Upf2-Mediated Nonsense-Mediated Degradation Pathway Involved in Genetic Compensation of TrpA1 Knockout Mutant Silkworm (Bombyx mori).

Genetic mutations leading to premature termination codons are known to have detrimental effects. Using the Lepidoptera model insect, the silkworm (Bombyx mori), we explored the genetic compensatory response triggered by mutations with premature termination codons. Additionally, we delved into the molecular mechanisms associated with the nonsense-mediated mRNA degradation pathway. CRISPR/Cas9 technology was utilized to generate a homozygous bivoltine silkworm line BmTrpA1-/- with a premature termination. Transcript levels were assessed for the BmTrpA paralogs, BmPyrexia and BmPainless as well as for the essential factors Upf1, Upf2, and Upf3a involved in the nonsense-mediated mRNA degradation (NMD) pathway. Upf2 was specifically knocked down via RNA interference at the embryonic stage. The results comfirmed that the BmTrpA1 transcripts with a 2-base deletion generating a premature termination codon in the BmTrpA1-/- line. From day 6 of embryonic development, the mRNA levels of BmPyrexia, BmPainless, Upf1, and Upf2 were significantly elevated in the gene-edited line. Embryonic knockdown of Upf2 resulted in the suppression of the genetic compensation response in the mutant. As a result, the offspring silkworm eggs were able to hatch normally after 10 days of incubation, displaying a non-diapause phenotype. It was observed that a genetic compensation response does exist in BmTrpA1-/-B. mori. This study presents a novel discovery of the NMD-mediated genetic compensation response in B. mori. The findings offer new insights into understanding the genetic compensation response and exploring the gene functions in lepidopteran insects, such as silkworms.

RNA N6-methyladenosine of DHAPAT and PAP involves in regulation of diapause of Bombyx mori via the lipid metabolism pathway.

Environment-induced epigenetics are involved in diapause regulation, but the molecular mechanism that epigenetically couples nutrient metabolism to diapause regulation remains unclear. In this study, we paid special attention to the significant differences in the level of N6-adenosine methylation (m6A) of dihydroxyacetone phosphate acyltransferase (DHAPAT) and phosphatidate phosphatase (PAP) genes in the lipid metabolism pathway of the bivoltine silkworm (Bombyx mori) strain Qiufeng developed from eggs incubated at a normal temperature (QFHT, diapause egg producer) compared to those from eggs incubated at a low temperature (QFLT, non-diapause egg producer). We knocked down DHAPAT in the pupal stage of the QFLT group, resulting in the non-diapause destined eggs becoming diapausing eggs. In the PAP knockdown group, the colour of the non-diapause destined eggs changed from light yellow to pink 3 days after oviposition, but they hatched as normal. Moreover, we validated that YTHDF3 binds to m6A-modified DHAPAT and PAP mRNAs to promote their stability and translation. These results suggest that RNA m6A methylation participates in the diapause regulation of silkworm by changing the expression levels of DHAPAT and PAP and reveal that m6A epigenetic modification can be combined with a lipid metabolism signal pathway to participate in the regulation of insect diapause traits, which provides a clearer image for exploring the physiological basis of insect diapause.

Exorista sorbillans (Diptera: Tachinidae) parasitism shortens host larvae growth duration by regulating ecdysone and juvenile hormone titers in Bombyx mori (Lepidoptera: Bombycidae).

