recently, more and more evidence has suggested that the nucleolus is a stress sensor for changes in growth status and that snoRNAs may orchestrate the response to environmental stress through molecular interactions outside of the nucleus. We previously showed that a box C/D snoRNA Bm-15 had both nuclear and cytoplasmic location in BmN4 cell line of the silkworm, Bombyx mori. To further study the functional roles of Bm-15, changes in expression level and cellular location of Bm-15 were examined in BmN4 cells subjected to serum starvation and ultraviolet (UV) ray radiation. Results indicated that total RNA level of Bm-15 was unchanged after 24 h serum starvation, but exhibited 3-fold increases in the cytoplasm, and the nuclear-to-cytosolic distribution ratio was reduced from 5:1 to 2:1. Moreover, UV radiation also causes rapid decline in nuclear Bm-15 and progressive cytoplasmic accumulation with a percentage of 22% and 57% after 6 and 24 h UV radiation. UV treatment results in a dramatic decrease in Bm-15 nuclear-to-cytosolic ratio from 7:1 to 2:1 and 2:1 to 1:20 after 6 and 24 h UV radiation, respectively. We show here for the first time that box C/D snoRNAs can translocate from the nucleus to the cytoplasm under the abiotic stress of nutritional deficiency and UV radiation. The rapid translocation of snoRNAs from nucleus to cytoplasm may slow down the maturation of rRNAs and synthesis of ribosomes to enhance the stress resistance of cells.

piRBase: a comprehensive database of piRNA sequences.

PIWI-interacting RNAs are a class of small RNAs that is most abundantly expressed in animal germline. Substantial research is going on to reveal the functions of piRNAs in the epigenetic and post-transcriptional regulation of transposons and genes. To collect and annotate these data, we developed piRBase, a database assisting piRNA functional study. Since its launch in 2014, piRBase has integrated 264 data sets from 21 organisms, and the number of collected piRNAs has reached 173 million. The latest piRBase release (v2.0, 2018) was more focused on the comprehensive annotation of piRNA sequences, as well as the increasing number of piRNAs. In addition, piRBase release v2.0 also contained the potential information of piRNA targets and disease related piRNA. All datasets in piRBase is free to access, and available for browse, search and bulk downloads at http://www.regulatoryrna.org/database/piRNA/.

First report of Enterobacter hormaechei with respiratory disease in calves.

BACKGROUND: Enterobacter hormaechei is commonly considered a causative pathogen for nosocomial infections and it does not usually cause diseases in animals. However, researchers have recently dissociated the pathogenic Enterobacter hormaechei from foxes and piglets. Here, the Enterobacter hormaechei was first found to be associated with respiratory disease in unweaned calves in China. CASE PRESENTATION: A 2-month-old calf was severely sick and diagnosed with respiratory infection by a rural veterinarian, and it died 5 days after treatment with penicillin G. The lung sample was then run through histopathological analysis and pathogen isolation. The sequence analysis and biochemical tests results showed the isolated bacterium strain to be Enterobacter hormaechei, and drug sensitivity tests showed resistance to all ß-lactam antimicrobials and sensitivity to quinolones. Thickened alveoli septum, inflammatory cell infiltration, and erythrocyte diapedesis around the pulmonary alveoli septum were visible in lung histopathological sections. One week later, at the same farm, another calf showed similar clinical signs, and the Enterobacter hormaechei strain was isolated from its nasal discharge; after a week of treatment with enrofloxacin, as suggested by the results of drug sensitivity tests, this calf fully recovered. CONCLUSIONS: To the best of our knowledge, this is the first case report of calves with respiratory disease that was associated with E. hormaechei, and multi-drug resistance was observed in isolates.

Small nucleolar RNA Sf-15 regulates proliferation and apoptosis of Spodoptera frugiperda Sf9 cells.

