A hairpin RNA construct residing in an intron efficiently triggered RNA-directed DNA methylation in tobacco
A hairpin RNA construct residing in an intron efficiently triggered RNA-directed DNA methylation in tobacco
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DOI：
10.1111/j.1365-313x.2009.04003.x
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发表时间：
2009-12-01
期刊：
影响因子：
7.2
通讯作者：
Wassenegger, Michael
中科院分区：
文献类型：
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作者：
Dalakouras, Athanasios;Moser, Mirko;Wassenegger, Michael
到目前为止，由直接引入表达盒的靶启动子的反向重复序列（IR）组成的常规发夹RNA（hpRNA）构建体已用于介导从头DNA甲基化。这种结构的转录物类似于mRNA分子，并且可能被输出到细胞质。hpRNA在细胞质和细胞核中的存在可以解释转录后基因沉默（PTGS）和RNA指导的DNA甲基化（RdDM）的同时激活。我们假设通过在细胞核中保留hpRNA，可以实现仅RdDM的有效诱导。因此，我们向烟草中引入了含有内含子的转基因，其中插入了靶启动子的IR。内含子hpRNA启动高度特异性的顺式和反式甲基化，但不诱导PTGS。未检测到甲基化扩散到hpRNA和靶DNA之间同源性区域侧翼的序列中。内含子hpRNA的高效甲基化指导活性可能表明内含子的先前未被认识的作用，可能在转录水平上调节基因表达。
P>So far, conventional hairpin RNA (hpRNA) constructs consisting of an inverted repeat (IR) of target promoters directly introduced into an expression cassette have been used to mediate de novo DNA methylation. Transcripts of such constructs resemble mRNA molecules, and are likely to be exported to the cytoplasm. The presence of hpRNAs in the cytoplasm and the nucleus may account for the simultaneous activation of post-transcriptional gene silencing (PTGS) and RNA-directed DNA methylation (RdDM). We hypothesized that by retaining hpRNAs in the nucleus, efficient induction of only RdDM may be achieved. Thus, we introduced into tobacco a transgene containing an intron into which an IR of a target promoter was inserted. The intronic hpRNA initiated highly specific cis- and trans-methylation, but did not induce PTGS. No spreading of methylation into sequences flanking the region of homology between the hpRNA and the target DNA was detectable. The efficient methylation-directing activity of the intronic hpRNA may indicate a previously unrecognized role of introns, potentially regulating gene expression at the transcriptional level.