The tachinid fly, Exorista sorbillans, is a notorious ovolarviparous endoparasitoid of the silkworm, Bombyx mori, causing severe damage to silkworm cocoon industry. Silkworm larvae show typically precocious wandering behavior after being parasitized by E. sorbillans; however, the underlying molecular mechanism remains unexplored. Herein, we investigated the changes in the levels of 20-hydroxyecdysone (20E) and juvenile hormone (JH) titer, and they both increased in the hemolymph of parasitized silkworms. Furthermore, we verified the expression patterns of related genes, which showed an upregulation of 20E signaling and biosynthesis genes but a significant downregulation of ecdysone oxidase (EO), a 20E inactivation enzyme, in parasitized silkworms. In addition, related genes of the JH signaling were activated in parasitized silkworms, while related genes of the JH degradation pathway were suppressed, resulting in an increase in JH titer. Notably, the precocious wandering behavior of parasitized silkworms was partly recoverable by silencing the transcriptions of BmCYP302A1 or BmCYP307A1 genes. Our findings suggest that the developmental duration of silkworm post parasitism could be shortened by regulation of 20E and JH titers, which may help silkworm to resist the E. sorbillans infestation. These findings provide a basis for deeper insight into the interplay between silkworms and E. sorbillans and may serve as a reference for the development of a novel approach to control silkworm myiasis.

m6A-dependent mevalonate kinase in juvenile hormone synthesis pathway regulates the diapause process of bivoltine silkworm (Bombyx mori).

BACKGROUND: Research has shown that epigenetic modification are involved the regulation of diapause in bivoltine silkworms (Bombyx mori), but it remains unclear how epigenetic modification in response to environmental signals precisely to regulate the diapause processing of bivoltine B. mori. METHODS AND RESULTS: In this study, the diapause terminated eggs of bivoltine B. mori, Qiufeng (QF) were divided into two groups: a QFHT group incubated at 25 °C with a natural day/night cycle to produce diapause eggs, and a QFLT group incubated at 16.5 °C in darkness to produce non-diapause eggs. On the 3rd day of the pupal stage, the total RNAs of the eggs were extracted and their N6-adenosine methylation (m6A) abundances were analyzed to explore the effects of m6A methylation on diapause in the silkworm. The results showed that 1984 m6A peaks are shared, 1563 in QFLT and 659 in QFHT. The m6A methylation level of the QFLT group was higher than that of the QFHT one in various signaling pathways. The m6A methylation rate of mevalonate kinase (MK) in the insect hormone synthesis pathway was significantly different between the two groups. The knockdown of MK by RNA interference in the pupae of QFLT resulted in females laying diapause eggs rather than non-diapause eggs after mating. CONCLUSIONS: m6A methylation involves in the diapause regulation of bivoltine B. mori by changing the expression levels of MK. This result provides a clearer image of the environmental signals on the regulation of diapause in bivoltine silkworms.

Cordyceps cicadae polysaccharides inhibit human cervical cancer hela cells proliferation via apoptosis and cell cycle arrest.

The present study presented the extraction and purification of polysaccharides from artificially cultured Cordyceps cicadae and wild Cordyceps cicadae by pre-soaking ultrasonic water extraction. The effects of different concentrations of polysaccharides on proliferation and cytotoxicity of Hela cells were detected by MTT and LDH methods. The results showed that the proliferation of Hela cells was inhibited by polysaccharides treatment (25 µg/mL-1600 µg/mL). The results of flow cytometry further confirmed that polysaccharides blocked the cell cycle in the S phase and promoted apoptosis. RT-qPCR and Western Blot were used to study the mRNA and protein expression of genes related to cell cycle and apoptosis signaling pathway. The results showed that polysaccharides treatment inhibited the expression of Cyclin E, Cyclin A and CDK2 and up regulated the expression of P53. Further, activation of Caspase cascade reaction, up regulation of death receptor, and the ratio of pro-apoptotic factor/anti-apoptotic factors, thus caused the cell cycle arrest and induced the apoptosis. The above research results lay a foundation for extending the anti-cancer effects of natural plant resources with low toxicity and high efficiency.

A 14-amino acids deletion in BmShadow results to non-moult on the 2nd instar in the bivoltine silkworm, Bombyx mori.