BACKGROUND: Small nucleolar RNAs (snoRNAs) function in guiding 2'-O-methylation and pseudouridylation of ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). In recent years, more and more snoRNAs have been found to play novel roles in mRNA regulation, such as pre-mRNA splicing or RNA editing. In our previous study, we found a silkworm C/D box snoRNA Bm-15 can interact with Notch receptor gene in vitro. To further study the function of Bm-15, we cloned its homolog Sf-15 from Spodoptera frugiperda and investigate the function of Sf-15 in Sf9 cells. RESULTS: We showed that knocking down of Sf-15 can inhibit the proliferation, then induce apoptosis of insect S. frugiperda Sf9 cells, but the results were reversed when Sf-15 was overexpressed. De novo sequencing of transcriptome of Sf9 cells showed that the expression of 21 apoptosis-related genes were increased upon Sf-15 repression. Further analysis showed that a Ca2+-induced cell death pathway gene Cn (PPP3C, the serine/threonine-protein phosphatase 2B catalytic subunit), was significantly increased upon Sf-15 depression but decreased when Sf-15 was overexpressed, which indicated that Cn might be a potential target of Sf-15. CONCLUSIONS: We conclude that C/D box snoRNA Sf-15 can participate in apoptosis through regulating the expression of Ca2+-induced cell death pathway gene Cn in Sf9 cells. This is the first time that we found snoRNAs exhibiting dual functions in insect, which reveals a novel layer of ncRNA modulation in cell growth and death.

Characterization of newly emerged NADC30-like strains of porcine reproductive and respiratory syndrome virus in China.

Different strains of porcine reproductive and respiratory syndrome virus (PRRSV) have emerged and circulated in different regions of mainland China since 1996, particularly after 2006. In 2012, NADC30-like PRRSV was first isolated in Henan Province. By 2016, it had spread to most provinces in China. In the present study, the whole genomes (excluding the poly(A) tails) of 13 newly emerged NADC30-like PRRSV strains were sequenced and analyzed. Furthermore, the pathogenicity of SD53-1603, one of the 13 PRRSV strains, was assessed. Phylogenetic analysis showed that these 13 newly emerged NADC30-like PRRSV strains, together with some reference strains, formed a new subgroup (subgroup 5), characterized by a predicted 131-amino-acid deletion in the nonstructural protein (NSP) 2. However, low levels of whole-genome similarity and a wide variety of recombination patterns complicated the classification of the NADC30-like PRRSV isolates. Interestingly, almost all of the recombination breakpoints found in these 13 PRRSV isolates and other NADC30-like PRRSV isolates occurred in genes encoding NSPs and/or minor structural proteins. In addition, piglets infected with the newly emerged NADC30-like strain SD53-1603 displayed clear clinical respiratory symptoms and underwent typical pathological changes. The findings may be useful for elucidating the characteristics and epidemic status of NADC30-like PRRSV in China.

Novel polymerase spiral reaction assay for the visible molecular detection of porcine circovirus type 3.

BACKGROUND: Porcine circovirus type 3 (PCV3) is a newly emerging circovirus that might be associated with porcine dermatitis and nephropathy syndrome, reproductive failure, and cardiac and multisystemic inflammation. To aid the prevention and control of the infectious disease caused by PCV3, we developed a novel isothermal amplification assay using polymerase spiral reaction (PSR), which allows the visual detection of preserved strains and clinical samples. RESULTS: This assay precisely amplified the PCV3 genome with the use of a water bath at 62 °C for 50 min. The detection limit was found to be 1.13 × 102 copies/µL by gel electrophoresis or with the use of a visible dye (an indicator comprising phenol red and cresol red). No cross-reaction with other porcine infectious viruses was observed. The detection results for 23 PCV3-positive samples by PSR were in accordance with loop-mediated isothermal amplification (LAMP) assay. The detection rate of the PSR assay for PCV3 positivity of clinical samples was 68/97, which was higher than LAMP assay (67/97). CONCLUSIONS: These results indicated that the PSR assay provides an accurate and rapid method for the detection of PCV3 with high sensitivity and specificity. It is particularly suited for use in a simple laboratory setting without a thermal cycler or gel electrophoresis equipment.

