The Arabidopsis HYL1 Protein and the Role of Small RNAs in Stress Physiology.

近年来，非常小的19-25个核苷酸的RNA，被称为microRNA(MiRNA)和siRNA(SiRNA)，已经成为真核生物主要遗传调控机制的关键组成部分。虽然动物和植物在小RNA调节机制和生物发生方面都有相似之处，但细节明显不同。在植物中，miRNAs和siRNAs主要针对互补的RNAs进行破坏，而在动物中，miRNAs主要调节翻译。植物的21-22个核苷酸的miRNAs在分生组织的功能、叶片的极性、花的特性和时机以及根和维管的发育中都是重要的。较长的24-25个核苷酸的siRNAs参与病毒和病原体抗性、转基因和转座子沉默以及异染色质的形成。MiRNAs是由基因组中编码的前体(pri-miRNAs)的短茎环片段衍生而来的，这些基因要么作为单独的基因编码，要么在常规基因的转录本中，有时在内含子中。SiRNA是由更长的、主要是双链的RNA分子衍生而来的，这些RNA分子要么是内源产生的，要么是外源引入的。虽然最初被认为是独立的系统，但已经描述了涉及miRNA和siRNA的调节机制。拟南芥miRNAs的生物发生涉及一种名为Dicerlike 1(DCL1)的RNAseIII家族酶、一种名为Hypontic LEAVES1(HYL1)的dsRNA结合蛋白(HYL1)和一种名为Huenancer1(HEN1)的RNA甲基转移酶。它还可能需要由最近发现的锯齿(SE)基因编码的蛋白质。本实验室以前的工作证实，HYL1蛋白是将pri-miRNA加工成前miRNA所必需的。本项目的总体目标是通过以下3个具体目标了解HYL1蛋白在miRNA生物发生中的功能：1)表征HYL1复合体，2)在体外重组miRNA加工，3)研究pri-miRNA加工和miRNA基因的调控机制。实验室的最新进展使鉴定miRNA前体加工所涉及的蛋白质的可能方法得以扩展。通过共表达GFP融合蛋白，DCL1和HYL1被证实共定位于小的核周体，这些小体不同于含有RNA沉默机制的类似的卡哈尔小体，被称为微处理器中心。最近一些分子工具的构建使该项目处于有利地位，可以在体外重建miRNA加工，快速分析正确的miRNA加工所需的RNA结构要求，并确定各种蛋白质在pri-miRNA加工中的功能。最后，将研究编码miRNAs的基因本身的调节。这个项目将扩大我们对涉及植物小RNA的基因调控的一个极其重要的方面的理解。对HYL1蛋白的研究将加深我们对miRNA在植物中生物发生的基本理解，而调控研究将加深我们对miRNA调控层次的理解。更广泛的影响。基于小RNA介导机制的病毒抗性已经被证明在农业中有价值。更多地了解小RNA机制可能会产生有益的应用，促进基于诱导的小RNA机制的开发，这些机制可能允许植物在不影响其生产力的情况下承受比目前更极端的环境条件。

Very small 19-25 nucleotide RNAs, termed microRNA (miRNA) and silencing RNA (siRNA), have emerged in recent years as key components of major genetic regulatory mechanisms in eukaryotic organisms. While there are similarities between animals and plants both in the small RNA regulatory mechanisms and in their biogenesis, the details differ markedly. In plants, both miRNAs and siRNAs primarily target complementary RNAs for destruction, while in animals miRNAs largely modulate translation. The 21-22 nt miRNAs of plants are important in meristem function, leaf polarity, floral identity and timing, as well as root and vascular development. The longer 24-25 nt siRNAs are involved in viral and pathogen resistance, transgene and transposon silencing, and heterochromatin formation. MiRNAs are derived from short stem-loop segments of precursors (pri-miRNAs) encoded in the genome either as separate genes or within the transcripts of conventional genes, sometimes in introns. SiRNAs are derived from longer, largely double-stranded RNA molecules that are either produced endogenously or introduced exogenously. Although originally thought to be separate systems, regulatory mechanisms have been described that involve both miRNA and siRNA. The biogenesis of miRNAs in Arabidopsis involves an RNAseIII-family enzyme called Dicerlike1 (DCL1), a dsRNA-binding protein called Hyponastic Leaves1 (HYL1) and an RNA methyl transferase called HUA enchancer1 (HEN1). It may also require a protein encoded by the recently identified SERRATE (SE) gene.Previous work in this laboratory established that the HYL1 protein is required for processing pri-miRNA to pre-miRNA. The overall goal of this project is to understand the function of the HYL1 protein in miRNA biogenesis through the following specific 3 objectives: 1) characterizing the HYL1 complex, 2) reconstituting miRNA processing in vitro, and 3) investigating the mechanism of pri-miRNA processing and regulation of miRNA genes. Recent progress in the laboratory has allowed an expansion in possible approaches to identifying the proteins involved in miRNA precursor processing. By co-expressing GFP fusion proteins, it was established that DCL1 and HYL1 co-localize to small perinucleolar bodies that are distinct from the similar Cajal bodies that contain the RNA silencing machinery, designated Microprocessor centers. The recent construction of a number of molecular tools puts the project in a good position to reconstitute miRNA processing in vitro, to rapidly analyze the RNA structural requirements for correct miRNA processing and to define the functions of various proteins in pri-miRNA processing. Finally, the regulation of genes encoding the miRNAs themselves will be investigated.Intellectual merit. This project will enlarge our understanding of an extremely important aspect of gene regulation involving small RNAs in plants. Studies on the HYL1 protein will enhance our basic understanding of miRNA biogenesis in plants, while the regulatory studies will deepen our understanding of the miRNA regulatory hierarchy. Broader impact. Viral resistance based on small RNA-mediated mechanisms has already proven valuable in agriculture. Greater understanding of small RNA mechanisms is likely to generate beneficial applications, facilitating the development of inducible small RNA-based mechanisms that may allow plants to withstand more extreme environmental conditions than they can at present without compromising their productivity.