The Bombyx mori Shadow gene (BmShadow) belongs to the superfamily of cytochrome P450 genes. To elucidate the function of the BmShadow gene and its association with diapause, we employed the CRISPR/Cas9 system to knock out the BmShadow gene in the bivoltine strain Qiufeng. The mutant (BmShadow-/-) was obtained in G2, exhibiting a 42-base deletion corresponded exactly to the amino acids regions from positions 155 to 168. The larvae of BmShadow-/- cannot moult at the pre-moulting stage of the 2nd instar. When the BmShadow-/- larvae were fed with 20E analogue at the late stage of the 2nd instar, they were rescued and developed into the 3rd instar. Rescue experiments indicated that the 20E concentration of BmShadow-/- larvae was significantly lower than that in WT larvae, and the 20E concentration of BmShadow-/- larvae which fed 20E analogue was restored to normal levels. Interestingly, the BmShadow-/- larvae could not moult on the 1st instar when they hatched from eggs after being stored at 5 °C for 40 days or after hibernation, suggesting that the 20E transported from the mother was partially consumed in the diapause maintenance phase. Our study confirmed that BmShadow is involved in 20E synthesis and a 14-amino acids region from position 155 to 168 was essential for its function, also there appears to be no other compensation pathway in vivo, which offered an important potential target locus for the control of silkworm development and the biological control of agricultural and forestry pests.

[Advances in m 6A modification and its regulation of viral replication].

N 6-methyladenosine (m 6A) is a prevalent modification of RNA in eukaryotes and plays an important role in the process of mRNA translocation, stabilization and translation. m 6A exerts different influences on the viral replication cycle, and both viral replication and host immune response to the virus are affected by m 6A. In this review, we summarize recent studies on the mechanism of m 6A modification and its effects on viral replication and host immune response, in order to provide a reference for epigenetic regulation in the viral life cycle.

Transcriptome sequencing reveals potential mechanisms of diapause preparation in bivoltine silkworm Bombyx mori (Lepidoptera: Bombycidae).

In the bivoltine strain of the silkworm, Bombyx mori, embryonic diapause is induced transgenerationally as a maternal effect. Progeny diapause ability is determined by the environmental condition such as temperature and lightness that mothers experience during their own embryonic development. Diapause preparation is a crucial phase of this process; diapause-destined individuals undergo a series of preparatory events before the entry into developmental arrest. However, the molecular regulatory mechanisms of diapause preparation have largely remained unknown. In the present study, we sequenced the transcriptome of bivoltine silkworm Qiufeng's ovaries resulted in laying of diapause destined or non-diapause eggs, using high-throughput RNA-Seq technology. Differential expression analyses identified 183 genes with higher expression, and 106 with lower expression under diapause-inducing conditions. GO and KEGG analysis revealed that the enrichment of several functional terms related to peroxisome, glycerolipid metabolism, steroid biosynthesis, longevity regulating pathway - multiple species, three signaling transductions, insect hormone biosynthesis, and cytoskeleton components. We conducted a detailed comparison of transcript profile data of ovaries from diapause-inducing and non-diapause conditions, the results imply up-regulation of peroxisomal metabolism, triacylglycerides accumulation, cryoprotectant production, and ecdysteroid biosynthesis in diapause-inducing group. Differential expression of genes related to actin cytoskeleton implies the occurrence of shifts in cellular structure and composition between diapause-inducing and non-diapause-inducing groups. The Hippo and FOXO signaling pathways may play an important role in preparing for entering diapause. This study provides an insight into the molecular events of insect diapause, in particular for the preparatory phase.

bmo-miR-0001 and bmo-miR-0015 down-regulate expression of Bombyx mori fibroin light chain gene in vitro.

Based on bioinformatic analysis, we selected two novel microRNAs (miRNAs), bmo-miR-0001 and bmo-miR-0015, from high-throughput sequencing of the Bombyx mori larval posterior silk gland (PSG). Firstly, we examined the expression of bmo-miR-0001 and bmo-miR 12 different tissues of the 5th instar Day-3 larvae of the silkworm. The results showed that the expression levels of both bmo-miR-0001 and bmo-miR-0015 were obviously higher in the PSG than in other tissues, implying there is a spatio-temporal condition for bmo-miR-0001 and bmo-miR-0015 to regulate the expression of BmFib-L. To test this hypothesis, we constructed pri-bmo-miR-0001 expressing the plasmid pcDNA3.0 and pri-bmo-miR-0015 expressing the plasmid pcDNA3.0 [ie1-egfp-pri-bmo-miR-0015-SV40]. Finally, the BmN cells were harvested and luciferase activity was detected. The results showed that luciferase activity was reduced significantly (P<0.05) in BmN cells co-transfected by pcDNA3.0 [ie1-egfp-pri-bmo-miR-0001-SV40] or pcDNA3.0 with pGL3.0 [A3-luc-Fib-L-3'UTR-SV40], suggesting that both bmo-miR-0001 and bmo-miR-0015 can down-regulate the expression of BmFib-L in vitro.

