The N-terminal double-stranded RNA binding domains of Arabidopsis HYPONASTIC LEAVES1 are sufficient for pre-microRNA processing
The N-terminal double-stranded RNA binding domains of Arabidopsis HYPONASTIC LEAVES1 are sufficient for pre-microRNA processing
复制标题
拟南芥下叶的 N 端双链 RNA 结合域1足以进行 microRNA 前处理
DOI：
10.1105/tpc.106.048637
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发表时间：
2007-03-01
期刊：
影响因子：
11.6
通讯作者：
He, Yuke
中科院分区：
文献类型：
--
作者：
Wu, Feijie;Yu, Lin;He, Yuke
拟南芥HYL1是一种微小RNA(MiRNA)生物发生蛋白，它含有两个N-末端双链RNA结合域(DsRBD)、一个可能的核定位位点(NLS)和一个可能的蛋白质-蛋白质相互作用结构域。在植物miRNA的生物发生过程中，HYL1与Disher-like 1的相互作用对于miRNA初级转录本的高效和精确处理是重要的。为了确定HYL1的各个结构域在miRNA加工和miRNA导向的表型中的作用，我们将一系列HYL1缺失结构转入hyl1缺失突变体。含有dsRBD1和dsRBD2的N-末端区域完全挽救了hyl1的突变表型，触发了miR166和miR160的积累，导致靶基因的mRNA水平降低。对转基因植株中含有HYL1的复合体的体内生化分析表明，HYL1的N端dsRBD足以加工miRNA前体和产生成熟的miRNA。瞬时和稳定表达分析表明，推测的NLS结构域确实是核定位信号，而含有dsRBDS的N-末端区域并不局限于核。我们认为，N-末端dsRBD完成了整个HYL1的功能，从而在miRNA加工和miRNA指导的靶基因沉默中发挥了重要作用。
Arabidopsis thaliana HYPONASTIC LEAVES1 (HYL1) is a microRNA (miRNA) biogenesis protein that contains two N-terminal double-stranded RNA binding domains (dsRBDs), a putative nuclear localization site (NLS), and a putative protein-protein interaction domain. The interaction of HYL1 with DICER-LIKE1 is important for the efficient and precise processing of miRNA primary transcripts in plant miRNA biogenesis. To define the roles of the various domains of HYL1 in miRNA processing and the miRNA-directed phenotype, we transferred a series of HYL1 deletion constructs into hyl1 null mutants. The N-terminal region containing dsRBD1 and dsRBD2 completely rescued the mutant phenotype of hyl1, triggering the accumulation of miR166 and miR160 and resulting in reduced mRNA levels of the targeted genes. In vivo biochemical analysis of the HYL1-containing complexes from the transgenic plants revealed that the N-terminal dsRBDs of HYL1 were sufficient for processing miRNA precursors and the generation of mature miRNA. Transient and stable expression analysis demonstrated that the putative NLS domain was indeed the nuclear localization signal, whereas the N-terminal region containing the dsRBDs was not restricted to the nucleus. We suggest that the N-terminal dsRBDs fulfill the function of the whole HYL1 and thus play an essential role in miRNA processing and miRNA-directed silencing of targeted genes.