RUI: Regulation of Gene Expression by the Nonsense-mediated mRNA Decay Pathway of Yeast

RUI：酵母无义介导的 mRNA 衰变途径对基因表达的调节

• 批准号: 0091300
• 负责人: Jeffrey Dahlseid
• 金额: $ 31.7万
• 依托单位: Saint Olaf College
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0091300&HistoricalAwards=false
• 关键词: RUI; Regulation; Gene; Expression; Nonsense

0091300 Jeffrey DahlseidNonsense-mediated mRNA decay (NMD) accelerates the degradation of mRNAs that undergo premature translation termination due to a nonsense mutation. NMD exists in all eukaryotes thus far examined, from yeast to man, and safeguards cells against the accumulation of potentially deleterious protein fragments encoded by so-called nonsense mRNAs. In budding yeast, the UPF1, UPF2, and UPF3 genes are required for NMD. These genes also affect the accumulation of hundreds of wild-type mRNAs, which suggests that NMD is an important part of the natural cellular repertoire for regulating wild-type gene expression. The primary objective of this research is to study the role of NMD in regulating the expression of wild-type genes. NMD affects the mRNA accumulation of several wild-type genes that encode proteins involved in chromosome transmission and stability. The mRNAs for CTF13, which encodes an essential protein of the CBF3 kinetochore complex, two additional kinetochore proteins, and five proteins that affect telomere function are elevated in upf mutant yeast strains. The three specific aims of this research are to determine if NMD directly affects the stability of these mRNAs, to characterize the recognition and degradation of any that are wild-type mRNA targets of NMD, and to investigate potential coordinate regulation of wild-type gene expression by NMD. Analysis of expression from promoter-reporter gene fusions and measurement of mRNA decay and transcription rates will be used to determine whether NMD directly affects mRNA stability or exerts indirect influence upon mRNA transcription. Decay of many wild-type yeast mRNAs involves deadenylation followed by decapping and 5'-3' exonucleolysis, whereas nonsense mRNAs bypass deadenylation but then undergo the same decay. Analysis of deadenylation rates and susceptibility to decapping and 5'-3' exonucleolysis will be used to determine whether NMD degrades wild-type mRNAs through deadenylation-dependent or -independent decapping and 5'-3' exonucleolysis or some other mechanism. Sequences or structures necessary for recognition of wild-type mRNAs by NMD will be identified using deletion mutations and gene fusions. The possibility that kinetochore- and/or telomere-related genes may be coordinately regulated by NMD will be investigated through analysis of mRNA from synchronized cell cultures and genetic approaches to identify putative regulatory genes, which may encode mRNA targets of NMD. This project serves as an important starting point to increase understanding of the cellular role of NMD in regulating the expression of wild-type genes and, ultimately, the mechanism for recognition and decay of specific wild-type mRNAs by NMD. In summary, protein molecules are functional components of nearly all the molecular processes in biological systems. Instructions for protein formation are stored in the DNA of genes and are provided as a chemical information intermediate, known as messenger RNA (mRNA), when genes are expressed. To achieve normal growth and development, cells must regulate the expression of genes. Selectively degrading the mRNA of a wild-type gene is an important mechanism for cells to regulate its expression. This research aims to increase understanding of the cellular role for specialized mRNA degradation pathways in regulating wild-type gene expression and the mechanisms involved in recognizing specific wild-type mRNAs for selective degradation.

0091300 Jeffrey Dahlseid 无义介导的 mRNA 衰减 (NMD) 会加速由于无义突变而导致过早翻译终止的 mRNA 的降解。 NMD 存在于迄今为止检查的所有真核生物中，从酵母到人类，并保护细胞免受所谓无义 mRNA 编码的潜在有害蛋白质片段的积累。 在芽殖酵母中，NMD 需要 UPF1、UPF2 和 UPF3 基因。 这些基因还影响数百种野生型 mRNA 的积累，这表明 NMD 是调节野生型基因表达的天然细胞库的重要组成部分。 本研究的主要目的是研究NMD在调节野生型基因表达中的作用。 NMD 影响多个野生型基因的 mRNA 积累，这些基因编码参与染色体传递和稳定性的蛋白质。 CTF13 的 mRNA 在 upf 突变酵母菌株中升高，CTF13 编码 CBF3 着丝粒复合物的必需蛋白、另外两种着丝粒蛋白和影响端粒功能的五种蛋白。 这项研究的三个具体目标是确定 NMD 是否直接影响这些 mRNA 的稳定性，表征 NMD 野生型 mRNA 靶标的识别和降解，并研究 NMD 对野生型基因表达的潜在协调调节。 启动子-报告基因融合的表达分析以及 mRNA 衰减和转录率的测量将用于确定 NMD 是否直接影响 mRNA 稳定性或对 mRNA 转录产生间接影响。 许多野生型酵母 mRNA 的衰变涉及去腺苷化，然后脱帽和 5'-3' 核酸外切，而无义 mRNA 绕过去腺苷化，但随后经历相同的衰变。 脱腺苷化率以及对脱帽和 5'-3' 核酸外切的敏感性的分析将用于确定 NMD 是否通过脱腺苷化依赖或不依赖的脱帽和 5'-3' 核酸外切或某些其他机制降解野生型 mRNA。 NMD 识别野生型 mRNA 所需的序列或结构将通过缺失突变和基因融合来鉴定。 将通过分析来自同步细胞培养物的 mRNA 和遗传方法来研究着丝粒和/或端粒相关基因可能被 NMD 协调调节的可能性，以鉴定可能编码 NMD mRNA 靶标的假定调节基因。 该项目是加深对 NMD 在调节野生型基因表达中的细胞作用以及最终 NMD 识别和降解特定野生型 mRNA 的机制的理解的重要起点。 总之，蛋白质分子是生物系统中几乎所有分子过程的功能成分。 蛋白质形成的指令存储在基因的 DNA 中，并在基因表达时作为化学信息中间体（称为信使 RNA (mRNA)）提供。 为了实现正常的生长和发育，细胞必须调节基因的表达。 选择性降解野生型基因的mRNA是细胞调节其表达的重要机制。 本研究旨在加深对特定 mRNA 降解途径在调节野生型基因表达中的细胞作用以及识别特定野生型 mRNA 进行选择性降解的机制的了解。