Characteristics Of EccDNAs In Silk Gland Of Silkworm Bombyx Mori And Functions Of EccDNA^fib-L

The silkworm,Bombyx mori,with a clear genetic background,is a model species of Lepidoptera and the most important economic insect.Silk glands are considered to be the most efficient organ for protein synthesis in known tissues.Silk protein synthesis is the most important economic character of silkworm,which determines the yield and quality of silk.The efficient synthesis and regulation mechanism of silk protein has been the most important research content in silkworm biology,the mechanism of silk protein synthesis and regulation has been explored from the perspectives of silkworm genomics,transcriptomics and proteomics of silk gland,silk gland development,regulation of silk gland cell size,efficient endoreduplication of silk gland cell,gender and silk protein synthesis,silk protein gene transcription and translation,and a lot of meaningful research progress has been made.However,silk protein synthesis is interactively regulated by multiple network pathways at multiple levels,and the regulatory mechanism remains unclear.Extrachromosomal circular DNA（eccDNA）is single-stranded or double-stranded circular DNA derived from chromosome but independent of chromosome DNA.Recent studies have shown that eccDNAs exist widely in cells and are involved in tumorigenesis and cell ageing in a special way,such as increasing oncogene copy number by eccDNAs carrying oncogenes and promoting oncogene amplification through autonomous replication of eccDNAs,and leads to increasing in plasticity and instability of oncogene.In addition,eccDNAs function in accelerating adaptive evolution in plants,gene dosage compensation,enhancing gene expression and autonomous transcriptional activity.To date,only the eccDNAs of model organism Drosophila have been reported among insects.In this study,the eccDNAs in the silk gland of the silkworm was determined by Circle-Seq,the expression profile and size of eccDNAs and distribution of eccDNAs on different chromosomes were investigated,the characteristics of the flanking sequences of the breaking point of eccDNAs were analyzed,the functions of eccDNAs host genes of were annotated,the eccDNAs carrying silk protein genes and transcription factor genes related to silk protein synthesis were screened,and the characteristics of silk gland eccDNAs were preliminarily defined.Furthermore,an eccDNA^fib-L from fibroin light chain（fib-L）gene was focused,and expression pattern of eccDNA^fib-L,the effect of eccDNA^fib-L on fib-L gene expression,and interaction of eccDNA^fib-L with mi RNA to regulate gene expression,transcription of eccDNA^fib-L was studied.Finally,the effect of flanking sequences of eccDNA^fib-L on the formation of eccDNA^fib-L was explored.The main results are as follows:1 Characteristics of eccDNAs in silk gland of silkwormA total of 35,346 eccDNAs with split reads ranging from 5-128,212 were identified by Circle-Seq of the posterior silk gland of the silkworm.The size distribution of eccDNAs was in the region of 32-13,569,549 bp,but most of eccDNAs（33,315,accounted for 94.25%）were less than 1 kb in size.The statistical results of distribution of eccDNAs on the silkworm chromosomesshowed that the number of eccDNAs from chromosome 11 was the largest（1,831,accounting for 5.18%）,the number of eccDNAs from chromosome 2 was the lowest（666,accounting for 1.88%）.Analysis of the average length of the base sequence,which is required to form an eccDNA on each chromosome revealed that the length was the largest（an average of15,660 bp）and 1,223 eccDNAs were formed for chromosome 24（19,152,483 bp in length）.The length was the smallest（an average of 11,243 bp）and 1,831 eccDNAs were formed for chromosome 11（20,585,495 bp in length）.In the same chromosome,the distribution of eccDNAs was different,indicating that there were hotspots for formation of eccDNA in silkworm genome.Based on sequence characteristics,eccDNAs can be classified into intergenic eccDNAs,exonic eccDNAs,intronic eccDNAs,and intronic-exonic eccDNAs.Most of eccDNAs were intergenic eccDNAs（20,701,58.58%）,followed by exonic eccDNAs（13,521,38.25%）,and the least for intron-exonic eccDNAs（105,0.30%）.According to the classification and size of eccDNAs,the conservation analysis of 10 bp of the flanking sequence at upstream and downstream of 5’-,3’-breaking point showed that there are directly repeated sequences between the flanking sequences of the 5’-and 3’-breaking points,while the upstream and downstream sequences of the breaking point have palindromic sequence-like features in intergenic eccDNA.The flanking sequences of breaking point of eccDNAs from intron,exon,and intron-exon exhibit AT or CG rich features.Characteristics of flanking sequences of breaking point changed with length of eccDNAs.The 6 bp of sequences close to breaking point of eccDNAs whose length are larger than 1,000 bp tend to ATTTTA or TAAAAT,CGGCCC or GCCGGG,the flanking sequences of breaking point for eccDNAs whose length are smaller than 1,000 bp were similar with that of intergenic eccDNAs.The functional annotation showed that the eccDNAs host genes were mainly enriched in GO items such as response to stimulus,membrane and binding,and enriched in KEGG pathways such as the c AMP signaling pathway,Apelin signaling pathway and Insulin resistance.In order to verify the reliability of the Circle-Seq