Overview of research on Bombyx mori microRNA.

MicroRNAs (miRNAs) constitute some of the most significant regulatory factors involved at the post-transcriptional level after gene expression, contributing to the modulation of a large number of physiological processes such as development, metabolism, and disease occurrence. This review comprehensively and retrospectively explores the literature investigating silkworm, Bombyx mori L. (Lepidoptera: Bombicidae), miRNAs published to date, including discovery, identification, expression profiling analysis, target gene prediction, and the functional analysis of both miRNAs and their targets. It may provide experimental considerations and approaches for future study of miRNAs and benefit elucidation of the mechanisms of miRNAs involved in silkworm developmental processes and intracellular activities of other unknown non-coding RNAs.

Differentially expressed genes in the ovary of the sixth day of pupal "Ming" lethal egg mutant of silkworm, Bombyx mori.

The "Ming" lethal egg mutant (l-em) is a vitelline membrane mutant in silkworm, Bombyx mori. The eggs laid by the l-em mutant lose water, ultimately causing death within an hour. Previous studies have shown that the deletion of BmEP80 is responsible for the l-em mutation in silkworm, B. mori. In the current study, digital gene expression (DGE) was performed to investigate the difference of gene expression in ovaries between wild type and l-em mutant on the sixth day of the pupal stage to obtain a global view of gene expression profiles using the ovaries of three l-em mutants and three wild types. The results showed a total of 3,463,495 and 3,607,936 clean tags in the wild type and the l-em mutant libraries, respectively. Compared with those of wild type, 239 differentially expressed genes were detected in the l-em mutant, wherein 181 genes are up-regulated and 58 genes are down-regulated in the mutant strain. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis results showed that no pathway was significantly enriched and three pathways are tightly related to protein synthesis among the five leading pathways. Moreover, the expression profiles of eight important differentially expressed genes related to oogenesis changed. These results provide a comprehensive gene expression analysis of oogenesis and vitellogenesis in B. mori which facilitates understanding of both the specific molecular mechanism of the 1-em mutant and Lepidopteran oogenesis in general.

Proteomic analysis of the phenotype of the scaleless wings mutant in the silkworm, Bombyx mori.

A scaleless wing mutant of silkworm, Bombyx mori, has much fewer scales than wild type (WT). The scaleless phenotype was associated with tracheal system developmental deficiency and excessive apoptosis of scale cells. In this study, the wing discs proteins of WT and scaleless during pupation were studied using 2-DE and mass spectrometry. Of the 99 identified protein spots, four critical differentially expressed proteins between WT and scaleless were further verified using Q-PCR. At the first day of pupation (P0) in WT, imaginal disk growth factor (IDGF) was upregulated, whereas actin-depolymerizing factor 1 (ADF1) and profilin (PFN), which associated with cellular motility and cytoplasmic extension, were downregulated. We speculated their coaction counteracts the correct organization of the tracheal system in wing disc. Thiol peroxiredoxin (TPx) was upregulated in scaleless at P0, but its mRNA higher expression occurred in the day before pupation (S4). TPx could inhibit the formation of hydrogen peroxide, preventing the release of cytochrome C and activation of the caspase family protease. Its higher expression in scaleless was responsible for the apoptosis of scale cells delayed. The results provide further evidence that the scaleless phenotype was related to the tracheal system developmental deficiency and excessive apoptosis of scale cells.

