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上核糖体的运动并调节基因表达的机制。精氨酸衰减肽（AAP）由指定真菌精氨酸（Arg）生物合成酶的mrna的5'-先导区uORF编码；它减少了对Arg的基因表达。在粗神经孢子菌和酿酒酵母体内以及体外，利用真菌、植物和动物提取物观察到aap介导的调节作用。新生的AAP和Arg导致核糖体停滞。当AAP作为uORF时，核糖体在终止步骤停止。停滞的核糖体阻断扫描核糖体，通过减少核糖体进入下游阅读框来降低基因表达。AAP也作为一个内部多肽结构域来阻止参与延伸的核糖体。我们的数据导致了一种调控模型，其中AAP在核糖体中采用一种构象，与Arg一起干扰a位点的解码，或者干扰对延伸或终止至关重要的另一个步骤。AAP调控的第二个方面是它控制mRNA的稳定性。无义介导的mRNA衰变（NMD）对酿酒葡萄球菌CPA1和草葡萄球菌arg-2 mRNA水平均有影响。aap介导的迟滞促进CPA1 mRNA的NMD。在没有aap介导的阻滞的情况下，CPA1 mRNA的NMD可以通过增加核糖体占用uORF来促进。这些数据支持一种调控模型，其中核糖体响应Arg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破坏CPA1 mRNA的稳定性。aap介导的失速与NMD之间的这种联系为评估导致NMD的顺式和反式因素提供了独特的机会。与许多具有医学、农业和经济重要性的真菌一样，N. crassa与S. cerevisiae不同，它具有elF4AIII、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的NMD，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(a)将新生的AAP与核糖体交联以研究它们之间的相互作用(b)通过将其与其他已知构象的新生链进行比较来评估AAP在核糖体中的构象，从而阐明核糖体延迟的机制；(c)通过2D-NMR直接检测AAP结构以及Arg对AAP结构的影响。2. 利用酵母CPA1的调控表达来测试NMD的人造utr模型的关键方面，并追踪AAP介导的核糖体停滞触发NMD的新观察结果。3. 通过分析神经孢子菌NMD和EJC因子在翻译和mRNA代谢中的功能丧失所导致的表型，利用新菌株和现有菌株确定神经孢子菌NMD和EJC因子在翻译和mRNA代谢中的功能。