The Role of HEN1 in MicroRNA Biogenesis in Arabidopsis

微小RNA（miRNAs）是存在于多种真核生物中的长度通常为21-25个核苷酸的单链RNA，在正常生长和发育期间充当基因表达的序列特异性调节剂。 miRNA是由一种名为Dicer的RNA酶III从较长的转录物中加工出来的，这些转录物折叠成茎环结构，其中miRNA位于双链区域。 除了Dicer，还有两类分子功能未知的蛋白质Argonaute和HEN 1与miRNA的生物发生有关。 许多miRNA在时间或空间上的表达受到限制，这表明miRNA的生物合成在发育过程中受到严格的调控。 然而，关于miRNA生物合成的途径或调控知之甚少。本研究的目的是使用分子遗传学和生物化学方法剖析HEN 1在miRNA生物合成中的作用。 特别是，这项研究将遵循以前观察的线索，例如在dicer突变体中潜在的miRNA前体的积累和hen 1 -1突变体中异质性miRNA物种的存在，以探索HEN 1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HEN 1的分子功能，一个先锋蛋白质与同源物从后生动物，在miRNA的生物合成，也提供了深入了解在植物中的miRNA的生物合成过程，如所涉及的步骤处理初级转录到成熟的miRNA和处理步骤的亚细胞位置。

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.