Visual detection of porcine epidemic diarrhea virus using a novel reverse transcription polymerase spiral reaction method.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) is a major etiological agent of porcine epidemic diarrhea around the world. Point-of-care testing in the field is lacking owing to the requirement for a simple, robust field applicable test that does not require professional laboratory equipment. The aim of this study was to establish a novel reverse transcription polymerase spiral reaction (RT-PSR) assay for the rapid detection of porcine epidemic diarrhea virus (PEDV). For the assay, a specific RT-PSR primer pair was designed against a conserved region in PEDV ORF3. RESULTS: The RT-PSR was optimized, and PEDV could be detected after a 50 min incubation at 62 °C, in addition to the 15 min required for reverse transcription. No cross-reaction with other porcine infectious viruses was observed. This new method for PEDV detection was 10 times more sensitive than the conventional reverse transcription-polymerase chain reaction (RT-PCR) assay. The positive rates for 65 clinical samples using the new RT-PSR assay and the conventional RT-PCR assay were 58.46% (38/65) and 53.84% (35/65), respectively. In the RT-PSR assay, the addition of a mixture of dyes allowed a positive reaction to be directly observed by the naked eye. CONCLUSIONS: These results indicate that this RT-PSR assay is capable of accurately detecting PEDV, and has the advantages of high specificity and sensitivity for the detection of PEDV.

Identification and functional characterization of intermediate-size non-coding RNAs in maize.

BACKGROUND: The majority of eukaryote genomes can be actively transcribed into non-coding RNAs (ncRNAs), which are functionally important in development and evolution. In the study of maize, an important crop for both humans and animals, aside from microRNAs and long non-coding RNAs, few studies have been conducted on intermediate-size ncRNAs. RESULTS: We constructed a homogenized cDNA library of 50-500 nt RNAs in the maize inbred line Chang 7-2. Sequencing revealed 169 ncRNAs, which contained 58 known and 111 novel ncRNAs (including 70 snoRNAs, 27 snRNAs, 13 unclassified ncRNAs and one tRNA). Forty of the novel ncRNAs were specific to the Panicoideae, and 24% of them are located on sense-strand of the 5' or 3' terminus of protein coding genes on chromosome. Target site analysis found that 22 snoRNAs can guide to 38 2'-O-methylation and pseudouridylation modification sites of ribosomal RNAs and small nuclear RNAs. Expression analysis showed that 43 ncRNAs exhibited significantly altered expression in different tissues or developmental stages of maize seedlings, eight ncRNAs had tissue-specific expression and five ncRNAs were strictly accumulated in the early stage of leaf development. Further analysis showed that 3 of the 5 stage-specific ncRNAs (Zm-3, Zm-18, and Zm-73) can be highly induced under drought and salt stress, while one snoRNA Zm-8 can be repressed under PEG-simulated drought condition. CONCLUSIONS: We provided a genome-wide identification and functional analysis of ncRNAs with a size range of 50-500 nt in maize. 111 novel ncRNAs were cloned and 40 ncRNAs were determined to be specific to Panicoideae. 43 ncRNAs changed significantly during maize development, three ncRNAs can be strongly induced under drought and salt stress, suggesting their roles in maize stress response. This work set a foundation for further study of intermediate-size ncRNAs in maize.

Characterization of a Pestivirus H isolate originating from goats.

Natural Pestivirus H infections in cattle have been reported worldwide; however, only a few cases of Pestivirus H have been described in non-bovine ruminants such as goats. A new Pestivirus H HN1507 strain was isolated from an infected goat in 2015 and the genome sequence was determined. The full-length genome sequence was 12,556 nucleotides. Phylogenetic analysis, based on the complete genome and Npro fragments, revealed that the isolate belonged to Pestivirus H and was closely related to strains from Italy. Two unique amino acid substitutions were found in the C-terminal of the E2 protein. To the best of our knowledge, this is first report determining the complete genome of a Pestivirus H strain from goat.

Complete genomic characteristics and pathogenic analysis of the newly emerged classical swine fever virus in China.

