Identification And Analysis Of CircRNAs In Silkworm,Bombyx Mori

Circular RNA?CircRNA?is single strand RNA with closed loop structure.CircRNA is the by-product of post transcriptional modification of gene which contains exons.Recently,thousands of circRNAs were identified in mouse and human cells and their function is involved in the expression regulation of parental gene,mi RNA sponge and the biomarker of some diseases.However,there is no report in Bombyx mori.In this study,we systematically identified and analyzed the circularRNA from Bombyx mori using high-throughput sequencing and bioinformatics prediction,providing a reference for the identification and research of new RNA molecules in silkworm and even insect.Firstly,high-throughput sequencing method was used to identify and analyze the circRNA from Bombyx mori at a large scale.The total RNAs were extracted from silkworm,and then rRNA was removed from them by the kit.After high throughput sequencing,a total of 112271320 sequencing read were obtained.From these obtained reads,9778 and 1659 circRNAs were predicted using mapsplice and findcirc softwares,respectively.A total of 1598 circRNAs were existed in both of these two predicted circRNA datasets,which were regarded as the potential circRNA candidates.molecular identification of silkworm.We further designed the specific PCR primer pairs,and identified 11 circRNAs from 17 predited circRNA candidates with the highest SRPBM.In these eleven identified circRNAs,BmcircR₃621,BmcircR₈955,BmcircR₈926,BmcircR₆123,BmcircR₈349,BmcircR₁198,BmcircR₉535,Bmcirc R₆215 and BmcircR₇937 were confirmed as the circular topology structure in the silkworm and the circularization sites were aslo consistent with the predicted results.However,the circularization sites of BmcircR₂196 and BmcircR₆226 were not consistent with the predicted results.We further identified and analyzed circRNAs from silkworm using bioinformatics method.Based on the characteristics of circRNA,the program CircRNApredictor.pl was developed for the prediction of circRNA using perl language.A total of 12955 circRNA candidates were predicted from high-throughput sequencing reads and silkworm genome.The mapping of reads and circRNA candidates showed that the flank of the mapped reads of the 5' and 3' terminus of many circRNA candidates were out of the mapping,furthermore,the unmapped flank of the mapped reads of 5' terminus were the same as the 3' terminus of circRNA candidates,similarly,the unmapped flank of the mapped reads of 3' terminus were the same as the 5' terminus of circRNA candidates,confirming the circularization of these circRNA candidates because 5' and 3' terminus of them were overlapped.Additionally,we found many circRNA candidates were homologs.Interestedly,Interestingly,the mapping analysis of circRNA candidates and genome showed that circRNA candidate BmCirc2091935 contained 7 similar sequence motifs with the size of the 780 bp,and there is a 67 nt fragment beteween each motif.The total RNAs were treated with RNase R and then PCR idenfication was performed.The corresponding fragments of BmCirc2091935 were amplified successfully,confirming the circularization of it.Furthermore,Northern Blot showed that BmCirc2091935 also contained 7 isoforms of circularization.The difference of sequence length between each isoform is speculated to be the multiple of the motif length.Thus,we speculated that BmCirc2091935 might contain variable motifs by alternative splicing,forming different isoforms of circularization.There is no report about this kind of circRNA,and the mechanism of circularization and function remain to be further studied.