The Role of HEN1 in MicroRNA Biogenesis in Arabidopsis

MicroRNAs （miRNAs）是一种单链rna，长度通常为21-25个核苷酸，存在于多种真核生物中，是正常生长发育过程中基因表达的序列特异性调控因子。mirna由一种名为Dicer的RNAse III酶处理，从较长的转录本折叠成茎环结构，mirna位于双链区域。除了Dicer外，Argonaute和HEN1这两类分子功能未知的蛋白也参与了miRNA的生物发生。许多miRNA的表达在时间或空间上受到限制，这表明miRNA的生物发生在发育过程中受到严格调控。然而，对miRNA生物发生的途径和调控知之甚少。本研究的目的是利用分子遗传学和生化方法剖析HEN1在miRNA生物发生中的作用。特别是，本研究将遵循先前观察的线索，例如dicer突变体中潜在miRNA前体的积累以及HEN1 -1突变体中异质miRNA物种的存在，以探索HEN1在miRNA生物发生中的潜在作用，例如miRNA前体或miRNA的产生，稳定或核输出。这项研究将揭示HEN1在miRNA生物发生中的分子功能，以及对植物中miRNA生物发生过程的了解，例如将初级转录物加工成成熟miRNA的步骤和加工步骤的亚细胞位置。HEN1是后生动物中具有同源物的先驱蛋白。

MicroRNAs (miRNAs), single-stranded RNAs of usually 21-25 nucleotides in length found in diverse eukaryotes, act as sequence-specific regulators of gene expression during normal growth and development. miRNAs are processed, by an RNAse III enzyme named Dicer, from longer transcripts that fold into stem-loop structures with the miRNAs being in the double-stranded regions. In addition to Dicer, two classes of proteins of unknown molecular functions, Argonaute and HEN1, have been implicated in miRNA biogenesis. The temporally or spatially restricted expression of many miRNAs suggests that miRNA biogenesis is tightly regulated during development. However, little is known about the pathway or the regulation of miRNA biogenesis. The goal of this research is to dissect the role of HEN1 in miRNA biogenesis using molecular genetic and biochemical approaches. In particular, this research will follow clues from previous observations, such as the accumulation of a potential miRNA precursor in a dicer mutant and the presence of heterogeneous miRNA species in the hen1-1 mutant to probe the potential roles of HEN1 in miRNA biogenesis, such as production, stabilization, or nuclear export of miRNA precursors or miRNAs. This investigation will shed light on the molecular function of HEN1, a pioneer protein with homologs from metazoans, in miRNA biogenesis and also provide insights into the poorly understood miRNA biogenesis process in plants, such as the steps involved to process the primary transcript to the mature miRNA and the subcellular locations of the processing steps.