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

本研究的重点是一种新的剪接系统，被认为是真核细胞中常见的，但迄今为止只有一个确定的mRNA底物的例子被确定。这项工作的主要目标是开发一种系统的方法来识别这种独特拼接系统的附加底物。这种新的剪接机制首次被确定为酵母应激反应信号通路的一个组成步骤。该途径的关键步骤，称为未折叠蛋白反应（UPR），是编码转录激活子（称为Hac1p）的mRNA的调节剪接。结果是调节Hac1p激活因子的水平，而Hac1p激活因子反过来又控制同源应激反应基因的表达。这种剪接事件不是由传统的mRNA剪接装置介导的。相反，两个不同的基因产物Ire1p和Trl1p分别作为剪接内切酶和RNA连接酶进行剪接反应。值得注意的是，Ire1p是一种跨膜受体，通过调节自身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。广泛的证据表明，在真核生物中存在UPR功能的保守性，包括酵母Hac1 mRNA可以在哺乳动物细胞中准确剪接，重组人Ire1p可以准确切割酵母Hac1 RNA。基于这种功能的保守性，我们假设：(1)在所有真核生物中，受调节的剪接可能是UPR信号通路的一个组成步骤。因此，Hac1同源物可能存在于多种真核细胞中。(2)除了UPR应答外，该剪接系统可能具有多种基因调控功能。因此，多种mrna的调节剪接可能由该系统介导。这些假设可以通过系统地鉴定和表征来自各种生物体的剪接底物来验证。在选择/扩增方法中使用Trl1p作为亲和配体将作为进行这种系统分析的一种手段进行测试。实验目标如下：首先，微阵列分析将作为一种综合手段，用于表征在选择/扩增过程中富集的酵母菌mrna群体。其次，将评估这种方法在另一种生物体中的应用，以及同源与异源Trl1基因产物作为亲和试剂的效用。选择白色念珠菌用于这一目的是基于适当的基因组资源的可用性，其与酵母菌的中间系统发育关系，以及该生物中剪接机制的保存证据。这项探索性研究的结果将有可能评估这种剪接系统作为真核细胞中基本应激反应机制的意义，并评估这种新型剪接途径的其他基因调控功能的潜力。

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.