Efficient Generation of diRNAs Requires Components in the Posttranscriptional Gene Silencing Pathway.
Efficient Generation of diRNAs Requires Components in the Posttranscriptional Gene Silencing Pathway.
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DOI：
10.1038/s41598-017-00374-7
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发表时间：
2017-03-22
影响因子：
4.6
通讯作者：
Zhu JK
中科院分区：
文献类型：
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作者：
Miki D;Zhu P;Zhang W;Mao Y;Feng Z;Huang H;Zhang H;Li Y;Liu R;Zhang H;Qi Y;Zhu JK
据报道，双链断裂（DSB）诱导的小rna （diRNAs）是通过rna导向的DNA甲基化途径产生的，并在拟南芥的DSB修复中起作用。然而，关于dirna的生物发生和功能仍然存在重要的问题。在这里，我们使用CRISPR/Cas9或talen触发的dsb来表征拟南芥和水稻中的diRNAs。我们发现21-nt diRNAs是由CRISPR/Cas9靶向的35S启动子：：GU-US报告基因产生的。出乎意料的是，高效的diRNA产生需要转基因的Pol II转录，并且diRNA的积累水平与转基因的表达水平相关。在拟南芥或水稻的内源基因中，未检测到CRISPR/Cas9或talen诱导的dsb中的diRNAs。我们还发现，已知参与转录后基因沉默的DCL4和RDR6是生成dirna所必需的。我们的研究结果表明，DSB对于有效的diRNA生成是必要的，但不是充分的，并且高水平的diRNA对于DSB修复不是必需的。
It has been reported that double-stranded break (DSB)-induced small RNAs (diRNAs) are generated via the RNA-directed DNA methylation pathway and function in DSB repair in Arabidposis. However, important questions remain regarding the biogenesis and function of diRNAs. Here, we used CRISPR/Cas9- or TALEN-triggered DSBs to characterize diRNAs in Arabidopsis and rice. We found that 21-nt diRNAs were generated from a 35S promoter::GU-US reporter transgene targeted by CRISPR/Cas9. Unexpectedly, Pol II transcription of the transgene was required for efficient diRNA production and the level of diRNA accumulation correlated with the expression level of the transgene. diRNAs were not detected from CRISPR/Cas9- or TALEN-induced DSBs within the examined endogenous genes in Arabidopsis or rice. We also found that DCL4 and RDR6 that are known to be involved in posttranscriptional gene silencing were required to generate diRNAs. Our results suggest that DSBs are necessary but not sufficient for efficient diRNA generation and a high level of diRNAs is not necessary for DSB repair.