Response Of CircRNAs Expression To BmCPV Infection And The Function Of CircRNA₅655 In Silkworm,Bombyx Mori

With the development of RNA-seq technology,circular RNA?circRNA?has been pushed into the public view as a new type of non-coding RNA.CireRNA produced from a typical spliceosome by "head-to-tail" back splicing,and it has tissue and developmental stage specificity.To date,some functions of circRNA have been identified,which can act as miRNA sponges to regulate target gene expression,participate in the regulation of transcriptional processes,compete with classical splicing of self-mRNA,interact with proteins,and have the potential to encode proteins Silkworm is a typical representative of lepidopteran insects and has important agricultural and economic significance.Bombyx mori cytoplasmic polyhedosis virus?BmCPV?can specifically infect the midgut epithelial cells of the silkworm,causing silkworm midgut polyhedrosis,which is harmful to the sericulture industry.CircRNA plays an important role in regulating gene expression,growth and pathogenesis in eukaryotes,but the infection response of circRNAs to BmCPV and the function of cireRNAs in silkworm are not knownIn this study,high-throughput sequencing was used to determine the expression profiles of silkworm circRNAs in normal midgut tissues?control?and BmCPV-infected midgut tissues?test?.9753 and 7475 circRNAs were detected from the control and test samples,respectively,of which 6085 were co-expressed circRNAs.646 and 737 circRNAs were specifically expressed in the control and test samples,respectively.A total of 3638 circRNAs were differentially expressed,of which 400 circRNAs showed differential expression fold change)2.0?p<0.05,FDR<0.05?,294 were up-regulated and 106 were down-regulated after viral infection.The main functions of the differentially expressed circRNAs parental genes were largely annotated by GO and KEGG enrichment analysis.Based on the correlation analysis between differential expression of circRNAs and microRNA?miRNA?binding sites,a circRNA-miRNA interaction network was constructed to infer the interaction between 13 miRNAs and 193 circRNAs.bmo-miR-3389-5p,bmo-miR-745-3p and bmo-miR-3262 were associated with 30,34 and 34 circRNAs,respectively.CircRNA₈115,circRNA₉444,circRNA₄553,circRNA₀827 and circRNA₆649 contained 6,5,4,4 and 4 miRNA binding sites,respectively.We further found that the alternative cyclization of circRNAs is a common feature of silkworm,and the junction sites of many silkworm circRNAs are flanked by the typical GT/AG splicing signal.The silkworm circRNA₅655 is 563 bp and is cyclized by the 9-13 exon of Bombyx mori histone-lysine N-methyltransferase eggless?LOC101742950?gene.In order to investigate the function and mechanism of the silkworm circRNA₅655,RT-qPCR results showed that the transcription level of the parental gene LOC101742950 was significantly decreased after overexpression of circRNA₅655,but the expression of histone methylation was increased by Western blotting.The results of immunofluorescence showed that circRNA₅655 was distributed in both nucleus and cytoplasm,while circRNA₅655 was colocalized with silkworm NF90 protein in a sequence-independent manner.It was confirmed that cireRNA₅655 can act as a bmo-miR-3391-5p sponge to activate the downstream target gene Bombyx mori histone deacetylase Rpd3?XM₀04931383.2?expression by circRNA-miRNA pull down and dual fluorescein reporter system.RT-qPCR was used to detect the expression of apoptosis-related genes in overexpressing circRNA₅655.It was found that overexpression of circRNA₅655 resulted in the expression levels of Bombyx mori p53-like protein mRNA?KC243147.1?,Bombyx mori signal transduction and transcription?NM₀01163916.1?and Bombyx mori survivin-1 gene?XM₀12688679.2?were down-regulated?P<0.05?.Flow cytometry revealed that overexpression of circRNA₅655 promoted cell proliferation and inhibited apoptosis.It was found by circRNA-protein pull down and mass spectrometry that circRNA₅655 may interact with proteins such as mitochondrial thiamine pyrophosphate carrier,succinate dehydrogenase assembly factor 2-B,and dynein ? chain.In addition,we also found that overexpression of circRNA₅655 inhibited BmCPV proliferation and replication,but decreased with the increase of circRNA₅655 concentration.Bioinformatics analysis revealed that circRNA₅655 has a m6A methylation modification site?GGACC?and contains an open reading frame ORF1,which spanning the circRNA₅655 back splicing site,possibly encoding the protein circR-P122 containing 122 amino acid residues.The specific small peptide RRNSVRTNASARSRFAC was synthesized and rabbit polyclonal antiserum was prepared.Western Blotting could detect the expression of circR-P122 protein in the midgut of silkworm.Immunofluorescence of the BmN cells and the silkworm midgut tissue showed that circR-P122 was mainly localized in the cytoplasm.Western blotting results showed that the level of histone methylation was inhibited after overexpression of circR-P122 protein.