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RIP-seq of BmAgo2-associated small RNAs reveal various types of small non-coding RNAs in the silkworm, Bombyx mori

Zuoming Nie1, Fang Zhou1, Dan Li1, Zhengbing Lv1, Jian Chen1, Yue Liu2, Jianhong Shu1, Qing Sheng1, Wei Yu1, Wenping Zhang1, Caiying Jiang1, Yuhua Yao1, Juming Yao3, Yongfeng Jin4 and Yaozhou Zhang1*

Author Affiliations

1 College of Life Sciences, Zhejiang Sci-Tech University, Hanghzou 310018, China

2 Zhejiang Economic & Trade Polytechnic, Hangzhou 310018, China

3 College of Materials and Textile, Zhejiang Sci-Tech University, Hangzhou 310018, China

4 College of Life Sciences, Zhejiang University, Hangzhou 310058, China

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BMC Genomics 2013, 14:661  doi:10.1186/1471-2164-14-661

Published: 28 September 2013



Small non-coding RNAs (ncRNAs) are important regulators of gene expression in eukaryotes. Previously, only microRNAs (miRNAs) and piRNAs have been identified in the silkworm, Bombyx mori. Furthermore, only ncRNAs (50-500nt) of intermediate size have been systematically identified in the silkworm.


Here, we performed a systematic identification and analysis of small RNAs (18-50nt) associated with the Bombyx mori argonaute2 (BmAgo2) protein. Using RIP-seq, we identified various types of small ncRNAs associated with BmAGO2. These ncRNAs showed a multimodal length distribution, with three peaks at ~20nt, ~27nt and ~33nt, which included tRNA-, transposable element (TE)-, rRNA-, snoRNA- and snRNA-derived small RNAs as well as miRNAs and piRNAs. The tRNA-derived fragments (tRFs) were found at an extremely high abundance and accounted for 69.90% of the BmAgo2-associated small RNAs. Northern blotting confirmed that many tRFs were expressed or up-regulated only in the BmNPV-infected cells, implying that the tRFs play a prominent role by binding to BmAgo2 during BmNPV infection. Additional evidence suggested that there are potential cleavage sites on the D, anti-codon and TψC loops of the tRNAs. TE-derived small RNAs and piRNAs also accounted for a significant proportion of the BmAgo2-associated small RNAs, suggesting that BmAgo2 could be involved in the maintenance of genome stability by suppressing the activities of transposons guided by these small RNAs. Finally, Northern blotting was also used to confirm the Bombyx 5.8 s rRNA-derived small RNAs, demonstrating that various novel small RNAs exist in the silkworm.


Using an RIP-seq method in combination with Northern blotting, we identified various types of small RNAs associated with the BmAgo2 protein, including tRNA-, TE-, rRNA-, snoRNA- and snRNA-derived small RNAs as well as miRNAs and piRNAs. Our findings provide new clues for future functional studies of the role of small RNAs in insect development and evolution.

Bombyx mori; BmAgo2-associated small RNAs; RIP-seq; tRFs; BmNPV