BACKGROUND: Classical swine fever (CSF) is one of the most devastating and highly contagious viral diseases in the world. Since late 2014, outbreaks of a new sub-genotype 2.1d CSF virus (CSFV) had caused substantial economic losses in numbers of C-strain vaccinated swine farms in China. The objective of the present study was to explore the genomic characteristics and pathogenicity of the newly emerged CSFV isolates in China during 2014-2015. RESULTS: All the new 8 CSFV isolates belonged to genetic sub-genotype 2.1d. Some genomic variations or deletions were found in the UTRs and E2 of these new isolates. In addition, the pathogenicity of HLJ1 was less than Shimen, suggesting the HLJ1 of sub-genotype 2.1d may be a moderated pathogenic isolate and the C-strain vaccine can supply complete protection. CONCLUSIONS: The new CSFV isolates with unique genomic characteristics and moderate pathogenicity can be epidemic in many large-scale C-strain vaccinated swine farms. This study provides the information should be merited special attention on establishing prevention and control policies for CSF.

ORF1a of highly pathogenic PRRS attenuated vaccine virus plays a key role in neutralizing antibody induction in piglets and virus neutralization in vitro.

BACKGROUND: Currently, porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important viral pathogens in swine in most countries, especially China. Two PRRSV attenuated live vaccine strains (HuN4-F112 and CH-1R) are currently widely used in China. Our previous study showed that HuN4-F112, but not CH-1R, induced high anti-nucleocapsid (N) antibody and neutralizing antibody (NA) titers. Additionally, sera from HuN4-F112 inoculated pigs induced low cross neutralization of CH-1R. METHODS: In the present study, 6 chimeric viruses through exchanging 5' untranslated region (UTR) + open reading frame (ORF)1a, ORF1b, and ORF2-7 + 3'UTR between HuN4-F112 and CH-1R were constructed and rescued based on the infectious clones of rHuN4-F112 and rCH-1R. The characteristics of these viruses were investigated in vitro and vivo. RESULTS: All the three fragments, 5'UTR + ORF1a, ORF1b, and ORF2-7 + 3'UTR, could affect the replication efficiencies of rHuN4-F112 and rCH-1R in vitro. Additionally, both 5'UTR + ORF1a and ORF2-7 + 3'UTR affected the anti-N antibody and NA responses targeting rHuN4-F112 and rCH-1R in piglets. CONCLUSIONS: The 5'UTR + ORF1a region of HuN4-F112 played a key role in inducing NAs in piglets. Furthermore, we confirmed for the first time that ORF1a contains a neutralization region. This study provides important information that can be used for further study of the generation of anti-PRRSV NAs.

Increased stability of heterogeneous ribonucleoproteins by a deacetylase inhibitor.

Splicing factors are often influenced by various signaling pathways, contributing to the dynamic changes of protein isoforms in cells. Heterogeneous ribonucleoproteins (hnRNPs) regulate many steps of RNA metabolism including pre-mRNA splicing but their control by cell signaling particularly through acetylation and ubiquitination pathways remains largely unknown. Here we show that TSA, a deacetylase inhibitor, reduced the ratio of Bcl-x splice variants Bcl-xL/xS in MDA-MB-231 breast cancer cells. This TSA effect was independent of TGFß1; however, only in the presence of TGFß1 was TSA able to change the splicing regulators hnRNP F/H by slightly reducing their mRNA transcripts but strongly preventing protein degradation. The latter was also efficiently prevented by lactacystin, a proteasome inhibitor, suggesting their protein stability control by both acetylation and ubiquitination pathways. Three lysines K87, K98 and K224 of hnRNP F are potential targets of the mutually exclusive acetylation or ubiquitination (K(Ac/Ub)) in the protein modification database PhosphoSitePlus. Mutating each of them but not a control non-K(Ac/Ub) (K68) specifically abolished the TSA enhancement of protein stability. Moreover, mutating K98 (K98R) and K224 (K224R) also abolished the TSA regulation of alternative splicing of a Bcl-x mini-gene. Furthermore, about 86% (30 of 35) of the multi-functional hnRNP proteins in the database contain lysines that are potential sites for acetylation/ubiquitination. We demonstrate that the degradation of three of them (A1, I and L) are also prevented by TSA. Thus, the deacetylase inhibitor TSA enhances hnRNP F stability through the K(Ac/Ub) lysines, with some of them essential for its regulation of alternative splicing. Such a regulation of protein stability is perhaps common for a group of hnRNPs and RNA metabolism.

The C-terminus of DSX(F5) protein acts as a novel regulatory domain in Bombyx mori.

