Control of arg-2 Gene Expression in Neurospora

脉孢菌中 arg-2 基因表达的控制

• 批准号: 7435302
• 负责人: MATTHEW Steven SACHS
• 金额: $ 32.01万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7435302
• 关键词: Adopted; Affect; Agriculture; Anabolism; Arginine; Bacteria; Complex; Data; Economics; Ejaculation; Enzymes; Exons; Gene Expression; Genetic Translation; Goals; Homologous Gene; In Vitro; Initiator Codon; Lead; Link; Mammals; Mediating; Medical; Messenger RNA; Metabolism; Modeling; Molecular; Molecular Analysis; Molecular Conformation; Movement; Mutation; Neurospora; Neurospora crassa; Nonsense Codon; Nonsense-Mediated Decay; Open Reading Frames; Peptides; Phenotype; Plant Viruses; Plants; Proteins; Reading Frames; Regulation; Regulator Genes; Ribosomes; Saccharomyces cerevisiae; Scanning; Site; Specific qualifier value; Structure; Terminator Codon; Testing; Translations; Untranslated Regions; Work; Yeasts; animal extract; arginine attenuator peptide; crosslink; follow-up; fungus; in vivo; loss of function; mRNA Decay; mRNA Stability; novel; peptide structure; polypeptide; response

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.

描述（由申请人提供）：这项工作的目标是了解上游开放阅读框（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 5'-前导区中的 uORF 编码，指定真菌精氨酸 (Arg) 生物合成酶；它减少对 Arg 的反应的基因表达。在粗糙脉孢菌和酿酒酵母中均观察到 AAP 介导的调节，在体外则使用真菌、植物和动物提取物观察到 AAP 介导的调节。新生的 AAP 和 Arg 导致核糖体停滞。当 AAP 作为 uORF 发挥作用时，核糖体在终止步骤停止。停滞的核糖体阻碍核糖体扫描，通过减少核糖体进入下游阅读框来减少基因表达。 AAP 还充当内部多肽结构域，以阻止参与延伸的核糖体。我们的数据得出了一个调控模型，其中 AAP 在核糖体中采用一种构象，该构象与 Arg 一起干扰 A 位点的解码，或干扰对延伸或终止至关重要的另一个步骤。 AAP 调节的第二个方面是它控制 mRNA 的稳定性。 S. cerevisiae CPA1 和 N. crassa arg-2 mRNA 水平均受到无义介导的 mRNA 衰减 (NMD) 的影响。 AAP 介导的停滞促进 CPA1 mRNA 的 NMD。在没有 AAP 介导的停滞的情况下，uORF 核糖体占据的增加可以促进 CPA1 mRNA 的 NMD。这些数据支持一种调节模型，其中核糖体响应于 Arg 而停滞在 uORF 终止密码子处，通过增加 NMD 对无义密码子识别的程度来破坏 CPA1 mRNA 的稳定性。 AAP 介导的停滞和 NMD 之间的这种联系为评估有助于 NMD 的顺式和反式作用元件提供了独特的机会。与许多具有医学、农业和经济重要性的真菌一样，粗糙脉孢菌与酿酒酵母不同，它与 eF4AIII、Y14 和 Magoh 等外显子连接复合蛋白具有明显的同源性，这些蛋白参与哺乳动物体内的 NMD 参与，并参与激活与其相关的 mRNA 的翻译。具体目标如下： 1. 通过以下方式分析 AAP 的结构，阐明核糖体停滞的机制：(a) 将新生 AAP 与核糖体交联以检查它们的相互作用 (b) 评估 AAP 在核糖体中的构象通过将其与具有已知构象的其他新生链进行比较； (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 CPA1 的调节表达来测试 NMD 的人造 UTR 模型的关键方面，并跟进 AAP 介导的核糖体停滞触发 NMD 的新观察结果。 3. 使用新的和现有的粗糙脉孢菌菌株，通过分析因这些功能丧失而导致的表型，确定脉孢菌 NMD 和 EJC 因子在翻译和 mRNA 代谢中的功能。