- In Bombyx mori, autophagy is induced by 20E in the larval fat body. - Results: RNA libraries from the fat body of B. - 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. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - mori, 15 Atgs have been identified in the genome [9, 10], and include two Atg4 homologs, Atg4B and Atg4-like, but their function are still not characterized.. - In the fat body, the ecdysone receptor (EcR) is necessary for the induction of autophagy by 20E in Drosophila melanoga- ster and B. - In the last decades, advances in transcriptome sequencing have led to the identification of a large number of lncRNAs in various eukaryotic organisms using new technologies and bio- informatics methods [17–22], but there are still few. - The fat body is an organ which is important for nutrient storage and energy metabolism in insects. - It plays critical roles in the regulation of insect metamorphosis by co- ordinating different hormones and nutritional signals [33, 34]. - Previous studies have shown that the injection of 20E into actively feeding larvae up-regulates Atg genes and reduces TORC1 activity, inducing autophagy in the fat body [10]. - In this study, the lncRNAs associated with the 20E response in the fat body of B. - To the best of our know- ledge, our study was the first to identify lncRNA that might be involved in the 20E-induced autophagy in silk- worm. - Genome-wide identification of lncRNAs using RNA-seq We first estimated the 20E-induced autophagy in the fat body from the 2-day-old 5th instar larvae using Lyso- Tracker Red staining. - However, in the 20E-induced sam- ples, the staining increased in fat body 2–12 h post- injection with 20E (h.p.i.20E) but became less intense at 24 h.p.i.20E (Fig. - The expression of the autophagy- related genes, Atg1 and Atg8, in the fat body at 2, 6, 12, and 24 h.p.i.20E showed that their expression did not change at 2 h.p.i.20E, but had increased significantly at 6 and 12 h.p.i.20E, then decreased at 24 h.p.i.20E (Fig. - LincRNAs are tran- scripts located in the intergenic regions between two protein-coding genes. - A total of 1035 lncRNAs and 14,622 mRNAs were ob- tained from the fat body of silkworm (Additional file 3:. - These results provided an overview of transcriptional changes in the expression of lncRNAs in the silkworm fat body in response to 20E treatment.. - In total, 166 differen- tially expressed lncRNAs and 3041 mRNAs were de- tected in silkworm fat body after 20E injection (Fig. - mori fat body by LysoTracker Red staining (red, magnification 40 x, the scale is 50 μ m). - 2 Expression analysis of Atg1 (a) and Atg8 (b) in 20E treated fat body by qRT-PCR. - 3 The computational pipeline for identifying lncRNAs from RNA-seq data of silkworm fat body and their classification. - C_6h), 878 upregulated and 1177 downregulated mRNAs were detected in the T_2h vs.. - C_2h group, and 755 upregulated and 972 downregu- lated mRNAs in the T_6h vs. - 0.05) in the T_2h vs. - However, there was no significant enrichment of GO terms in the T_6h vs.. - KEGG analysis [45] indicated that 94 pathways were enriched in cis-regula- tory target genes of lncRNAs in the two groups. - The most enriched pathways included “Phosphatidylinositol signaling system,” “Ribosome biogenesis,” and “Glyoxylate and dicar- boxylate metabolism.” The pathways “Hippo signaling path- way – fly,” “Notch signaling pathway,” and “Wnt signaling pathway” were common among the top 20 enriched path- ways (Additional file 6: Table S6).. - c Expression level indicated by log10(FPKM + 1) in the lncRNAs and mRNAs. - C_2h group, and none in the T_6h vs. - Twenty GO terms associ- ated with metabolism or biosynthesis were enriched in the category Biological Process, two in Cell Com- ponent and two in Molecular Function (Additional file 7: Table S7). - KEGG analysis showed 98 pathways enriched in trans-acting target genes of lncRNAs in the two groups. - “Fatty acid biosynthesis,” “Citrate cycle,” and “Prote- asome.” In the two groups, “Fatty acid biosynthesis,”. - “Fatty acid metabolism,” and “Citrate cycle” were the most enriched pathways among the downregulated target genes, while “Proteasome” and “Lysosome” were the most enriched pathways in the upregulated target genes. - common pathway among the top 20 enriched path- ways in the T_6h vs. - Functional analysis of mRNA in silkworm fat body. - Seven GO terms were significantly enriched in the Biological Process and Molecular Function categories in the T_2h vs. - Sixteen GO terms were significantly enriched in the T_6h vs. - KEGG analysis showed 113 pathways enriched in the differentially expressed mRNAs in the two groups. - The most enriched pathways of mRNAs in the two groups were “ Citrate cycle,. - 6 Hierarchical clustering of the differentially expressed lncRNAs (a) and mRNAs (b) in T_2h vs. - 7 qRT-PCR validation of selected lncRNAs and mRNAs in 20E treated fat body. - Since Atg4B is an essential protein involved in autoph- agy, to further study the function of lncRNA in 20E- induced autophagy, we focused on LNC_000560 and its target gene Atg4B to explore the regulatory function of lncRNAs in the silkworm. - We further analyzed the ex- pression profiles of LNC_000560 and Atg4B in the fat body at 2, 6, 12, and 24 h.p.i.20E, at different developmen- tal stages and in different tissues of silkworm larvae. - 000560 and Atg4B showed highly similar expression pat- terns in the fat body. - The expression of LNC_000560 in larval tissues was high in the epidermis and fat body, and relatively low in other tissues, with no expression in the hemolymph or midgut (Fig. - 000560 and Atg4B, we performed RNAi experiment to knockdown the expression of LNC_000560 in the fat body of silkworm larvae. - Compared with dsEGFP