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

微小RNA （microRNA, miRNA）和沉默RNA （silencing RNA, siRNA）是近年来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调控机制和生物发生上有相似之处，但在细节上却有明显的不同。在植物中，mirna和sirna都主要针对互补rna进行破坏，而在动物中，mirna主要调节翻译。21- 22nt mirna在植物分生组织功能、叶片极性、花的身份和时间以及根和维管发育中起重要作用。较长的24- 25nt sirna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形成。mirna来源于前体的短茎环片段（pri- mirna），这些前体在基因组中编码为单独的基因或在常规基因的转录本中，有时在内含子中。sirna来源于较长的双链RNA分子，这些分子要么是内源性产生的，要么是外源性引入的。虽然最初认为它们是独立的系统，但已经描述了涉及miRNA和siRNA的调节机制。拟南芥mirna的生物发生涉及一种rnaseiii家族酶Dicerlike1 (DCL1)，一种dsrna结合蛋白Hyponastic Leaves1 （HYL1）和一种RNA甲基转移酶HUA增强酶1 （HEN1）。它也可能需要一种由最近发现的SERRATE （SE）基因编码的蛋白质。本实验室之前的工作证实，将pri-miRNA加工成pre-miRNA需要HYL1蛋白。本项目的总体目标是通过以下3个具体目标来了解HYL1蛋白在miRNA生物发生中的功能：1)表征HYL1复合物；2)体外重构miRNA加工；3)研究pri-miRNA加工和miRNA基因调控的机制。最近在实验室的进展已经允许扩展可能的方法来识别参与miRNA前体加工的蛋白质。通过共表达GFP融合蛋白，我们确定DCL1和HYL1共定位于小的核周小体，这些小体不同于含有RNA沉默机制的类似Cajal小体，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良好的位置，可以在体外重建miRNA加工，快速分析正确miRNA加工的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mirna的基因的调控。知识价值。这个项目将扩大我们对植物中涉及小rna的基因调控的一个极其重要的方面的理解。对HYL1蛋白的研究将增强我们对植物miRNA生物发生的基本认识，而对miRNA调控体系的研究将加深我们对miRNA调控体系的认识。更广泛的影响。基于小rna介导机制的病毒抗性已被证明在农业中很有价值。对小RNA机制的深入了解可能会产生有益的应用，促进基于诱导小RNA的机制的发展，使植物能够承受比目前更极端的环境条件而不影响其生产力。

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.