Yeast eLF2A: A Suppressor of Internal Initiation

描述(由申请人提供)： 最近的观察表明，Ure2p的表达有两种方式。主要的翻译形式是依赖于上限的翻译启动。然而，在“应激”条件下，一种较短形式的Ure2p通过IRES表达，该IRES包含在Ure2 mRNA的编码区(对应于蛋白质的N末端区域)。一个明显不相关的发现是发现了酵母与人类eIF2A基因的同源物，这对酵母的生长不是必需的。然而，当在eIF2A缺失菌株中监测到较小形式的Ure2p蛋白的表达(通过融合到LacZ的报告系统)时，flacZ的表达增加了6到10倍。在最简单的术语中，eIF2A似乎是在Ure2mRNA中发现的IRES功能的抑制者。为了更全面地评估这些发现，我们提出了以下4个问题： 1.在Ure2p mRNA中，哪些顺式作用序列是允许内部启动所必需的？ 2.还有哪些酵母mRNAs是通过内部启动而启动的？ 3.如何通过共价修饰或蛋白质含量来调节内部引发的控制？ 4.eIF2A的哪个部分需要抑制内部启动，它在这一过程中与哪些蛋白质相互作用？ 鉴于普遍观察到，在许多系统中，“应力”导致了帽子非依赖性的启动(即内部启动)，我们认为我们已经发现了一个理想的模型系统来研究从上限依赖性翻译到帽子非依赖性翻译的转换。预计酵母系统中定义的机制将具有广泛的适用性，并可能为哺乳动物系统提供洞察力，在哺乳动物系统中，已有大量关于帽非依赖性翻译(热休克、细胞凋亡、病毒感染等)的报道。

DESCRIPTION (provided by applicant): Recent observations indicate that the expression of Ure2p occurs in two manners. The predominant form is via cap-dependent initiation of translation. However, under conditions of "stress", a shorter form of Ure2p is expressed via an IRES that is contained within the coding region of the Ure2 mRNA (corresponding to the Nterminal region of the protein). An apparently unrelated finding has been the identification of the yeast homolog to the human eIF2A gene which is non-essential for yeast growth. However, when the expression of the smaller form of the Ure2p protein (via a reporter system fused to lacZ) was monitored in the eIF2A deletion strain, expression oflacZ was increased 6 to 10-fold. In the simplest of terms, eIF2A would appear to be a suppressor of the IRES function found in the Ure2 mRNA. We propose the following 4 questions to more fully evaluate these findings: 1. What are the cis-acting sequences in Ure2p mRNA that are required to allow internal initiation? 2. What other yeast mRNAs are initiated via internal initiation? 3. How is control of internal initiation regulated, through covalent modification or protein content? 4. What portion of eIF2A is required to suppress internal initiation and with what proteins does it interact in this process? Given the general observation that in many systems, "stress" leads to cap-independent initiation (i.e. internal initiation), we feel that we have discovered an ideal model system in which to study the switch from capdependent to cap-independent translation. It is anticipated that the mechanism defined in the yeast system will have broad applicability and will likely provide insights into mammalian systems where there have been numerous reports on the utilization of cap-independent translation (heat shock, apoptosis, viral infection, etc.)