The regulation of silkworm fibroin L chain production by miRNA-965 and miRNA-1926 in insect cells.

MicroRNAs (miRNAs) are an abundant class of approximately 22-nucleotide (nt)-short noncoding RNA molecules present in the genomes of all multicellular organisms that act through base pairing to partially complementary sequences of the 3'untranslated region (UTR) of targeted mRNAs. Using bioinformatic approach, we found that the 3'UTR of the Fibroin L chain (Fib-L) mRNA matches perfectly the nucleotides 2-8 at the 5' end of the miRNA-965 and miRNA-1926. These two miRNAs might act as regulators of Fib-L gene expression at the post-transcriptional level. To examine whether Fib-L is directly targeted by miRNA-965 and miRNA-1926 in vitro, miRNA expression vectors and target reporter vector with 3'UTR of Fib-L were constructed respectively. Two vectors were co-transfected into Sf21cells. The luciferase assay showed that miRNA-965 and miRNA-1926 may down regulate the expression of Fib-L via complementary interaction with the target sites in 3'UTR.

Construction of baculovirus expression vector of miRNAs and its expression in insect cells.

MicroRNAs (miRNAs) are endogenous small non-protein coding RNAs that play important regulatory roles in animals and plants by binding to target transcripts for cleavage or translational repression. The miR-9a is very conservative in animals from flies to humans. Studies indicated that miR-9a is involved in the regulation of neurogenesis in animals. In our study, the baculovirus expression system was used to transcribe a recombinant vector containing miR-9a for further analysis the function ofmiR-9a. The sequence ofpre-miR-9a from silkworm DNA was first cloned into the donor pFastBac. The enhanced green fluorescent protein (EGFP) was used as reporter gene. The recombinant donor plasmid pFastBac-miR-9a was transformed into E.coli DH10Bac/AcNPV forming Bacmid-9a which was transfected into insect cells with cational lipofectin. The transcription of mature miR-9a was detected by Real-time PCR. The results show the recombinant Bacmid-9a was successfully constructed and effectively transcribed miR-9a in infected Sf21 insect cells.

Differential expression of microRNA-2b with potential target coding P25 in the fifth instar larvae posterior silk gland of the silkworm.

MicroRNAs (miRNAs) are a class of non-protein coding small RNAs that regulate a gene expression at the post-transcriptional level. Using in silico screening, we found that the 3'-untranslated regions of the P25 gene mRNA are perfectly complementary to nucleotides 2-8 at the 5' end of the miRNA-2b (miR-2b). The expression of miR-2b and the P25 gene in posterior silk gland of the fifth instar larval silkworm was investigated using real-time PCR detection method. The results indicated that expression of the P25 gene was very high in the posterior silk gland during the fifth instar larvae, whereas a level of miR-2b sharply decreased until reaching the lowest one on the 8th day. The expression patterns of miR-2b and P25 gene indicate that miR-2b might act as a fine-tuning regulator of expression of the P25 gene at the post-transcriptional level.

Construction and detection of expression vectors of microRNA-9a in BmN cells.

MicroRNAs (miRNAs) are small endogenous RNAs molecules, approximately 21-23 nucleotides in length, which regulate gene expression by base-pairing with 3' untranslated regions (UTRs) of target mRNAs. However, the functions of only a few miRNAs in organisms are known. Recently, the expression vector of artificial miRNA has become a promising tool for gene function studies. Here, a method for easy and rapid construction of eukaryotic miRNA expression vector was described. The cytoplasmic actin 3 (A3) promoter and flanked sequences of miRNA-9a (miR-9a) precursor were amplified from genomic DNA of the silkworm (Bombyx mori) and was inserted into pCDNA3.0 vector to construct a recombinant plasmid. The enhanced green fluorescent protein (EGFP) gene was used as reporter gene. The Bombyx mori N (BmN) cells were transfected with recombinant miR-9a expression plasmid and were harvested 48 h post transfection. Total RNAs of BmN cells transfected with recombinant vectors were extracted and the expression of miR-9a was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot. Tests showed that the recombinant miR-9a vector was successfully constructed and the expression of miR-9a with EGFP was detected.

