Small RNAs in skeletogenesis

描述(由申请人提供)：小RNA(SRNAs)构成一个长达30个核苷酸的非编码调控RNA家族。两类主要的内源性小RNA，microRNAs(MiRNAs)和内源性小干扰RNAs(SiRNAs)分别通过结合靶mRNAs和引导染色质沉默机制来负性调节基因的表达。这些sRNA已被证明在植物、蠕虫、苍蝇和斑马鱼等各种生物的不同生物学过程中发挥关键作用。然而，尽管存在大量的miRNAs，但对sRNAs在哺乳动物中的作用的了解有限，更不用说在哺乳动物的骨骼组织中了。该项目的目标是首先确定sRNAs是否在活体骨骼组织中发挥重要的生理作用，其次确定sRNAs在骨骼组织中的表达模式和水平。在目标I中，我们将使用Cre-/oxP重组系统在小鼠软骨中删除编码DICER的基因，这是一种产生成熟miRNA和siRNA所必需的核糖核酸酶。将对缺失DICER的小鼠的骨骼异常进行分析，以确定miRNAs和siRNAs丢失的影响。这项实验应该确定依赖Dester的sRNAs在骨骼发育过程中的生理作用。在AIM II中，我们将分析miRNAs的表达模式和水平，miRNAs是哺乳动物中的主要sRNAs群体。我们将使用miRNA阵列分析进行筛选，使用定量聚合酶链式反应和原位杂交来验证和进一步鉴定从筛选中选择的每个miRNA。AIM II的实验应该为软骨中miRNA的表达提供一个基线数据库，这将有助于形成关于特定miRNAs及其靶基因在骨骼发育中作用的假设。了解sRNAs在骨骼组织中的作用不仅将揭示基因表达的另一个水平的调节，而且可能为疾病条件下的基因干预提供新的治疗靶点。

DESCRIPTION (provided by applicant): Small RNAs (sRNAs) constitute a family of regulatory non-coding RNAs of up to 30 nucleotides in length. Two major classes of endogenous small RNAs, microRNAs (miRNAs) and endogenously occurring small interfering RNAs (siRNAs) negatively regulate gene expression by binding target mRNAs and by guiding chromatin silencing machineries, respectively. These sRNAs have been demonstrated to play critical roles in diverse biological processes in various organisms such as plants, worms, flies, and zebrafish. However, despite the abundant presence of miRNAs, knowledge about roles of sRNAs in mammals, let alone in mammalian skeletal tissues, is limited. The goals of this proposed project are first, to determine whether sRNAs play physiologically important roles in skeletal tissues in vivo, and second, to determine expression patterns and levels of sRNAs in skeletal tissues. In Aim I, we will delete the gene encoding Dicer, an RNase essential for generation of mature miRNA and siRNA, in mouse cartilage using the Cre-/oxP recombination system. Skeletal abnormalities of mice missing Dicer will be analyzed to define the effect of loss of miRNAs and siRNAs. This experiment should establish physiological roles of Dicer-dependent sRNAs during skeletal development. In Aim II, we will analyze expression patterns and levels of miRNAs, the predominant population of sRNAs in mammals. We will use miRNA array analysis for screening, quantitative PCR and in situ hybridization for validation and further characterization of each miRNA selected from the screening. Experiments in Aim II should provide a baseline database for miRNA expression in cartilage, which would help to form hypotheses about roles of specific miRNAs and their target genes in skeletal development. Understanding roles of sRNAs in skeletal tissues would not only reveal another level of regulation of gene expression, but also may provide novel therapeutic targets for genetic intervention in disease conditions.