The Roles of MicroRNAs in Plant Development

描述（由申请人提供）：了解植物或动物的发育需要了解从干细胞分化到所有不同细胞类型和组织所需的协调基因表达程序的调控电路。小的非编码rna，被称为microrna (mirna)，最近被认为是这种调节回路的组成部分。mirna是一种微小的rna，长度约为22个核苷酸，被认为使用rna干扰途径的元件来转录后下调蛋白质编码基因的表达。在动物中发现了许多miRNA基因，它们占总基因的0.5%到1%。在植物中也发现了一些mirna，与动物mirna相比，已经通过计算确定了几乎所有这些植物mirna的调控靶点。本提案的合作研究计划侧重于植物miRNAs及其预测靶点，以了解miRNAs在植物发育过程中调节基因表达的作用为广泛而长期的目标。提出的研究的具体目的是：1)检查破坏miRNA调节的表型后果。2)目标mrna的空间表达与miRNA功能定位的相关性。3)鉴定更多的植物mirna和候选靶点。在目标1和目标2的实验中，将构建miRNA调控中断或异位的转基因植物，并进行分子（例如，原位杂交，分子报告）和形态计量学分析。这些实验将确定适当的miRNA功能的生物学重要性，并确定miRNA靶点的发育作用。他们还将阐明任何发育异常的分子基础，特别是研究mirna在清除女儿细胞系的关键调控转录本中的假设作用。在aim #3的实验中，将通过cDNA克隆和随后的计算分析来鉴定额外的mirna及其靶标。由于对这类新发现的基因知之甚少，人们普遍猜测它们在人类中可能起作用的过程。就像之前对植物中rna介导的基因沉默的研究为理解动物中类似的过程提供了信息一样，鉴定植物mirna并研究它们的生物学作用将为理解mirna在人类中的作用以及它们的功能障碍如何导致疾病提供重要的见解和试剂。

DESCRIPTION (provided by applicant): Understanding plant or animal development requires knowing the regulatory circuitry that orchestrates the gene-expression programs needed to differentiate from a stem cell to all the different cell-types and tissues. Small non-coding RNAs, known as microRNAs (miRNAs), have recently been implicated as components of this regulatory circuitry. MiRNAs are tiny RNAs, about 22 nucleotides in length, that are thought to use elements of the RNA-interference pathway to posttranscriptionally down-regulate the expression of protein-coding genes. Many miRNA genes have been found in animals, where they comprise between 0.5 and 1% of the total genes. Some miRNAs have also been found in plants, and in contrast to the animal miRNAs, regulatory targets have been computationally identified for nearly all of these plant miRNAs. The collaborative research plan of this proposal focuses on the plant miRNAs and their predicted targets, with the broad, long-term objective of understanding the roles of miRNAs in regulating gene expression during plant development. The specific aims of the proposed research are: 1) To examine the phenotypic consequences of disrupting miRNA regulation. 2) To correlate the spatial expression of target mRNAs with the localization of miRNA function. 3) To identify additional plant miRNAs and candidate targets. In experiments of aims #1 and #2, transgenic plants with disrupted or ectopic miRNA regulation will be constructed and subjected to molecular (e.g., in situ hybridizations, molecular reporters) and morphometric analyses. These experiments will determine the biological importance of proper miRNA function and identify developmental roles for miRNA targets. They also will elucidate the molecular bases of any developmental abnormalities, specifically investigating the hypothesized role of miRNAs in clearing key regulatory transcripts from daughter-cell lineages. In experiments of aim #3, additional miRNAs and their targets will be identified by cDNA cloning and subsequent computational analyses. Because so little is known about this newly identified class of genes, speculation is rife on the processes in which they might function in humans. In the same way that previous work on RNA-mediated gene silencing in plants has informed the understanding similar processes in animals, identifying plant miRNAs and studying their biological roles will provide important insights and reagents for understanding the roles of miRNAs in humans and how their dysfunction might contribute to disease.