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


RESULTS
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.


CONCLUSIONS
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.
College of Life Sciences, Zhejiang Sci-Tech University, Hanghzou 310018, China. yaozhou@chinagene.com.
Zuoming
Z
Nie
Fang
F
Zhou
Dan
D
Li
Zhengbing
Z
Lv
Jian
J
Chen
Yue
Y
Liu
Jianhong
J
Shu
Qing
Q
Sheng
Wei
W
Yu
Wenping
W
Zhang
Caiying
C
Jiang
Yuhua
Y
Yao
Juming
J
Yao
Yongfeng
Y
Jin
Yaozhou
Y
Zhang
D000818:Animals; D060565:Argonaute Proteins / Q000378:metabolism*; D012831:Bombyx / Q000235:genetics*; D002460:Cell Line; D004251:DNA Transposable Elements / Q000235:genetics; D047468:Immunoprecipitation / Q000379:methods*; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D017924:Nucleopolyhedroviruses / Q000235:genetics; D012313:RNA / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012340:RNA, Ribosomal, 5.8S / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D020537:RNA, Small Nucleolar / Q000235:genetics / Q000378:metabolism; D058727:RNA, Small Untranslated / Q000235:genetics / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics
D060565:Argonaute Proteins; D004251:DNA Transposable Elements; D035683:MicroRNAs; D012340:RNA, Ribosomal, 5.8S; D034741:RNA, Small Interfering; D020537:RNA, Small Nucleolar; D058727:RNA, Small Untranslated; D012313:RNA; D012343:RNA, Transfer