College of Life Sciences, Zhejiang Sci-Tech University, Hanghzou 310018, China.
BMC Genomics. 2013 Sep 28;14:661. doi: 10.1186/1471-2164-14-661.
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.
小非编码 RNA（ncRNA）是真核生物基因表达的重要调控因子。以前，只有 microRNAs（miRNAs）和 piRNAs 在蚕，Bombyx mori 中被鉴定出来。此外，只有中间大小的 ncRNAs（50-500nt）在蚕中被系统地鉴定出来。
在这里，我们进行了系统的鉴定和分析与 Bombyx mori Argonaute2（BmAgo2）蛋白相关的小 RNA（18-50nt）。使用 RIP-seq，我们鉴定了与 BmAGO2 相关的各种类型的小 ncRNA。这些 ncRNA 表现出多模态的长度分布，在20nt、27nt 和~33nt 处有三个峰，包括 tRNA、转座元件（TE）、rRNA、snoRNA 和 snRNA 衍生的小 RNA 以及 miRNAs 和 piRNAs。tRNA 衍生片段（tRFs）的丰度极高，占 BmAgo2 相关小 RNA 的 69.90%。Northern 印迹证实，许多 tRFs 仅在 BmNPV 感染的细胞中表达或上调，这表明 tRFs 在 BmNPV 感染过程中通过与 BmAgo2 结合发挥重要作用。进一步的证据表明，tRNA 的 D、反密码子和 TψC 环上可能存在潜在的切割位点。TE 衍生的小 RNA 和 piRNAs 也占 BmAgo2 相关小 RNA 的很大比例，表明 BmAgo2 可能通过抑制这些小 RNA 指导的转座子的活性来参与维持基因组稳定性。最后，还使用 Northern 印迹证实了 Bombyx 5.8s rRNA 衍生的小 RNA 的存在，证明了各种新型小 RNA 存在于蚕中。
使用 RIP-seq 方法结合 Northern 印迹，我们鉴定了与 BmAgo2 蛋白相关的各种类型的小 RNA，包括 tRNA、TE、rRNA、snoRNA 和 snRNA 衍生的小 RNA 以及 miRNAs 和 piRNAs。我们的研究结果为未来研究小 RNA 在昆虫发育和进化中的作用提供了新的线索。
