CSIRO Agriculture and Food, Clunies Ross Street, Canberra, ACT 2610, Australia.
School of Chemistry & Molecular Bioscience, University of Wollongong, Wollongong, NSW, 2522, Australia.
Nat Commun. 2022 Jul 7;13(1):3926. doi: 10.1038/s41467-022-31641-5.
Hairpin RNA (hpRNA) transgenes are the most successful RNA interference (RNAi) method in plants. Here, we show that hpRNA transgenes are invariably methylated in the inverted-repeat (IR) DNA and the adjacent promoter, causing transcriptional self-silencing. Nucleotide substitutions in the sense sequence, disrupting the IR structure, prevent the intrinsic DNA methylation resulting in more uniform and persistent RNAi. Substituting all cytosine with thymine nucleotides, in a G:U hpRNA design, prevents self-silencing but still allows for the formation of hpRNA due to G:U wobble base-pairing. The G:U design induces effective RNAi in 90-96% of transgenic lines, compared to 57-65% for the traditional hpRNA design. While a traditional hpRNA transgene shows increasing self-silencing from cotyledons to true leaves, its G:U counterpart avoids this and induce RNAi throughout plant growth. Furthermore, siRNAs from G:U and traditional hpRNA show different characteristics and appear to function via different pathways to induce target DNA methylation.
发夹 RNA (hpRNA) 转基因是植物中最成功的 RNA 干扰 (RNAi) 方法。在这里，我们表明 hpRNA 转基因在反向重复 (IR) DNA 和相邻启动子中总是被甲基化，导致转录自我沉默。在有义序列中发生核苷酸取代，破坏了 IR 结构，阻止了内在的 DNA 甲基化，从而导致更均匀和持久的 RNAi。用胸腺嘧啶核苷酸替代所有胞嘧啶核苷酸，在 G:U hpRNA 设计中，可防止自我沉默，但仍允许由于 G:U 摆动碱基配对而形成 hpRNA。与传统的 hpRNA 设计相比，G:U 设计在 90-96%的转基因系中诱导有效的 RNAi，而在传统的 hpRNA 设计中为 57-65%。虽然传统的 hpRNA 转基因从子叶到真叶显示出越来越强的自我沉默，但它的 G:U 对应物避免了这种情况，并在整个植物生长过程中诱导 RNAi。此外，来自 G:U 和传统 hpRNA 的 siRNA 表现出不同的特征，似乎通过不同的途径诱导靶 DNA 甲基化来发挥作用。
