Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
Gene. 2021 Feb 5;768:145314. doi: 10.1016/j.gene.2020.145314. Epub 2020 Nov 18.
The BmN-4 cell line, originated from the silkworm Bombyx mori ovary, possesses endogenous small interfering RNA (siRNA) and PIWI-interacting RNA (piRNA) pathways. We performed CRISPR/Cas9-mediated genome editing of Ago2 and Siwi, which are the core factors for siRNA and piRNA pathways, respectively, to understand the importance of the two distinct small RNA pathways in this cell line. We found that approximately half of the alleles contained loss-of-function mutations in both Ago2- and Siwi-mutated cells. The mutated cells grew at a slower rate compared to the control cells, strongly suggesting that the siRNA and piRNA pathways are both crucial for the normal growth of BmN-4 cells. The amounts of piRNAs decreased markedly in the Siwi-mutated cells, but global de-repression of transposable elements was not observed. Although the RNA amount of latently infected RNA virus, Bombyx mori macula-like virus (BmLV), increased in both Ago2- and Siwi-mutated cells, the siRNA and piRNA pathways showed a bias toward targeting BmLV genomic and subgenomic RNA, respectively. These results indicate the common, specific, and crucial roles of the two small RNA pathways in B. mori cultured cells.
BmN-4 细胞系源自家蚕卵巢，具有内源性小干扰 RNA (siRNA) 和 PIWI 相互作用 RNA (piRNA) 途径。我们使用 CRISPR/Cas9 介导的基因组编辑技术对 Ago2 和 Siwi 进行编辑，Ago2 和 Siwi 分别是 siRNA 和 piRNA 途径的核心因子，以了解这两个不同的小 RNA 途径在家蚕细胞系中的重要性。我们发现，大约一半的等位基因在 Ago2 和 Siwi 突变细胞中都含有功能丧失突变。与对照细胞相比，突变细胞的生长速度较慢，强烈表明 siRNA 和 piRNA 途径对于 BmN-4 细胞的正常生长都是至关重要的。在 Siwi 突变细胞中，piRNAs 的含量明显下降，但并未观察到转座元件的全局去抑制。尽管潜伏感染的 RNA 病毒——家蚕斑点状病毒 (BmLV) 的 RNA 量在 Ago2 和 Siwi 突变细胞中都增加，但 siRNA 和 piRNA 途径分别表现出对 BmLV 基因组和亚基因组 RNA 的靶向偏好。这些结果表明，这两个小 RNA 途径在家蚕培养细胞中具有共同的、特异的和关键的作用。