Recombinant canine parvovirus-like particles express foreign epitopes in silkworm pupae.

The capsid structural protein VP2 of canine parvovirus (CPV) can self-assemble into highly organized virus-like particles (VLPs) and retain major immunoreactivity. In this study, different recombinant baculoviruses that expressed varying fusion proteins of the CPV VP2 protein with the T cell determinant and/or the linear virus-neutralizing epitope of rabies virus (RV) were generated. Infection with these baculoviruses changed BmN cell morphology and inhibited their proliferation as well as damaged silkworms and pupae. However, infection with these baculoviruses induced high levels of recombinant protein expression in silkworms and pupae. More importantly, these fusion proteins self-assembled VLPs with properties similar to CPV virions and retained their VP2-specific immunoreactivity, but some retained their RV-specific immunoreactivity. Interestingly, only one fusion protein, T-VP2, maintained its haemagglutination activity. These data indicated that these insertions and replacements in the loop 2 of VP2 did not interfere with the formation of VLP, and silkworms and pupae could act as a low-costing bioreactor for the production of heterologous proteins. Therefore, our findings may provide a new framework for the development of subunit vaccines against RV and CPV.

Genomic analysis of silkworm microRNA promoters and clusters.

MicroRNAs (miRNAs) are endogenous single-stranded RNAs of 18-22 nt in length, which can regulate the complementary mRNAs at the post-transcriptional level by cleavage or repression of translation of the target mRNAs. Studies have shown that the majority of animal miRNAs are transcribed from independent transcription units, and someare transcribed together with their host genes. However, the nature of the primary transcript of intergenic miRNAs remains unknown. Silkworm (Bombyx mori) miRNAs are representative of those of the Lepidoptera insects and many of them are conserved in Caenorhabditis elegans and other animal species. To date, little is known about the transcriptional regulation of silkworm miRNA genes. We performed the genomic analysis on the silkworm miRNA transcripts around the promoter region including the transcription start site (TSS) and the TATA-box, and on the organization of the miRNA cluster. In 73 pre-miRNAs from the silkworm 131 promoters were detected via a bioinformatics approach. Among them the portion of non-conserved promoters is greater than that of the conserved ones. The genomic organization of pre-miRNAs of the silkworm was globally analyzed and it was determined that 11 of them we reorganized into five clusters. Sequence alignment showed that paralogs existed for some of the miRNAs in the cluster. These results may increase the understanding of the specific sequences upstream of the pre-miRNAs and of the functional implications of miRNA clusters in the silkworm.

Biological functions of microRNAs: a review

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MicroRNAs (miRNAs) are a recently discovered family of endogenous, noncoding RNA molecules approximately 22 nt in length. miRNAs modulate gene expression post-transcriptionally by binding to complementary sequences in the coding or 3Œ untranslated region of target messenger RNAs (mRNAs). It is now clear that the biogenesis and function of miRNAs are related to the molecular mechanisms of various clinical diseases, and that they can potentially regulate every aspect of cellular activity, including differentiation and development, metabolism, proliferation, apoptotic cell death, viral infection and tumorgenesis. Here, we review recent advances in miRNA research, and discuss the diverse roles of miRNAs in disease (AU)

Biological functions of microRNAs: a review.

MicroRNAs (miRNAs) are a recently discovered family of endogenous, noncoding RNA molecules approximately 22 nt in length. miRNAs modulate gene expression post-transcriptionally by binding to complementary sequences in the coding or 3' untranslated region of target messenger RNAs (mRNAs). It is now clear that the biogenesis and function of miRNAs are related to the molecular mechanisms of various clinical diseases, and that they can potentially regulate every aspect of cellular activity, including differentiation and development, metabolism, proliferation, apoptotic cell death, viral infection and tumorgenesis. Here, we review recent advances in miRNA research, and discuss the diverse roles of miRNAs in disease.