Expression and function of the let-7 RNA in C. elegans

产品说明：22个核苷酸的let-7 RNA基因是一个新发现的非编码RNA基因大家族的创始成员，称为microRNA（miRNAs），似乎存在于所有多细胞生物中。let-7 RNA基因被确定为一个重要的调节发育时间的线虫的遗传研究。let-7 RNA序列，参与其表达的蛋白质，包括RNA干扰（RNAi）途径中的参与者，以及由该RNA控制的基因在动物生殖发育中广泛保守。该提案的长期目标是阐明let-7 RNA如何在C.希望这一发现有助于揭示let-7在人类健康和发育中的作用。此外，这项研究将有助于对新发现的miRNA基因的生物起源和目的的一般理解。为了验证let-7 RNA基因的表达和功能需要特定的RNA序列和蛋白质因子来产生这种发育调节因子的假设，将追求以下具体目标：1.表征产生22 nt产物的let-7 RNA转录物的序列。(A)将确定let-7 RNA初级转录物和加工中间体的序列。(B)调节加工的let-7 RNA序列元件将通过将体外测定与体内功能分析相结合来定义。2.鉴定参与let-7 RNA表达的基因。(A)将表征和鉴定在正向遗传筛选中分离的突变，该筛选旨在鉴定与dcr-1（将约70 nt pre-let-7 RNA加工为22 nt形式的RNA酶）一起发挥功能的基因。(B)通过在特定基因已被RNAi灭活的提取物中进行加工反应，检测含有核糖核酸酶、RNA解旋酶或RNA结合结构域的候选基因在let-7 RNA成熟中的作用。3.确定介导let-7依赖性调节特定靶点的因子。(A)与let-7 RNA一起发挥功能的基因将使用受let-7调控的敏感报告基因来揭示。(B)将使用体外合成的RNA探针和C.甘草提取物

DESCRIPTION: The 22 nucleotide let-7 RNA gene is a founding member of a large family of newly identified non-coding RNA genes, called microRNAs (miRNAs), which appear to be present in all multicellular organisms. The let-7 RNA gene was identified by genetic studies in the nematode Caenorhabditis elegans as an important regulator of developmental timing. The let-7 RNA sequence, proteins involved in its expression, including players in the RNA interference (RNAi) pathway, and genes controlled by this RNA are broadly conserved across animal phylogeny. The long-term goal of this proposal is to elucidate how the let-7 RNA is expressed and functions in C. elegans with the expectation that this insight will help reveal the role of let-7 in human health and development. Additionally, this research will contribute to the general understanding of the biogenesis and purpose of the populous family of newly discovered miRNA genes. To test the hypothesis that expression and function of the let-7 RNA gene requires specific RNA sequences and protein factors to produce this regulator of development, the following specific aims will be pursued: 1. Characterize the sequence of let-7 RNA transcripts that give rise to the 22nt product. (A) The sequence of the let-7 RNA primary transcript and processing intermediates will be determined. (B) The let-7 RNA sequence elements that regulate processing will be defined by coupling in vitro assays to in vivo functional analyses. 2. Identify genes involved in the expression of let-7 RNA. (A) Mutations isolated in a forward genetic screen aimed at identifying genes that function with dcr-1, the RNase that processes the approximate 70nt pre-let-7 RNA to the 22nt form, will be characterized and identified. (B) Candidate genes containing ribonuclease, RNA helicase or RNA binding domains will be tested for roles in let-7 RNA maturation by performing processing reactions in extracts where specific genes have been inactivated by RNAi. 3. Determine the factors that mediate let-7-dependent regulation of specific targets. (A) Genes that function with let-7 RNA will be uncovered using a sensitive reporter that is regulated by let-7. (B) The association of factors with the lin-41 3'UTR sequences regulated by let-7 will be characterized using in vitro synthesized RNA probes and C. elegans extracts.