SCREEN FOR SMALL MOLECULAR INHIBITORS OF MAMMALIAN SPLICEOSOMES

描述（由申请人提供）：本项目的目的是鉴定哺乳动物细胞中前体mRNA剪接体剪接的小分子抑制剂。剪接是哺乳动物基因表达过程中一个重要且高度调控的步骤。目前还没有已知的小分子剪接抑制剂。为了鉴定这些化合物，我们将采用以前发表的双基因报告基因检测前mRNA剪接的高通量配置。我们将为哺乳动物细胞中发现的主要112依赖性剪接途径和次要U12依赖性剪接途径开发双报告质粒。为了验证高通量筛选的结构，我们将在我们机构可用的小分子文库上对其进行测试。似乎抑制剪接的化合物的二次筛选将包括对含有不含内含子的构建体的细胞的测试和RT-PCR测定以评价化合物对体内内含子保留的影响。将在对两种剪接体剪接机制中的每一种特异性的体外剪接测定中进一步测试仅对含内含子的报告基因具有抑制作用的化合物的剪接抑制作用。初级和次级测定将鉴定在体内环境中抑制剪接体剪接的小分子化合物。这些化合物的潜在用途包括1）哺乳动物细胞中剪接体剪接途径的生物化学研究，2）完整细胞、组织和生物体中剪接体剪接下调的体内结果的研究，3）高等真核生物中两类剪接体内含子在基因表达和基因功能中可能的不同作用的研究。公共卫生相关性：基因的调节表达是人类生长、发育、正常和病理功能以及身体对内外环境变化的反应的核心。基因调控的一个重要方面是通过RNA剪接从基因的初级转录本中去除内含子。这项提议是为了发现可以直接影响剪接过程的药物样化合物。这些化合物将提高我们研究和理解剪接过程的能力，并且还可能具有用于研究或治疗疾病的有用生物学特性。

DESCRIPTION (provided by applicant): The goal of this project is to identify small molecule inhibitors of spliceosomal splicing of pre-mRNAs in mammalian cells. Splicing is an essential and highly regulated step in mammalian gene expression. There are currently no known small molecule inhibitors of splicing. To identify such compounds, we will adapt a previously published dual gene reporter assay for pre-mRNA splicing to a high-throughput configuration. We will develop dual reporter plasmids for both the major 112-dependent splicing pathway and the minor U12-dependent splicing pathway found in mammalian cells. In order to validate the comstructs for high- throughput screening, we will test them on a small molecule library available in our institution. Secondary screens of compounds which appear to inhbit splicing will include tests on cells containing constructs which do not contain introns and RT-PCR assays to evaluate the effect of the compounds on intron retention in vivo. Compounds that are inhibitory only to intron-containing reporters will be further tested for splicing inhibition in in vitro splicing assays specific to each of the two spliceosomal splicing mechanisms. The primary and secondary assays will identify small molecule compounds that inhibit spliceosomal splicing in an in vivo context. Potential uses of such compounds include 1) the biochemical investigation of the splliceosomal splicing pathways in mammalian cells, 2) investigations of the in vivo consequences of down regulation of spliceosomal splicing in intact cells, tissues and organisms, 3) investigations of the possible different roles in gene expression and gene function of the two classes of spliceosomal introns in higher eukaryotes. Public health relevance: The regulated expression of genes is central to human growth, development, normal and pathological functioning and the response of the body to changes in the internal and external environment. An important point of gene regulation is the removal of introns from the primary transcripts of genes by RNA splicing. This proposal is to discover drug-like chemical compounds that can directly influence the process of splicing. Such compounds will improve our ability to study and understand the process of splicing and may also have useful biological properties for the study or treatment of disease.