Identification Of The BmAGO2-associated TE-siRNAs In Silkworm And Its Expression Regulation To Transposable Element Bm1645

Argonaute protein as the core element of RISC can combine with small RNAs, then splice or inhibit the translation of target genes. Small RNA is the important regulatory factor of many vital pathways. It plays an important role in the life activities, such as gene transcription, individual development, genetic and epigenetic regulation. Recent studies have found that Transposable element can produce functional small RNAs. And nearly half of the sequences are transposon genes in silkworm. In our previous studies, we analyzed the BmAG02-associated RNAs in BmN cells by RIP-seq. The results showed that there are a large number of TEs and TE-derived small RNAs(TE-siRNAs) identified in BmAG02-associated RNAs. The transposon Bm1645 is the most abundant TE associated with BmAG02, and Bm1645-derived small RNAs also appeared the most abundant, representing a small RNA pool. Based on previous researches, this study identified BmAG02-associated TE-siRNAs and TEs in fifth instar silkworm by RIP-seq. We further studied on the binding between Bm1645-derived TE-siRNAs and BmAG02, and the expression regulation of Bm 1645 by these TE-siRNAs.Firstly, we obtained endogenous BmAG02 protein complexes from fifth instar larvae by co-immunoprecipitation using anti-BmAG02 monoclonal antibody produced early, and then successfully separated the BmAG02-associated RNAs. The <50nt small RNAs and long RNAs were analysed using high-throughput sequencing and bioinformatics analysis, respectively. The result showed that there are also a large number of TEs and TE-siRNAs identified in BmAG02-associated RNAs in fifth instar larva of silkworm. The transposon Bm1645 is also the most abundant TE(RPKM=73982.57 ) associated with BmAG02, meanwhile, the abundance of TE-siRNA derived from Bm1645 ( 1814707) is also far greater than TE-siRNAs derived from other transposon. These results were accorded with the results in BmN cells previously identified. The above result further confirmed BmAG02 protein could combine with a great number of TEs and TE-siRNAs, especially transposon Bm1645 and Bm1645-derived TE-siRNAs. We speculated that Bm1645-derived TE-siRNAs could combine with BmAGO2 protein and regulate the expression of Bm1645.We chosed 6 BmAG02-associated TE-siRNAs derived from Bm1645, and performed the EMSA experiment to further identify the binding between TE-siRNAs and BmAG02. The results suggested that 4 of the 6 TE-siRNAs were identified to associate with BmAG02. These TE-siRNAs were TE-siRNA134, TE-siRNA610, TE-siRNA671, and TE-siRNA688. The experiment in vitro simulated the combination of the TE-siRNA and BmAG02. This result reveals that the small RNAs generated by Bm 1645 (TE) can associate with activated BmAG02 protein, suggesting Bm1645 TE-siRNAs might act to regulate Bm1645 expression in vivo. We further chosed the 4 TE-siRNAs to use for the dual-luciferase reporter assays to verify the TE-siRNA targeted sites in Bm1645. We cloned TE-siRNA targeted sites in Bm 1645 and their mutant sequences into the plasmid pIEx-1-Rluc-Luc (a dual luciferase plasmid constructed in our laboratory). These recombinant vectors and the TE-siRNAs synthesized in vitro were co-transfected into BmN cells. The dual luciferase reporter assays showed that these 4 TE-siRNAs could significantly inhibit the expression of the luciferase fused with the wild-type region containing TE-siRNA targeted sites in Bm1645(p<0.05) but did not affect the activity of luciferase fused with the mutant sequences. The result suggested that TE-siRNA134?TE-siRNA610?TE-siRNA671 and TE-siRNA688 can target the targeted sites in Bm 1645 to suppress the gene's expression.Finally, we confirmed that TE-siRNAs could regulate the expression of Bm1645. We transfected the TE-siRNAs synthesized in vitro into BmN cells, and detected the transcription level of Bm1645 by qRT-PCR. The transcription of Bm1645 was significantly down-regulated in BmN cells transfected with TE-siRNAs compared with the negative control (p<0.05 ) . The result further confirmed TE-siRNAs can bind to BmAG02 protein and regulate the expression of TEs. This study firstly identified TE-siRNA can combine with BmAG02 protein and regulate the expression of transposon mediated by BmAG02, laying the foundation for the identification and functional study of novel small RNAs in silkworm and even in insects.