con- trol, we found that the expression of LNC_000560 and Atg4B in fat body was significantly decreased after LNC_. - 000560 knockdown, which indicated that the expression of LNC_000560 was successfully down-regulated and it could indirectly participate in the regulation of autoph- agy by regulating its target gene Atg4B (Fig. - To identify lncRNAs involved in 20E-induced autophagy in the fat body, we chose the 2 and 6 h.p.i.20E experimental times for RNA-seq analysis.. - 8 Phylogenetic analysis of the ATG4 homologs from different species. - 10 Expression profile of LNC_000560 (a) and Atg4B (b) in 20E treated fat body of B. - 12 Expression of LNC_000560 and Atg4B after RNAi of LNC_000560 in fat body of 5th instar larvae of B. - The cis and trans method was used to predicted the function of the identified lncRNAs, we found 6493 lncRNA-mRNA pairs in the cis prediction and 42,797 lncRNA-mRNA pairs in the trans prediction.. - “Citrate cycle,” whereas, those enriched in upregulated genes were “Proteasome” and “Lysosome.” The insect fat body is the main organ involved in energy metabolism and is analogous to the adipose tissue and liver in verte- brates. - In the last dec- ade, several studies have found that the expression of many genes involved in energy metabolism is downregu- lated by 20E in the Drosophila midgut and fat body, blocking their metabolic activity for the initiation of metamorphosis [50, 51]. - Hence, we assume that the identi- fied lncRNAs may function mainly through the positive correlation target genes, and the downregulation of these genes may play a role in fat body development, and up- regulated genes may function mainly in the degradation of intracellular proteins via autophagy or ubiquitin pathway.. - Several lncRNAs have been identified in the silkworm, but only a few of them have so far been functionally characterized . - We se- lected LNC_000560 and LNC_000063 to verify the ex- pression of their target genes in the fat body. - The expression profiles of LNC_000560 and Atg4B were highly consistent in 20E-treated fat bodies and in the different developmental stages from larvae to adults.. - 000560 may be involved in the 20E-induced autophagy of the fat body by up-regulating its target gene Atg4B.. - In the current study, 1035 lncRNAs were identified from 12 RNA-seq libraries of the B. - The 20E levels in the 2-day-old 5th instar larvae are low, and the fat body is sensitive to 20E [57]. - mori fat body [10]. - RNA extraction, library construction and sequencing Total RNA from the silkworm’s fat body tissue at 2 and 6 h.p.i.20E was extracted using the Trizol reagent (Invi- trogen) and further purified with RNeasy kits (Qiagen).. - (3) transcripts that overlapped with any protein-coding exon in the sense orientation were filtered out. - The expres- sion of the autophagy-related genes, Atg1 and Atg8, in the fat body at 2, 6, 12, and 24 h.p.i.20E was evaluated using qRT-PCR. - After injection for 24 h, the fat body was dissected for qRT-PCR analysis.. - Common differentially expressed lncRNAs and mRNAs in the two groups.. - The significantly enriched GO terms detected in the two groups (cis).. - The top 20 enriched KEGG pathways in the two groups (cis).. - The significantly enriched GO terms detected in the two groups (trans).. - The top 20 enriched KEGG pathways in the two groups (trans).. - The significantly enriched GO terms of differentially expressed mRNAs in the two groups.. - The top 20 enriched KEGG pathways of differentially expressed mRNAs in the two groups.. - The primers of the selected lncRNAs and mRNAs.. - DL and LY assisted in the data analysis and revision. - The funding bodies had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.. - The datasets generated during the current study are available in the SRA database of the NCBI system with accession number of PRJNA672230 (https://. - 20-Hydroxyecdysone upregulates Atg genes to induce autophagy in the Bombyx fat body.. - Programmed autophagy in the Drosophila fat body is induced by ecdysone through regulation of the PI3K pathway. - Modes of cell death in the pupal perivisceral fat body tissue of the silkworm Bombyx mori L. - Identification and properties of 1,119 candidate lincRNA loci in the Drosophila melanogaster genome. - Hormonal and nutritional regulation of insect fat body development and function. - Fat Body Biology in the Last Decade. - Identification and characterization of a novel nuclear noncoding RNA, Fben-1, which is preferentially expressed in the. - Systematic Identification and Characterization of Long Non-Coding RNAs in the Silkworm, Bombyx mori. - BmncRNAdb: a comprehensive database of non-coding RNAs in the silkworm, Bombyx mori. - Identification and comparison of long non-coding RNAs in the silk gland between domestic and wild silkworms. - An antisense lncRNA functions in alternative splicing of Bmdsx in the silkworm, Bombyx mori. - Identification and characterization of a new long noncoding RNA iab-1 in the Hox cluster of silkworm, Bombyx mori identification of iab-1. - Larval fat body cells die during the early pupal stage in the frame of metamorphosis remodelation in Bombyx mori. - Fat body remodeling and homeostasis control in Drosophila. - 20-Hydroxyecdysone- upregulated proteases involved in Bombyx larval fat body destruction. - Developmental regulation of glycolysis by 20-hydroxyecdysone and juvenile hormone in fat body tissues of the silkworm, Bombyx mori. - Genome-wide regulation of innate immunity by juvenile hormone and 20- hydroxyecdysone in the Bombyx fat body. - accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. - Molecular cloning, characterization and expression analysis of ATG1 in the silkworm, Bombyx mori
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