The doublesex gene regulates the somatic sexual development of Bombyx mori by alternatively splicing into sex-specific splice forms. In our previous study, the splice form Bmdsx (F7) , which encodes the BmDSX(F5) protein, was found to be expressed in a female-specific manner and to contain a novel C-terminus. In this study, we aimed to investigate the role of this C-terminus. Two transgenic lines, L1 and L2, were constructed to ectopically express Bmdsx (F7) in males. Phenotype and W chromosome-specific polymerase chain reaction (PCR) analysis showed that developmental abnormalities and sex reversal did not occur. Moreover, the sex ratio was also normal. Quantitative PCR revealed that the expression levels of SP1 and Vg were upregulated in the fat body of transgenic males. Additionally, the expression level of PBP was downregulated in the antenna of transgenic males. The results suggested that the C-terminus of BmDSX(F5) functioned as a regulatory domain during regulation of downstream target gene expression and that BmDSX(F5) participated in the sexual development of somatic cells together with other DSX proteins in B. mori.

Ferritin from the Pacific abalone Haliotis discus hannai: Analysis of cDNA sequence, expression, and activity.

Ferritin plays an important role in iron homeostasis due to its ability to bind and sequester large amounts of iron. In this study, the gene encoding a ferritin (HdhFer2) was cloned from Pacific abalone (Haliotis discus hannai). The full-length cDNA of HdhFer2 contains a 5'-UTR of 121 bp, an ORF of 516 bp, and a 3'-UTR of 252 bp with a polyadenylation signal sequence of AATAAA and a poly(A) tail. It also contains a 31 bp iron-responsive element (IRE) in the 5'-UTR position, which is conserved in many ferritins. HdhFer2 consists of 171 amino acid residues with a predicted molecular weight (MW) ∼19.8 kDa and a theoretical isoelectric point (PI) of 4.84. The deduced amino acid sequence of HdhFer2 contains two ferritin iron-binding region signatures (IBRSs). HdhFer2 mRNA was detected in a wide range of tissues and was dominantly expressed in the gill. Infection with the bacterial pathogen Vibrio anguillarum significantly upregulated HdhFer2 expression in a time-dependent manner. Recombinant HdhFer2 (rHdhFer2) purified from Escherichia coli was able to bind ferrous iron in a concentration-dependent manner. In summary, these results suggest that HdhFer2 is a crucial protein in the iron-withholding defense system, and plays an important role in the innate immune response of abalone.

siRNA-Mediated BmAurora B Depletion Impedes the Formation of Holocentric Square Spindles in Silkworm Metaphase BmN4 Cells.

Silkworm ovary-derived BmN4 cells rely on chromatin-induced spindle assembly to form microtubule-based square mitotic spindles that ensure accurate segregation of holocentric chromosomes during cell division. The chromosome passenger protein Aurora B regulates chromosomal condensation and segregation, spindle assembly checkpoint activation, and cytokinesis; however, its role in holocentric organisms needs further clarification. This study examined the architecture and dynamics of spindle microtubules during prophase and metaphase in BmN4 cells and those with siRNA-mediated BmAurora B knockdown using immunofluorescence labeling. Anti-α-tubulin and anti-γ-tubulin antibodies revealed faint γ-tubulin signals colocalized with α-tubulin in early prophase during nuclear membrane rupture, which intensified as prophase progressed. At this stage, bright regions of α-tubulin around and on the nuclear membrane surrounding the chromatin suggested the start of microtubules assembling in the microtubule-organizing centers (MTOCs). In metaphase, fewer but larger γ-tubulin foci were detected on both sides of the chromosomes. This resulted in a distinctive multipolar square spindle with holocentric chromosomes aligned at the metaphase plate. siRNA-mediated BmAurora B knockdown significantly reduced the γ-tubulin foci during prophase, impacting microtubule nucleation and spindle structure in metaphase. Spatiotemporal BmAurora B expression analysis provided new insights into the regulation of this mitotic kinase in silkworm larval gonads during gametogenesis. Our results suggest that BmAurora B is crucial for the formation of multipolar square spindles in holocentric insects, possibly through the activation of γ-tubulin ring complexes in multiple centrosome-like MTOCs.