SGER: Strategy for Systematic Identification of Targets for a Novel Splicing Pathway

SGER：系统识别新型剪接途径靶标的策略

• 批准号: 0206374
• 负责人: Suzanne Sandmeyer
• 金额: $ 10万
• 依托单位: University of California-Irvine
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-15 至 2003-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0206374&HistoricalAwards=false
• 关键词: SGER; Strategy; Systematic; Identification; Targets

This research focuses on a novel splicing system thought to be common among eukaryotic cells but for which only a single example of a defined mRNA substrate has thus far been identified. The primary goal in this work is to develop a systematic means for identifying additional substrates for this unique splicing system. This novel splicing mechanism was first identified as a component step in a stress-response signaling pathway in the yeast Saccharomyces cerevisiae. A key step in this pathway, termed the Unfolded Protein Response (UPR), is regulated splicing of the mRNA encoding a transcriptional activator (termed Hac1p). The result is to modulate levels of the Hac1p activator which, in turn, controls expression of the cognate stress response genes. This splicing event is not mediated by the conventional mRNA splicing apparatus. Instead, two distinct gene products, Ire1p and Trl1p, act as splicing endonuclease and RNA ligase, respectively, to carry out the splicing reaction. Remarkably, the Ire1p is a transmembrane receptor that responds to changes in ligand binding by modulating its own splicing activity. Thus, splicing is truly a component step in the signaling cascade that constitutes this stress response pathway. Extensive evidence exists for conservation of UPR function among eukaryotes including the observations that yeast Hac1 mRNA can be accurately spliced in mammalian cells and recombinant human Ire1p accurately cleaves yeast Hac1 RNA. Based on this conservation of function, it is hypothesized that: (1) Regulated splicing may be a component step in UPR signaling pathways in all eukaryotes. Thus, Hac1 homologues may be found in a wide variety of eukaryotic cells. (2) This splicing system may serve a variety of gene regulatory functions other than the UPR response. Thus, regulated splicing of a variety of mRNAs may be mediated by this system. These hypotheses can be tested by the systematic identification and characterization of splicing substrates from a variety of organisms. The use of Trl1p as an affinity ligand in a selection/amplification approach will be tested as a means for conducting such a systematic analysis. The experimental goals are as follows. First, microarray analyses will be used as a comprehensive means for characterizing populations of Saccharomyces mRNAs enriched in a selection/amplification procedure. Second, application of this approach to another organism and the utility of homologous versus heterologous Trl1 gene products as affinity reagents will be assessed. Candida albicans has been chosen for this purpose based on the availability of appropriate genomic resources, its medial phylogenetic relationship to Saccharomyces, and on evidence for conservation of the splicing machinery in this organism. The results of this exploratory research will make it possible to evaluate the significance of this splicing system as a fundamental stress response mechanism among eukaryotic cells and to assess the potential for additional gene regulatory functions for this novel splicing pathway.

这项研究的重点是一种被认为在真核细胞中常见的新型剪接系统，但迄今为止只确定了一个特定mRNA底物的例子。 这项工作的主要目标是开发一种系统的方法，用于识别这种独特的剪接系统的其他底物。 这种新的剪接机制首先被确定为在酵母酿酒酵母的应激反应信号通路的组成步骤。 这一途径中的一个关键步骤，称为未折叠蛋白反应（UPR），是编码转录激活因子（称为Hac 1 p）的mRNA的调节剪接。 其结果是调节Hac 1 p激活剂的水平，而Hac 1 p激活剂反过来控制同源应激反应基因的表达。 这种剪接事件不是由常规的mRNA剪接装置介导的。 相反，两种不同的基因产物Ire 1 p和Trl 1 p分别作为剪接核酸内切酶和RNA连接酶来进行剪接反应。 值得注意的是，Ire 1 p是一种跨膜受体，通过调节自身的剪接活性来响应配体结合的变化。 因此，剪接确实是构成这种应激反应途径的信号级联中的一个组成步骤。 在真核生物中存在UPR功能保守的广泛证据，包括酵母Hac 1 mRNA可以在哺乳动物细胞中准确剪接和重组人Ire 1 p准确切割酵母Hac 1 RNA的观察结果。 基于这种功能的保守性，假设：（1）调节剪接可能是所有真核生物中UPR信号通路的组成步骤。 因此，Hac 1同源物可在多种真核细胞中发现。(2)这种剪接系统除了UPR反应外，还具有多种基因调控功能。 因此，多种mRNA的调节剪接可以由该系统介导。 这些假设可以通过系统鉴定和表征来自各种生物体的剪接底物来检验。 在选择/扩增方法中使用Trl 1 p作为亲和配体将作为进行此类系统分析的方法进行测试。 实验目标如下。首先，微阵列分析将被用作一个全面的手段，用于表征群体的酵母mRNA富集在一个选择/扩增程序。 其次，将评估这种方法应用于另一种生物体以及同源与异源Trl 1基因产物作为亲和试剂的效用。 基于合适的基因组资源的可用性、其与酵母菌属的中间系统发育关系以及该生物体中剪接机制保守的证据，选择白色念珠菌用于此目的。 这项探索性研究的结果将使人们有可能评估的意义，这种剪接系统作为一个基本的压力反应机制之间的真核细胞，并评估这种新的剪接途径的额外的基因调控功能的潜力。