Small RNAs in skeletogenesis

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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.