results,the PCR products of the junction site region of the eccDNAs were sequenced by Sanger,and the results showed that the flanking sequences of junction site of the 3 selected eccDNAs were consistent with the high-throughput sequencing results,indicating that the high-throughput sequencing results were reliable.Further,the silk protein genes and transcription factor genes related to silk protein synthesis carried by the eccDNAs were analyzed,the results showed that two eccDNAs carried the complete fibroin heavyg chain（fib-H）gene,20 eccDNAs contained partial sequences of the fib-L gene,and one eccDNA contained partial sequences of the P25protein gene.In addition,there are also some eccDNAs containing sequences of transcription factor genes related to silk protein synthesis,suggesting that these eccDNAs may be involved in the regulation of silk protein synthesis.These results indicated the presence of eccDNAs in the silk gland,and provided a new perspective for understanding the efficient synthesis of silk proteins.2 Identification and function of eccDNA^fib-LTo investigate the functions of eccDNAs,an eccDNA(named eccDNA^fib-L)from the 9,692,083-9,692,623 nt region of chromosome 14,which contained partial sequence of fib-L gene,was selected.The junction site and flanking sequence of eccDNA^fib-Ldetermined by Sanger sequencing were consistent with the high-throughput results,and the rolling circle amplification（RCA）products showed that eccDNA^fib-L was a ring molecule.Moreover,positive signals could be detected by in situ hybridization with oligonucleotide probe targeting the junction site in the posterior silk glands of the 5th instar silkworm,confirming the presence of eccDNA^fib-L in the silk gland.The q PCR results showed that the expression pattern of eccDNA^fib-L was similar to that of the fib-L gene,with the highest expression level in the silk gland,and the expression level of eccDNA^fib-L gradually increased with the development of the silk gland.Furtherly,to investigate the effect of eccDNA^fib-L on silk protein synthesis,eccDNA^fib-L was constructed in vitro.q PCR assays showed that the relative expression level of fib-L gene in silkworm injected with eccDNA^fib-L was increased compared with the control,indicating that eccDNA^fib-L could promote the expression of fib-L gene.In addition,bioinformatic predictions showed that eccDNA^fib-L has the potential to interact with mi RNA bmo-mi R-3318,and there is a bmo-mi R-3318 binding site in the 3’-untranslation region（UTR,2,636-2,660 nt）of the silkworm protein pangolin,isoforms A/H/I/S gene（Bmpan,LOC101742450）,so it is hypothesized that Bmpan gene expression can be regulated by interaction eccDNA^fib-L with bmo-mi R-3318.To validate this hypothesis,pull down experiment based on eccDNA^fib-L was carried out with a biotin-labeled probe targeting the eccDNA^fib-L junction site,the result showed that bmo-mi R-3318 could be recruited by eccDNA^fib-L.Subsequently,q RT-PCR confirmed that Bmpan gene expression level could be elevated by eccDNA^fib-L in a dose-dependent manner.Further,eccDNA^fib-L interacted with bmo-mi R-3318 to enhance the expression of the downstream target gene Bmpan was confirmed by dual-luciferase reporter gene system.eccDNAs can be transcripted in a classical transcription element independent manner.To investigate the transcription of eccDNA^fib-L,the RNAs extracted were used for RT-PCR after incubating eccDNA^fib-Lwith cytosolic proteins and nuclear protein,respectively,and sanger sequencing of the amplified products showed that sequences corresponding to the eccDNA^fib-L junction site could be found in the products amplified from the RNA samples extracted from the mixture of nuclear protein with eccDNA^fib-L,indicating that eccDNA^fib-L can be transcribed and occurs in the nucleus.3 Preliminary investigation of the formation mechanism of eccDNA^fib-LUsually,there is a short direct repeats sequence with 2-15 bp in the 5’-,3’-breaking point of eccDNAs.To explore the formation mechanism of eccDNA^fib-L,the primer pairs were designed based the sequences at 200 nt（150,100,50 nt）of upstream of the5’-breaking point and at 200 nt（150,100,50 nt）of downstream of the 3’-breaking point,respectively.The silkworm genomicDNA was used as a template for PCR,the correspoing products mini-200（mini-150,mini-100,mini-50）obtained were transfected into Spodoptera frugiperd Sf9,grass crap CIK,human ovarian cancer COV362 and B.mori Bm N cell lines,respectively.Sanger sequencing showed that eccDNA^fib-L could be generated in all transfected cells,implying that the formation mechanism of eccDNAs is evolutionarily conserved.Bm N cells were transfected with mini-150,mini-100,and mini-50,and the q PCR assay showed that the efficiency of forming eccDNA^fib-L decreased roughly in the order of mini-150,mini-100 and mini-50.There was a GAGT sequence in both 5’-and 3’-breaking point of eccDNA^fib-L,respectively,but only one GAGT sequence is retained in eccDNA^fib-L.mini-50 delete was constructed by deleting"GAGT"from the 5’-breaking point in mini-50.The results showed that the efficiency of forming eccDNA^fib-L in the transfected Bm N cells with mini-50 delete was lower than that in the transfected cells with mini-50.These results suggested that the formation efficiency of eccDNA^fib-L was associated with the flanking sequence of eccDNA^fib-L and the short repeat sequence"GAGT".