Identification, Evolution And Function Of A Class Of Atypical SINEs (BmSINE-A3B) In Silkworm

The domestic silkworm (Bombyx mori) genome contains40%repetitive sequences (repeats), and most of them belong to transposable elements (TEs). Short interspersed nuclear elements (SINEs), one category of the non-autonomous TEs, occupy up to4.7%of the silkworm genome. According to the structure and homology, the SINEs in the silkworm genome are divided into six families, BM1, BmSE, BmSINEl, BmSINE2, BmSINE3and BmSINE4. However, there are still many SINEs unidentified in the silkworm genome. We found a class of SINEs with atypical structure, named BmSINE-A3B. It has three A boxes, which is different from the typical SINEs. In this study, I systematically identified BmSINE-A3B elements and investigated their distribution in the silkworm genome and their functions. The major results are as follows:1. Identification of BmSINE-A3BWe identified506BmSINE-A3B copies in the silkworm genome based on structure and homology search method, which occupy-0.025%of the genome. The structure of the BmSINE-A3B elements has the head, body and tail of the typical SINE. However, it contains3A boxes in the head. We also searched for BmSINE-A3B in other four Lepidoptera insect genomes:Danaus plexippus, Heliconius melpomene, Manduca sexta and Plutella xylostella, but none of these genomes has BmSINE-A3B, indicating that BmSINE-A3B is the silkworm specific SINE.2. Distribution of BmSINE-A3B among chromosomes and nearby genesTo investigate the preference of BmSINE-A3B inserting to the silkworm genome, we counted the number of BmSINE-A3B at each chromosome and nearby genes. The results showed that BmSINE-A3B randomly inserted into chromosomes. In addition, we counted BmSINE-A3B insertions in the silkworm predicted genes and the both sides of them. The results revealed that the inserting pattern of BmSINE-A3B was no significant difference from the control, suggesting that it preferred to reside in the non-functional regions.3. The amplification time and evolution pattern of BmSINE-A3BWe then performed the inserting data and network analysis to estimate the expansion of BmSINE-A3B. The results revealed that BmSINE-A3B may experience dramatically expansion in recent2-3million years, and has become inactive in recent1million years.4. BmSINE-A3B regulates the expression of its neighbor genesTo test whether BmSINE-A3B insertion regulates the expression of nearby genes, we constructed an EGFP expression vector, in which BmSINE-A3B were inserted into the5’and3’terminal ends of the gene, respectively. Then these vectors were transfected into silkwom cells. We found that BmSINE-A3B could down-regulate the expression of its neighbore genes, no matter where its insertion site is. In addition, our results also showed that the inhibitory effect was stronger when the BmSINE-A3B inserted into the3’terminal of the nearby genes.5. BM1and BmSE regulates expression of its neighbor genesWe used the same method to test whether other silkworm SINEs (BM1and BmSE) may regulate the expression of their nearby genes. As the BmSINE-A3B, the BM1transposon also can down-regulate expression of its neighbor genes no matter it was inserted into5’terminal or3’terminal of the genes. BmSE can up-regulate the expression of its neighbor genes when it was inserted into5’terminal of the genes, in contrast, BmSE can down-regulate expression of its neighbor genes when it was inserted into3’terminal of these genes.This study identified a new SINE family in silkworm genome-BmSINE-A3B, which is a kind of atypical SINE with three A boxes. By comparative genomics analysis, we found that BmSINE-A3B only exists in the silkworm genome among the Lepidoptera. BmSINE-A3B randomly inserted into chromosomes and nearby genes, but could down-regulate the expression of their neighbor genes. In addition, we also make a preliminary study on the regulation patterns of other silkworm SINEs. In summary, this study provides the foundation for identifying atypical SINEs and father function studies of SINEs.