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. 2017 Feb 23;18(1):201.
doi: 10.1186/s12864-017-3598-5.

Transposable Element Bm1645 is a Source of BmAGO2-associated Small RNAs that affect its expression in Bombyx mori

Hongqiang Si  1   2 , Yunjie Cao  1   2 , Honglin Zhu  1   2 , Dan Li  1   2 , Zhengbing Lv  1   2 , Qing Sheng  1   2 , Zuoming Nie  3   4
Affiliations

Transposable Element Bm1645 is a Source of BmAGO2-associated Small RNAs that affect its expression in Bombyx mori

Hongqiang Si et al. BMC Genomics. .

Abstract

Background: A transposable element (TE) is a DNA fragment that can change its position within a genome. Transposable elements play important roles in maintaining the stability and diversity of organisms by transposition. Recent studies have shown that approximately half of the genes in Bombyx mori are TEs.

Results: We systematically identified and analyzed the BmAGO2-associated TEs, which exceed 100 in the B. mori genome. Additionally, we also mapped the small RNAs associated with BmAGO2 in B.mori. The transposon Bm1645 is the most abundant TE associated with BmAGO2, and Bm1645-derived small RNAs represent a small RNA pool. We determined the expression patterns of several Bm1645-derived small RNAs by northern blotting, and the results showed there was differential expression of multiple small RNAs in normal and BmNPV-infected BmN cells and silkworms from various developmental stages. We confirmed that four TE-siRNAs could bind to BmAGO2 using EMSA and also validated the recognition sites of these four TE-siRNAs in Bm1645 by dual-luciferase reporter assays. Furthermore, qRT-PCR analysis revealed the overexpression of the four TE-siRNAs could downregulate the expression of Bm1645 in BmN cells, and the transcription of Bm1645 was upregulated by the downregulation of BmAGO2.

Conclusions: Our results suggest Bm1645 functions as a source of small RNAs pool and this pool can produce many BmAGO2-associated small RNAs that regulate TE's expression.

Keywords: Bm1645; BmAGO2-associated small RNAs; TE-siRNA; TEs.

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Figures

Fig. 1
Fig. 1
Identification of TE-siRNAs by northern blotting in BmN cells and each phase of the silkworm. a Identification of TE-siRNAs by northern blotting in BmN cells. N: normal BmN cells, V: BmNPV-infected BmN cells. There were 7 of 13 TE-siRNAs detected in BmN cells. The detected TE-siRNAs were TE-siRNA134, TE-siRNA286, TE-siRNA413, TE-siRNA610, TE-siRNA649, TE-siRNA671 and TE-siRNA688. We used U6 as the control. There were obvious differences of the expression of TE-siRNA413 and TE-siRNA610 between the normal and virus-infected BmN cells. b Expression profile of TE-siRNAs in each phase of the silkworm by northern blotting. N: normal samples, V: BmNPV-infected samples. Only TE-siRNA156 could be detected in all four developmental stages of the Bombyx mori. The others were expressed in one, two, or three different stages
Fig. 2
Fig. 2
The mapping pattern of Bm1645 and 6 Bm1645-derived small RNAs. These 6 TE-siRNAs are reversely complementary to Bm1645 and show relatively high abundance. We chose these as our targets for subsequent experiments. The mapping figure was generated using the Tablet software
Fig. 3
Fig. 3
TE-siRNAs combine with activated BmAGO2 in vitro. We used the Control IRE/IRP System included in the kit as one positive control and the IRE/BmAGO2 and TE-siRNAs without proteins as the negative control. The shift of the positive control was a thick stripe. We detected 6 TE-siRNAs, and there were 4 detected with block stripes. The shifted TE-siRNAs were TE-siRNA134, TE-siRNA610, TE-siRNA671, and TE-siRNA688. On the contrary, TE-siRNA413 and TE-siRNA649 were not associated with BmAGO2
Fig. 4
Fig. 4
Validation of TE-siRNA targeted sites using dual-luciferase assays. The expression of the firefly luciferase fused with the wild-type sequences containing TE-siRNA targeted sites in Bm1645 was inhibited by the corresponding TE-siRNAs. But TE-siRNAs did not affect the activity of luciferase fused with the mutant sequences
Fig. 5
Fig. 5
The relative expression level of Bm1645. a The qRT-PCR analysis of Bm1645 in TE-siRNAs-transfected BmN cells. The expression was downregulated when the BmN cells were transfected with TE-siRNAs. A negative control was used as contrast. b The transcription level of Bm1645 affected by BmAGO2 knockdown in BmN cells. We used the RNA interference method to downregulate the expression of BmAGO2. The result shows the transcriptional level of Bm1645 was upregulated 1.66 times, as assessed by qRT-PCR. Each histogram bar represents the mean relative expression of the indicated transcripts, with at least three replicates for each bar. The asterisks (*, ** or ***) indicate the significant differences (P < 0.05, P < 0.01 or P < 0.001, respectively) compared with the relevant control with a two-tailed t-test
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References

    1. Liu J, Carmell MA, Rivas FV, Marsden CG, Thomson JM, Song JJ, Hammond SM, Joshua-Tor L, Hannon GJ. Argonaute2 is the catalytic engine of mammalian RNAi. Science. 2004;305(5689):1437–1441. doi: 10.1126/science.1102513. - DOI - PubMed
    1. Sasaki T, Shiohama A, Minoshima S, Shimizu N. Identification of eight members of the Argonaute family in the human genome. Genomics. 2003;82(3):323–330. doi: 10.1016/S0888-7543(03)00129-0. - DOI - PubMed
    1. Song JJ, Smith SK, Hannon GJ, Joshua-Tor L. Crystal structure of Argonaute and its implications for RISC slicer activity. Science. 2004;305(5689):1434–1437. doi: 10.1126/science.1102514. - DOI - PubMed
    1. Tahbaz N, Kolb FA, Zhang H, Jaronczyk K, Filipowicz W, Hobman TC. Characterization of the interactions between mammalian PAZ PIWI domain proteins and Dicer. EMBO Rep. 2004;5(2):189–194. doi: 10.1038/sj.embor.7400070. - DOI - PMC - PubMed
    1. Wang GH, Jiang L, Zhu L, Cheng TC, Niu WH, Yan YF, Xia QY. Characterization of Argonaute family members in the silkworm, Bombyx mori. Insect Sci. 2013;20(1):78–91. doi: 10.1111/j.1744-7917.2012.01555.x. - DOI - PubMed

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