Homology-independent discovery of replicating pathogenic circular RNAs by deep sequencing and a new computational algorithm
Homology-independent discovery of replicating pathogenic circular RNAs by deep sequencing and a new computational algorithm
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DOI：
10.1073/pnas.1117815109
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发表时间：
2012-03-06
影响因子：
11.1
通讯作者：
Ding, Shou-Wei
中科院分区：
文献类型：
--
作者：
Wu, Qingfa;Wang, Ying;Ding, Shou-Wei
通过深度测序方法发现病原体的一个常见挑战是识别患病组织样本中与已知病原体没有显着同源性的病毒或亚病毒病原体。在这里，我们报告了一种用于发现类病毒的同源独立方法，类病毒是一类独特的游离环状 RNA 亚病毒病原体，不编码蛋白质，已知仅感染植物。我们的方法包括使用独特的计算算法、逐步过滤重叠小RNA (PFOR) 来分析通过深度测序获得的受感染植物的总小RNA 序列。类病毒感染会触发类病毒衍生的重叠 siRNA 的产生，这些 siRNA 高密度地覆盖整个基因组。 PFOR 通过逐步消除非重叠小 RNA 和那些重叠但不能组装成直接重复 RNA 的小 RNA（在类病毒复制过程中由环状或多聚重复序列模板合成），保留用于基因组组装的类病毒特异性 siRNA。我们表明，来自两个已知家族的类病毒很容易被识别，并且它们的全长序列通过 PFOR 从受感染植物测序的小 RNA 组装而成。对葡萄文库的 PFOR 分析进一步鉴定出一种类病毒的环状 RNA，长度为 375 nt，与已知分子没有显着的序列同源性，并在正负极性的 RNA 中编码活性锤头核酶，这些核酶可能会自我裂解，从多聚体复制中间体中释放单体。讨论了同源无关方法在植物和动物物种中发现类病毒的潜在应用，其中 RNA 复制触发 siRNA 的生物发生。
A common challenge in pathogen discovery by deep sequencing approaches is to recognize viral or subviral pathogens in samples of diseased tissue that share no significant homology with a known pathogen. Here we report a homology-independent approach for discovering viroids, a distinct class of free circular RNA subviral pathogens that encode no protein and are known to infect plants only. Our approach involves analyzing the sequences of the total small RNAs of the infected plants obtained by deep sequencing with a unique computational algorithm, progressive filtering of overlapping small RNAs (PFOR). Viroid infection triggers production of viroid-derived overlapping siRNAs that cover the entire genome with high densities. PFOR retains viroid-specific siRNAs for genome assembly by progressively eliminating nonoverlapping small RNAs and those that overlap but cannot be assembled into a direct repeat RNA, which is synthesized from circular or multimeric repeated-sequence templates during viroid replication. We show that viroids from the two known families are readily identified and their full-length sequences assembled by PFOR from small RNAs sequenced from infected plants. PFOR analysis of a grapevine library further identified a viroid-like circular RNA 375 nt long that shared no significant sequence homology with known molecules and encoded active hammerhead ribozymes in RNAs of both plus and minus polarities, which presumably self-cleave to release monomer from multimeric replicative intermediates. A potential application of the homology-independent approach for viroid discovery in plant and animal species where RNA replication triggers the biogenesis of siRNAs is discussed.