Control of arg-2 Gene Expression in Neurospora

描述（由申请人提供）：这项工作的目的是了解由上游开放阅读框（uORF）编码的新生肽控制mRNA上核糖体运动并调节基因表达的机制。精氨酸衰减肽（AAP）由mRNA的5 '-前导区中的uORF编码，其指定真菌精氨酸（Arg）生物合成酶;其响应于Arg而降低基因表达。AAP介导的调节在粗糙脉孢菌和酿酒酵母中在体内和体外使用真菌、植物和动物提取物观察到。新生的AAP和Arg导致核糖体停滞。当AAP作为uORF起作用时，核糖体在终止步骤停止。停滞的核糖体阻碍了扫描核糖体，通过减少核糖体进入下游阅读框来降低基因表达。AAP还作为内部多肽结构域发挥功能，以阻止参与延伸的核糖体。我们的数据导致一个监管模型中，AAP采用的核糖体中的构象，与精氨酸，干扰解码在A-站点，或与另一个步骤的延长或终止至关重要。AAP调控的第二个方面是它控制mRNA的稳定性。两个S。cerevisiae CPA 1和N. crassa arg-2 mRNA水平受无义介导的mRNA衰减（NMD）的影响。AAP介导的停滞促进CPA 1 mRNA的NMD。在不存在AAP介导的停滞的情况下，CPA 1 mRNA的NMD可以通过增加uORF的核糖体占据来促进。这些数据支持一种调控模型，其中核糖体响应于Arg而在uORF终止密码子处停滞，通过增加NMD对无义密码子的识别程度来使CPA 1 mRNA不稳定。AAP介导的停滞与NMD之间的这种联系为评估促成NMD的顺式和反式作用因素提供了独特的机会。N. crassa，像许多具有医学、农业和经济重要性的真菌一样，但与S.酿酒酵母，具有eIF 4AIII、Y14和Magoh的明确同源物，这些外显子连接复合物蛋白参与哺乳动物中的NMD接合，并参与激活与其相关的mRNA的翻译。具体目标如下：1.通过分析AAP的结构来阐明核糖体停滞的机制，通过（a）将新生AAP与核糖体交联以检查它们的相互作用（B）通过将其与具有已知构象的其他新生链进行比较来评估核糖体中AAP的构象;（c）通过2D-NMR直接检查AAP结构以及Arg如何影响它。2.利用酵母CPA 1的调节表达来测试NMD的faux-UTR模型的关键方面，并跟踪AAP介导的核糖体停滞触发NMD的新观察结果。3.使用新的和现有的N。crassa菌株的基因组序列，以确定链孢菌NMD和EJC因子在翻译和mRNA代谢中的功能，通过分析这些功能丧失导致的表型。

DESCRIPTION (provided by applicant): The goal of this work is to understand the mechanisms by which a nascent peptide encoded by an upstream open reading frame (uORF) controls the movement of ribosomes on mRNA and regulates gene expression. The arginine attenuator peptide (AAP) is encoded by a uORF in the 5'-leader regions of mRNAs specifying a fungal arginine (Arg) biosynthetic enzyme; it reduces gene expression in response to Arg. AAP-mediated regulation is observed in vivo in both Neurospora crassa and Saccharomyces cerevisiae and in vitro, using fungal, plant and animal extracts. The nascent AAP and Arg cause the ribosome to stall. When the AAP functions as a uORF, the ribosome stalls at the termination step. The stalled ribosomes block scanning ribosomes, decreasing gene expression by reducing ribosomal access to the downstream reading frame. The AAP also functions as an internal polypeptide domain to stall ribosomes involved in elongation. Our data lead to a regulatory model in which the AAP adopts a conformation in the ribosome that, with Arg, interferes with decoding at the A-site, or with another step crucial for elongation or termination. A second aspect of regulation by the AAP is that it controls mRNA stability. Both S. cerevisiae CPA1 and N. crassa arg-2 mRNA levels are affected by nonsense-mediated mRNA decay (NMD). AAP-mediated stalling promotes NMD of the CPA1 mRNA. In the absence of AAP-mediated stalling, NMD of the CPA1 mRNA can be promoted by increased ribosome occupancy of the uORF. These data support a regulatory model in which ribosome stalling at the uORF termination codon in response to Arg destabilizes CPA1 mRNA by increasing the extent of nonsense codon recognition by NMD. This link between AAP-mediated stalling and NMD provides unique opportunities for assessing the cis- and trans-acting elements that contribute to NMD. N. crassa, like many fungi of medical, agricultural, and economic importance, but unlike S. cerevisiae, has clear homologs of elF4AIII, Y14, and Magoh, exon junction complex proteins which are involved in engaging NMD in mammals and are implicated in activating the translation of mRNAs with which they associate. Specific aims are as follows: 1. Elucidate the mechanism of ribosome stalling by analyzing the structure of the AAP through (a) cross-linking the nascent AAP to the ribosome to examine their interactions (b) assessing the conformation of the AAP in the ribosome by comparing it to other nascent chains with known conformations; (c) directly examining AAP structure and how Arg affects it by 2D-NMR. 2. Exploit the regulated expression of yeast CPA1 to test key aspects of the faux-UTR model of NMD and follow up on novel observations that AAP- mediated ribosome stalling triggers NMD. 3. Use new and existing N. crassa strains to determine the functions of Neurospora NMD and EJC factors in translation and mRNA metabolism through the analysis of the phenotypes that result from the loss of these functions.