Lee Jae Man, Kojin Yoshito, Tatsuke Tsuneyuki, Mon Hiroaki, Miyagawa Yoshitaka, Kusakabe Takahiro
Laboratory of Silkworm Sciences, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Fukuoka, Japan.
Insect Sci. 2013 Feb;20(1):69-77. doi: 10.1111/j.1744-7917.2012.01569.x. Epub 2012 Nov 6.
Long hairpin dsRNA transcribed from chromosomal DNA can induce RNA interference in Bombyx mori cells, although its gene silencing efficiency is lower than that of exogenously introduced double-stranded RNAs (dsRNAs). To solve this problem, we monitored the nuclear cytoplasmic translocation of the transcribed hairpin dsRNA and analyzed the processing efficiency into mature small interfering RNA (siRNA). Northern blot analysis revealed that the transcribed hairpin dsRNAs were spliced and transported into the cytoplasm, but were not effectively diced into siRNAs. Interestingly, RNAi with hairpin dsRNAs from genome-integrated IR transgene was stimulated by the coexpression of Escherichia coli RNase III, although this exogenous enzyme seemed to bring about nonspecific cleavage of cellular mRNA.
从染色体DNA转录而来的长链发夹状双链RNA（dsRNA）能够在家蚕细胞中诱导RNA干扰，尽管其基因沉默效率低于外源导入的双链RNA（dsRNA）。为了解决这个问题，我们监测了转录的发夹状dsRNA在细胞核与细胞质之间的转运，并分析了其加工成成熟小干扰RNA（siRNA）的效率。Northern印迹分析表明，转录的发夹状dsRNA被剪接并转运到细胞质中，但未有效切割成siRNA。有趣的是，来自基因组整合IR转基因的发夹状dsRNA的RNA干扰受到大肠杆菌RNase III共表达的刺激，尽管这种外源酶似乎会导致细胞mRNA的非特异性切割。
