RNA-directed DNA methylation involves co-transcriptional small-RNA-guided slicing of polymerase V transcripts in Arabidopsis.
RNA-directed DNA methylation involves co-transcriptional small-RNA-guided slicing of polymerase V transcripts in Arabidopsis.
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DOI：
10.1038/s41477-017-0100-y
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发表时间：
2018-03
期刊：
影响因子：
18
通讯作者：
Jacobsen SE
中科院分区：
文献类型：
--
作者：
Liu W;Duttke SH;Hetzel J;Groth M;Feng S;Gallego-Bartolome J;Zhong Z;Kuo HY;Wang Z;Zhai J;Chory J;Jacobsen SE
小rna在真核生物基因组中调控染色质修饰，如DNA甲基化和基因沉默。在植物中，rna定向DNA甲基化（RdDM）需要24个核苷酸（nt）的小rna （sirna）结合ARGONAUTE4 （AGO4）和目标基因组区域进行沉默。它还需要RNA聚合酶V （Pol V）转录的非编码RNA，这些非编码RNA可能作为结合AGO4/siRNA复合物的支架。在这里，我们利用改良的全局核运行（GRO）协议，然后进行深度测序，以捕获全基因组的Pol V新生转录本。我们发现了Pol V rna的独特特征，包括在第10位共有的尿嘧啶(U)。该尿嘧啶与许多ag4结合的24-nt sirna中发现的5 '腺嘌呤互补，在sirna缺陷突变体和ag4 /6/9三重突变体中被消除，这表明+10U特征是由于sirna介导的Pol V转录物共转录切割。在AGO4 /6/9中表达野生型AGO4能够恢复Pol V转录本的切片，但催化失活的AGO4突变体不能纠正切片缺陷。我们还发现，Pol V转录物切片需要鲜为人知的延伸因子SPT5L。这些结果强调了Pol V转录物切片在rna介导的转录基因沉默中的重要性，这在许多真核生物中是一个保守的过程。
Small RNAs regulate chromatin modifications such as DNA methylation and gene silencing across eukaryotic genomes. In plants, RNA-directed DNA methylation (RdDM) requires 24-nucleotide (nt) small RNAs (siRNAs) that bind ARGONAUTE4 (AGO4) and target genomic regions for silencing. It also requires non-coding RNAs transcribed by RNA POLYMERASE V (Pol V) that likely serve as scaffolds for binding of AGO4/siRNA complexes. Here we utilized a modified global nuclear run-on (GRO) protocol followed by deep sequencing to capture Pol V nascent transcripts genome-wide. We uncovered unique characteristics of Pol V RNAs, including a uracil (U) common at position 10. This uracil was complementary to the 5′ adenine found in many AGO4-bound 24-nt siRNAs and was eliminated in a siRNA-deficient mutant as well as in the ago4/6/9 triple mutant, suggesting that the +10U signature is due to siRNA-mediated co-transcriptional slicing of Pol V transcripts. Expressing wild-type AGO4 in ago4/6/9 was able to restore slicing of Pol V transcripts but a catalytically inactive AGO4 mutant did not correct the slicing defect. We also found that Pol V transcript slicing required the little understood elongation factor SPT5L. These results highlight the importance of Pol V transcript slicing in RNA-mediated transcriptional gene silencing, which is a conserved process in many eukaryotes.
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影响因子：
30.8
作者：
Noma, K;Sugiyama, T;Grewal, SIS
通讯作者：
Grewal, SIS
影响因子：
64.5
作者：
Li, Carey Fei;Pontes, Olga;Jacobsen, Steven E.
通讯作者：
Jacobsen, Steven E.
DOI：
10.1073/pnas.1420515111
发表时间：
2014-12-09
影响因子：
11.1
作者：
Groth, Martin;Stroud, Hume;Ausin, Israel
通讯作者：
Ausin, Israel
影响因子：
64.8
作者：
通讯作者：
--
影响因子：
10.5
作者：
Vo Ngoc L;Cassidy CJ;Huang CY;Duttke SH;Kadonaga JT
通讯作者：
Kadonaga JT