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知道协调从干细胞到所有不同细胞类型和组织所需的基因表达程序的调控电路。小的非编码RNA，称为microRNAs(MiRNAs)，最近被认为是这种调节电路的组成部分。MiRNAs是一种微小的RNAs，长度约为22个核苷酸，被认为利用RNA干扰途径的元件在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miRNAs，与动物的miRNAs相反，几乎所有这些植物的miRNAs都通过计算确定了调控靶点。这项建议的合作研究计划侧重于植物miRNAs及其预测的靶标，其广泛的长期目标是了解miRNAs在植物发育过程中调节基因表达的作用。拟议研究的具体目的是：1)检查干扰miRNA调控的表型后果。2)研究靶mRNAs的空间表达与miRNA功能定位的关系。3)识别更多的植物miRNAs和候选靶标。在AIMS#1和#2的实验中，将构建具有干扰或异位miRNA调控的转基因植株，并进行分子(例如，原位杂交、分子记者)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靶标的发育作用。他们还将阐明任何发育异常的分子基础，特别是研究miRNAs在清除子代细胞谱系中关键调控转录的假设作用。在AIM#3的实验中，将通过cDNA克隆和随后的计算分析来识别更多的miRNAs及其靶标。由于人们对这类新发现的基因知之甚少，因此人们对它们可能在人类体内发挥作用的过程进行了大量的猜测。就像以前关于植物RNA介导的基因沉默的工作为理解动物中类似的过程提供了信息一样，鉴定植物miRNAs并研究它们的生物学作用将为理解miRNAs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.