Yeast eLF2A: A Suppressor of Internal Initiation

描述（由申请人提供）： 最近的观察表明，Ure2p的表达以两种方式发生。主要形式是通过帽依赖性翻译起始。然而，在“应激”条件下，Ure2p的较短形式通过IRES表达，IRES包含在Ure2 mRNA的编码区（对应于蛋白质的N末端区域）内。一个明显无关的发现是鉴定出了人eIF2A基因的酵母同源物，该基因对酵母生长是非必需的。然而，当在eIF2A缺失菌株中监测较小形式的Ure2p蛋白（通过与lacZ融合的报告系统）的表达时，lacZ的表达增加了6至10倍。用最简单的术语来说，eIF2A似乎是Ure2 mRNA中发现的IRES功能的抑制剂。我们提出以下4个问题，以更全面地评估这些发现： 1. Ure2p mRNA中允许内部起始所需的顺式作用序列是什么？ 2.哪些其他酵母mRNA是通过内部起始启动的？ 3.如何通过共价修饰或蛋白质含量控制内部起始？ 4. eIF2A的哪一部分需要抑制内部起始，在此过程中它与哪些蛋白质相互作用？ 考虑到在许多系统中，“压力”导致帽非依赖性启动（即内部启动）的一般观察结果，我们认为我们已经发现了一个理想的模型系统，在其中研究从帽依赖性到帽非依赖性翻译的转换。预期在酵母系统中定义的机制将具有广泛的适用性，并且将可能提供对哺乳动物系统的深入了解，在哺乳动物系统中已经有许多关于利用帽非依赖性翻译（热休克、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.)