| Small RNA is a very important regμlatory factor for living in organisms,and it also plays animportant role in growth and development. Some small RNAs can regulate gene expressiondepending on RNA-induced silencing complex (RISC) which is formed by small RNAs andArgonaute (AGO) proteins. Via RISC, small RNAs will combine with target genes, then splice orinhibit the translation of target genes. We can isolate AGO protein and it’s associated small RNAs byco-immunoprecipitation method using anti-AGO antibody, and then validate the cooperationbetween small RNAs and their target genes using high-throughput sequencing, bioinformaticsanalysis and experimental verification. In our previous studies, we isolated the BmAGO2-associatedsmall RNAs (more than200nt) by co-immunoprecipitation. The high-throughput sequencing andbioinformatics analysis showed that transposon elements (TEs) derived small RNAs accounted for47.78%of total small RNAs. In this study, we further performed the identification and expressionanalysis for TE-siRNAs by Northern Blotting, up-regulated or down-regulated the expression ofAGO proteins by eukaryotic expression or RNA interference (RNAi), and then explored the TEsexpression level in this condition to clarify the regulation function of RISC to TEs.Firstly, using bioinformatics software, we mapped the BmAGO2-associated small RNAs with1,694TEs and obtained the matching map of BmAGO2-associated small RNAs and TEs in silkworm,indicating that these small RNAs are a new type of siRNA-like small RNAs derived from TEs andtherefore named it TE-siRNA. Using specific probes, TE-siRNAs were identified in normal andbaculovirus-infected BmN cells by Northern Blotting. The results showed that most TE-siRNAswere successfully detected in BmN cells, such as TE-siRNA413, TE-siRNA610, TE-siRNA479,TE-siRNA649, TE-siRNA134and TE-siRNA671. Except for TE-siRNA479, the other detectedTE-siRNAs are located in bm1645, indicating that TE bm1645may be a “pool” which produce smallRNAs. Compared with TE-siRNAs detected in normal BmN cells, a few TE-siRNAs wereup-regulated in baculovirus-infected BmN cells suggesting these TE-siRNAs may be related withvirus infection; however, the expression level of other TE-siRNAs are stable. We further identifiedthe expression of TE-siRNAs in normal and baculovirus-infected five instar larva, pupa, moth and normal eggs by Northern Blotting. The results showed that TE-siRNAs could be detected in differentdevelopment stages, however, the detected TE-siRNAs were not exactly the same in different stages,indicating that certain TE-siRNA only exist in a particular development stage. The differentexpression level of TE-siRNA in development stages of silkworm indicated that TE-siRNA mayexert specific function for gene regulation in specific development stages in silkworm and this needfurther studies. Additionally, certain TE-siRNAs were not detected; this maybe due to the lowexpression level of them.Similar to miRNA and siRNA pathway, TE-siRNA could associate with BmAGO2protein toregulate the expression of target genes. Therefore, we studied the effect of BmAGO2overexpressionon TEs gene transcription. After transfection with recombinant plasmid pIEX-1-BmAGO2to BmNcells, the transcription level of BmAGO2gene were detected to be up-regulated and the transcriptionlevel of TEs R1-3_BM, M19755, AK380858.1and Mariner-1-BM were detected to bedown-regulated. Lots of BmAGO2-associated small RNAs were derived from these down-regulatedTEs, indicating that RISC medicated by BmAGO2may combine with TE-siRNAs to regulate theexpression of these down-regulated TEs and the mechanism maybe the same with siRNA pathway,needing further study. BmAGO3and BmSIWI are the other two members of Argonaute proteinfamily. We also studied the effect of the down-regulation of BmAGO3and BmSIWI on TEs genetranscription. Using RNAi method, we firstly down-regulated the expression of BmAGO3andBmSIWI genes by transfecting BmAGO3-dsRNA and BmSIWI-dsRNA into BmN cells. Thetranscriptional level of TEs were then detected by qRT-PCR. The results suggested that thedown-regulation of BmAGO3could lead to the up-regulation of certain TEs, such as bm1157,AK380858.1and R1-3_BM; the down-regulation of BmSIWI could lead to the up-regulation ofcertain TEs, such as bm1157, AB480244.1, L1BM, AK380858.1, Mariner-1-BM, R1-3_BM andM19755, indicating that TE-siRNA may bind to different AGO proteins to exert the functions forregulation of TEs.In addition, we identified the expression profile of BmAGO2expressed by recombinantbaculovirus in infected BmN cells. The results showed that BmAGO2protein was expressed at ahigh level at12hours after transfection, and the expression level was continued to be up-regulated within72hours after transfection. This will lay the foundation for the studies on TE-siRNA functionmediated by BmAGO2through co-immunoprecipitation method. |
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