RNA group/Groupe ARN, Département de biochimie, Faculté de médecine et des sciences de santé, Université de Sherbrooke, Sherbrooke, QC, J1H 5N4, Canada.
BMC Mol Biol. 2010 Feb 16;11:16. doi: 10.1186/1471-2199-11-16.
It has been observed that following viroid infection, there is an accumulation of viroid-derived siRNAs in infected plants. Some experimental results suggest that these small RNAs may be produced by the plant defense system to protect it from infection, indicating that viroids can elicit the RNA-silencing pathways. The objective of this study is to identify in the peach latent mosaic viroid (PLMVd), a model RNA genome, the regions that are most susceptible to RNA interference machinery.
The RNA isolated from an infected tree have been used to sequence in parallel viroid species and small non-coding RNA species. Specifically, PLMVd RNAs were amplified, cloned and sequenced according to a conventional approach, while small non-coding RNAs were determined by high-throughput sequencing. The first led to the typing of 18 novel PLMVd variants. The second provided a library of small RNAs including 880 000 sequences corresponding to PLMVd-derived siRNAs, which makes up 11.2% of the sequences of the infected library. These siRNAs contain mainly 21-22 nucleotide RNAs and are equivalently distributed between the plus and the minus polarities of the viroid. They cover the complete viroid genome, although the amount varies depending on the regions. These regions do not necessarily correlate with the double-stranded requirement to be a substrate for Dicer-like enzymes. We noted that some sequences encompass the hammerhead self-cleavage site, indicating that the circular conformers could be processed by the RNA-silencing machinery. Finally, a bias in the relative abundance of the nature of the 5' nucleotides was observed (A, U >> G, C).
The approach used provided us a quantitative representation of the PLMVd-derived siRNAs retrieved from infected peach trees. These siRNAs account for a relatively large proportion of the small non-coding RNAs. Surprisingly, the siRNAs from some regions of the PLMVd genome appear over-represented, although these regions are not necessarily forming sufficiently long double-stranded structures to satisfy Dicer-like criteria for substrate specificity. Importantly, this large library of siRNAs gave several hints as to the components of the involved silencing machinery.
研究发现，感染类病毒后，植物中会积累类病毒衍生的 siRNA。一些实验结果表明，这些小 RNA 可能是植物防御系统产生的，以保护其免受感染，这表明类病毒可以引发 RNA 沉默途径。本研究的目的是鉴定在桃潜隐花叶病毒（PLMVd）中，一个模型 RNA 基因组，哪些区域最易受 RNA 干扰机制的影响。
从感染的树木中分离出的 RNA 已被用于同时对类病毒物种和小非编码 RNA 物种进行测序。具体来说，根据常规方法扩增、克隆和测序了 PLMVd RNA，同时通过高通量测序确定了小非编码 RNA。第一种方法导致了 18 种新型 PLMVd 变体的分型。第二种方法提供了一个小 RNA 文库，包括 880000 个对应于 PLMVd 衍生 siRNA 的序列，占感染文库序列的 11.2%。这些 siRNA 主要包含 21-22 个核苷酸的 RNA，并且在病毒的正负链上均匀分布。它们覆盖了完整的类病毒基因组，尽管不同区域的含量不同。这些区域不一定与双链结构作为 Dicer-like 酶的底物相关。我们注意到，一些序列包含锤头自我切割位点，表明环状构象可以被 RNA 沉默机制加工。最后，观察到 5'核苷酸性质的相对丰度存在偏差（A、U>>G、C）。
使用的方法为我们提供了从感染桃树中提取的 PLMVd 衍生 siRNA 的定量表示。这些 siRNA 占小非编码 RNA 的相对较大比例。令人惊讶的是，PLMVd 基因组的一些区域的 siRNA 似乎过度表达，尽管这些区域不一定形成足够长的双链结构以满足 Dicer-like 对底物特异性的标准。重要的是，这个大型 siRNA 文库为参与的沉默机制的组成部分提供了一些线索。
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