Targeted pre-mRNA modification and gene silencing

描述(由申请人提供)：这项探索性申请的目标是建立一种新颖、高效和极其有用的基因沉默技术。这项技术一旦完全建立起来，将为基础科学研究提供强有力的工具，并应用于临床研究。针对这一目标，我们提出了两个具体目标下的系统研究。我们在酿酒酵母中进行的初步实验表明，RNA引导的针对其分支点的肌动蛋白前mRNA的2‘-O-甲基化可以抑制肌动蛋白前mRNA的剪接，导致肌动蛋白mRNA的完全丧失，从而导致细胞死亡。在这一令人兴奋的结果的基础上，我们建议对酵母进行系统的研究，从而完善这项技术。具体地说，关于基因沉默的两个基本问题，即靶标特异性和技术可推广性，将被进一步仔细审查。我们将使用微阵列和基因拯救，通过将内含子较少的肌动蛋白基因引入含有引导RNA的细胞来解决底物专一性问题。为了解决这项技术是否可以推广的问题，我们将测试几个额外的含有内含子的酵母基因。此外，为了提供前mRNA底物中所有潜在靶点的完整光谱，我们计划设计指导RNA来修饰其他对剪接重要的不变核苷酸。我们相信，我们的系统分析将在酿酒酵母中产生一种可靠的新型基因沉默技术。(2)针对哺乳动物细胞的前mRNA修饰和基因沉默在我们对酿酒酵母研究的基础上，我们将把我们的基因沉默技术应用于哺乳动物细胞，哺乳动物细胞的基因包含许多内含子，其中许多内含子参与了选择性剪接。由于通过简单的基因序列检查很难识别分支点核苷酸(由于定义的一致序列松散)，我们将使用体外剪接系统来实验地映射每个感兴趣的前mRNA的分支点核苷酸。在确定了目标核苷酸之后，我们将评估我们的基因沉默方法的有效性，重点放在两个有趣的基因上：参与mRNA无义介导的衰退(NMD)的Upf3X和参与乳腺癌的ERα。我们在罗切斯特大学医学中心的邻近实验室对这两种基因进行了广泛的研究，并提供了试剂和分析方法，包括RNAi击倒分析，从而提供了在我们的方法和RNAi击倒方法之间进行直接比较的机会。我们相信，我们的新方法将在哺乳动物系统和酵母中被证明是有效和有用的。

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.