Efficient Generation of diRNAs Requires Components in the Posttranscriptional Gene Silencing Pathway.
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.
Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 210602, China. miki@sibs.ac.cn.
Daisuke
D
Miki
Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 210602, China.
Peiying
P
Zhu
Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 210602, China.
Wencan
W
Zhang
Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 210602, China.
Yanfei
Y
Mao
Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 210602, China.
Zhengyan
Z
Feng
Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 210602, China.
Huan
H
Huang
Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 210602, China.
Hui
H
Zhang
Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 210602, China.
Yanqiang
Y
Li
Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 210602, China.
Renyi
R
Liu
Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 210602, China.
Huiming
H
Zhang
Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing, 100084, China.
Yijun
Y
Qi
Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 210602, China.|Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN, 47907, USA.
Jian-Kang
0000-0001-5134-731X
JK
Zhu
D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism; D064113:CRISPR-Cas Systems; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D018390:Gene Targeting; D012275:Oryza / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D058727:RNA, Small Untranslated / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D043244:Ribonuclease III / Q000378:metabolism; D000069896:Transcription Activator-Like Effector Nucleases
D029681:Arabidopsis Proteins; D058727:RNA, Small Untranslated; C514575:RDR6 protein, Arabidopsis; D012324:RNA-Dependent RNA Polymerase; C504982:DCL4 protein, Arabidopsis; D000069896:Transcription Activator-Like Effector Nucleases; D043244:Ribonuclease III