Targeted pre-mRNA modification and gene silencing

描述（由申请人提供）：本探索性应用的目标是建立一种新型、高效且非常有用的基因沉默技术。这项技术一旦完全建立，将为基础科学研究和临床研究提供一个强有力的工具。为了实现这一目标，我们提出了两个具体目标下的系统研究。(1)使用酵母系统的靶向前mRNA修饰和基因沉默的详细分析酿酒酵母已经证明，靶向肌动蛋白前体mRNA在其分支点的RNA引导的2 '-O-甲基化腺苷可以抑制肌动蛋白前体mRNA剪接，导致肌动蛋白mRNA的完全丧失，从而导致细胞死亡。 基于这一令人兴奋的结果，我们建议对酵母进行系统研究，从而完善该技术。 具体而言，两个基本问题，基因沉默，即目标特异性和技术的普遍性，将进一步审查。我们将使用微阵列和基因拯救，通过将无内含子肌动蛋白基因引入含有指导RNA的细胞来解决底物特异性问题。为了解决该技术是否可以推广，我们将测试几个额外的含内含子的酵母基因。此外，为了提供前体mRNA底物中所有潜在靶位点的完整谱，我们计划设计指导RNA来修饰对剪接重要的其他不变核苷酸。我们相信，我们的系统分析将产生一个可靠的新的基因沉默技术在S。啤酒。(2)哺乳动物细胞中靶向的前体mRNA修饰和基因沉默在酿酒酵母中，我们将我们的基因沉默技术应用于哺乳动物细胞，其基因，除了少数例外，包含许多内含子，其中许多参与选择性剪接。由于分支点核苷酸难以在简单检查基因序列时鉴定（由于定义不严格的共有序列），我们将使用体外剪接系统对每个感兴趣的前mRNA进行实验性的分支点核苷酸作图。在确定了靶核苷酸后，我们将评估我们的基因沉默方法的有效性，重点是两个有趣的基因：Upf 3X，参与mRNA无义介导的衰变（NMD），和ER α参与乳腺癌。这两种基因在我们邻近的罗切斯特大学医学中心实验室进行了广泛的研究，并且试剂和测定，包括RNAi敲除测定，都是可用的，这为我们的方法和RNAi敲除方法之间的直接比较提供了机会。我们相信，我们的新方法将证明有效和有用的哺乳动物系统以及酵母。

DESCRIPTION (provided by applicant): The goal of this exploratory application is to establish a novel, highly effective, and extremely useful technique for gene silencing. This technique, once fully established, will offer a powerful tool for basic science research with application in clinical investigation. Toward this goal, we propose a systematic study under two specific aims. (1) Detailed analysis of targeted pre-mRNA modification and gene silencing using a yeast system Our preliminary experiments carried out in S. cerevisiae have demonstrated that RNA-guided 2'-O- methylation targeted to actin pre-mRNA at its branch point adenosine can inhibit actin pre-mRNA splicing, leading to a complete loss of actin mRNA and hence cell death. Building on this exciting result, we propose a systematic investigation in yeast, whereby we will perfect the technique. Specifically, two fundamental issues regarding gene silencing, namely target specificity and technique generalizability, will be further scrutinized. We will use microarrays and gene rescue by introducing an intron-less actin gene into the guide RNA- containing cells to address the substrate specificity issue. To address whether the technique can be generalized, we will test several additional intron-containing yeast genes. Furthermore, to provide a complete spectrum of all potential target sites in a pre-mRNA substrate, we plan to design guide RNAs to modify the other invariant nucleotides important for splicing. We believe that our systematic analysis will yield a reliable novel gene silencing technique in S. cerevisiae. (2) Targeted pre-mRNA modification and gene silencing in mammalian cells Building on our investigations in S. cerevisiae, we will apply our gene silencing technique to mammalian cells, whose genes, with few exceptions, contain a number of introns, many of which are involved in alternative splicing. Because the branch point nucleotide is difficult to identify upon simple inspection of a gene sequence (due to the loosely defined consensus sequence), we will use in vitro splicing systems to map the branch point nucleotide experimentally for each pre-mRNA of interest. Having identified the target nucleotide, we will then assess the effectiveness of our gene silencing method focusing on two interesting genes: Upf3X, involved in mRNA nonsense-mediated decay (NMD), and ERalpha involved in breast cancer. Both genes are extensively studied in our neighboring labs at the University of Rochester Medical Center, and reagents and assays, including RNAi knockdown assays, are available, offering an opportunity for a direct comparison between our method and the RNAi knockdown method. We believe that our novel method will prove effective and useful in mammalian systems as well as in yeast.