Small RNA deep sequencing reveals role for Arabidopsis thaliana RNA-dependent RNA polymerases in viral siRNA biogenesis.
Small RNA deep sequencing reveals role for Arabidopsis thaliana RNA-dependent RNA polymerases in viral siRNA biogenesis.
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DOI：
10.1371/journal.pone.0004971
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发表时间：
2009
期刊：
影响因子：
3.7
通讯作者：
Xie Z
中科院分区：
文献类型：
--
作者：
Qi X;Bao FS;Xie Z
RNA沉默作为一种重要的抗病毒防御机制在广泛的真核生物中发挥作用。在植物中，几类内源性小干扰RNA（siRNA）的生物发生需要RNA依赖性RNA聚合酶（RDR）活性。RDR家族蛋白的成员，包括RDR 1和RDR 6，也参与了抗病毒防御，尽管RDRs在病毒siRNA生物发生中的直接作用尚未得到证实。以烟草花叶病毒（TMV-Cg）十字花科侵染株系和拟南芥为模型系统，应用小RNA深度测序技术，分析了野生型植物和rdr突变体中病毒小RNA的表达谱。在早期感染阶段捕获了超过100，000个TMV-Cg特异性小RNA读段，大部分为21-（78.4%）和22-核苷酸（12.9%）大小，并且主要（79.9%）来自基因组正义RNA链，产生了植物病毒的第一个高分辨率小RNA图谱。TMV-Cg基因组含有多个高度可重复的小RNA生成热点，这些热点对应于没有明显的局部发夹形成能力的区域。值得注意的是，rdr 1和rdr 6突变体均表现出病毒小RNA产生水平的整体降低以及病毒小RNA群体中链偏差的降低，揭示了这些宿主RDRs在病毒siRNA生物发生中的重要作用。此外，信息学分析表明，大量的宿主基因可能被TMV-Cg衍生的siRNA靶向用于转录后沉默。其中两个预测的宿主靶点分别编码切割和多聚腺苷酸化特异性因子（CPSF 30）和一种类似于translocon相关蛋白α（TRAP α）的未知蛋白，在5′RACE检测的切割验证中得到阳性结果。我们的数据提出了病毒siRNA介导的病毒-宿主相互作用的有趣的可能性，可能有助于病毒的致病性和宿主特异性。
RNA silencing functions as an important antiviral defense mechanism in a broad range of eukaryotes. In plants, biogenesis of several classes of endogenous small interfering RNAs (siRNAs) requires RNA-dependent RNA Polymerase (RDR) activities. Members of the RDR family proteins, including RDR1and RDR6, have also been implicated in antiviral defense, although a direct role for RDRs in viral siRNA biogenesis has yet to be demonstrated. Using a crucifer-infecting strain of Tobacco Mosaic Virus (TMV-Cg) and Arabidopsis thaliana as a model system, we analyzed the viral small RNA profile in wild-type plants as well as rdr mutants by applying small RNA deep sequencing technology. Over 100,000 TMV-Cg-specific small RNA reads, mostly of 21- (78.4%) and 22-nucleotide (12.9%) in size and originating predominately (79.9%) from the genomic sense RNA strand, were captured at an early infection stage, yielding the first high-resolution small RNA map for a plant virus. The TMV-Cg genome harbored multiple, highly reproducible small RNA-generating hot spots that corresponded to regions with no apparent local hairpin-forming capacity. Significantly, both the rdr1 and rdr6 mutants exhibited globally reduced levels of viral small RNA production as well as reduced strand bias in viral small RNA population, revealing an important role for these host RDRs in viral siRNA biogenesis. In addition, an informatics analysis showed that a large set of host genes could be potentially targeted by TMV-Cg-derived siRNAs for posttranscriptional silencing. Two of such predicted host targets, which encode a cleavage and polyadenylation specificity factor (CPSF30) and an unknown protein similar to translocon-associated protein alpha (TRAP α), respectively, yielded a positive result in cleavage validation by 5′RACE assays. Our data raised the interesting possibility for viral siRNA-mediated virus-host interactions that may contribute to viral pathogenicity and host specificity.
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作者：
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