University of Sydney, Waterhouse Laboratory, Lvl 8 Biochemistry Bldg G08, Sydney, NSW, 2006, Australia.
Plant J. 2013 Nov;76(3):519-29. doi: 10.1111/tpj.12306. Epub 2013 Oct 3.
It is known that 22-nucleotide (nt) microRNAs (miRNAs) derived from asymmetric duplexes trigger phased small-interfering RNA (phasiRNA) production from complementary targets. Here we investigate the efficacy of 22-nt artificial miRNA (amiRNA)-mediated RNA silencing relative to conventional hairpin RNA (hpRNA) and 21-nt amiRNA-mediated RNA silencing. CHALCONE SYNTHASE (CHS) was selected as a target in Arabidopsis thaliana due to the obvious and non-lethal loss of anthocyanin accumulation upon widespread RNA silencing. Over-expression of CHS in the pap1-D background facilitated visual detection of both local and systemic RNA silencing. RNA silencing was initiated in leaf tissues from hpRNA and amiRNA plant expression vectors under the control of an Arabidopsis RuBisCo small subunit 1A promoter (SSU). In this system, hpRNA expression triggered CHS silencing in most leaf tissues but not in roots or seed coats. Similarly, 21-nt amiRNA expression from symmetric miRNA/miRNA* duplexes triggered CHS silencing in all leaf tissues but not in roots or seed coats. However, 22-nt amiRNA expression from an asymmetric duplex triggered CHS silencing in all tissues, including roots and seed coats, in the majority of plant lines. This widespread CHS silencing required RNA-DEPENDENT RNA POLYMERASE6-mediated accumulation of phasiRNAs from the endogenous CHS transcript. These results demonstrate the efficacy of asymmetric 22-nt amiRNA-directed RNA silencing and associated phasiRNA production and activity, in mediating widespread RNA silencing of an endogenous target gene. Asymmetric 22-nt amiRNA-directed RNA silencing requires little modification of existing amiRNA technology and is expected to be effective in suppressing other genes and/or members of gene families.
已知 22 个核苷酸（nt）的 microRNAs（miRNAs）来源于不对称双链体，触发互补靶标产生相移小干扰 RNA（phasiRNA）。在这里，我们研究了 22 个核苷酸的人工 miRNA（amiRNA）介导的 RNA 沉默相对于传统发夹 RNA（hpRNA）和 21 个核苷酸的 amiRNA 介导的 RNA 沉默的功效。由于广泛的 RNA 沉默会导致拟南芥中花色苷积累明显且非致死性丧失，因此选择查尔酮合酶（CHS）作为靶标。在 pap1-D 背景下过表达 CHS 有助于可视化检测局部和系统的 RNA 沉默。hpRNA 和 amiRNA 植物表达载体在拟南芥 RuBisCo 小亚基 1A 启动子（SSU）的控制下启动 RNA 沉默。在该系统中，hpRNA 表达在大多数叶片组织中触发 CHS 沉默，但在根或种皮中没有。同样，来自对称 miRNA/miRNA*双链体的 21 个核苷酸 amiRNA 表达在所有叶片组织中触发 CHS 沉默，但在根或种皮中没有。然而，来自不对称双链体的 22 个核苷酸 amiRNA 表达在大多数植物系的所有组织中，包括根和种皮，引发 CHS 沉默。这种广泛的 CHS 沉默需要 RNA 依赖性 RNA 聚合酶 6 介导的内源性 CHS 转录物 phasiRNA 的积累。这些结果证明了不对称 22 个核苷酸 amiRNA 指导的 RNA 沉默及其相关的 phasiRNA 产生和活性在介导内源性靶基因的广泛 RNA 沉默方面的功效。不对称 22 个核苷酸 amiRNA 指导的 RNA 沉默需要对现有的 amiRNA 技术进行很少的修改，预计在抑制其他基因和/或基因家族成员方面是有效的。
