The DNA/RNA-Dependent RNA Polymerase QDE-1 Generates Aberrant RNA and dsRNA for RNAi in a Process Requiring Replication Protein A and a DNA Helicase
The DNA/RNA-Dependent RNA Polymerase QDE-1 Generates Aberrant RNA and dsRNA for RNAi in a Process Requiring Replication Protein A and a DNA Helicase
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DOI：
10.1371/journal.pbio.1000496
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发表时间：
2010-10-01
期刊：
影响因子：
9.8
通讯作者：
Liu, Yi
中科院分区：
文献类型：
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作者：
Lee, Heng-Chi;Aalto, Antti P.;Liu, Yi
异常RNA(Arna)的产生是几种RNAi途径的第一步。Arna是如何产生的，并被RNA依赖的RNA聚合酶(RdRPs)特异性识别以产生双链RNA(DsRNA)，目前尚不清楚。我们之前的研究表明，在丝状真菌Neuropora中，Rdrp QDE-1是rDNA特异性Arna合成所必需的，这表明QDE-1在Arna的合成中可能是重要的。在这里，我们证明了重组QDE-1既是RdRP又是DNA依赖的RNA聚合酶(DdRP)。它的DdRP活性比RdRP活性强得多，并且存在于ssDNA模板上，而不是dsDNA模板上。我们进一步证明，复制蛋白A(RPA)是一种与QDE-1相互作用的单链DNA结合复合体，对于Arna的产生和基因沉默是必不可少的。体外重组分析表明，QDE-1可以从单链DNA中产生dsRNA，这一过程受到RPA的强烈促进。此外，QDE-1和RPA之间的相互作用需要RecQ DNA解旋酶QDE-3，它是人类Werner/Bloom综合征蛋白的同源物。综上所述，这些结果提示了一种新的脉孢子菌小RNA生物发生途径，以及一种在RNAi途径中产生Arna和dsRNA的新机制。
The production of aberrant RNA (aRNA) is the initial step in several RNAi pathways. How aRNA is produced and specifically recognized by RNA-dependent RNA polymerases (RdRPs) to generate double-stranded RNA (dsRNA) is not clear. We previously showed that in the filamentous fungus Neurospora, the RdRP QDE-1 is required for rDNA-specific aRNA production, suggesting that QDE-1 may be important in aRNA synthesis. Here we show that a recombinant QDE-1 is both an RdRP and a DNA-dependent RNA polymerase (DdRP). Its DdRP activity is much more robust than the RdRP activity and occurs on ssDNA but not dsDNA templates. We further show that Replication Protein A (RPA), a single-stranded DNA-binding complex that interacts with QDE-1, is essential for aRNA production and gene silencing. In vitro reconstitution assays demonstrate that QDE-1 can produce dsRNA from ssDNA, a process that is strongly promoted by RPA. Furthermore, the interaction between QDE-1 and RPA requires the RecQ DNA helicase QDE-3, a homolog of the human Werner/Bloom Syndrome proteins. Together, these results suggest a novel small RNA biogenesis pathway in Neurospora and a new mechanism for the production of aRNA and dsRNA in RNAi pathways.