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

无义介导的mRNA衰变（NMD）加速了由于无义突变导致的mRNA过早翻译终止的降解。NMD存在于迄今为止研究的所有真核生物中，从酵母到人类，它保护细胞免受由所谓的无义mrna编码的潜在有害蛋白质片段的积累。在出芽酵母中，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影响一些野生型基因的mRNA积累，这些基因编码参与染色体传递和稳定性的蛋白质。在upf突变型酵母菌株中，编码CBF3着丝点复合体的一种必需蛋白、另外两种着丝点蛋白和五种影响端粒功能的蛋白的CTF13 mrna水平升高。本研究的三个具体目的是确定NMD是否直接影响这些mRNA的稳定性，表征NMD对任何野生型mRNA靶点的识别和降解，以及研究NMD对野生型基因表达的潜在协调调节。通过分析启动子-报告基因融合表达以及测量mRNA衰减和转录率，将确定NMD是直接影响mRNA稳定性还是间接影响mRNA转录。许多野生型酵母mrna的衰变包括死基化，随后是脱帽和5‘-3’核外解，而无义mrna绕过死基化，但随后经历相同的衰变。通过分析去烯化率和对脱核和5‘-3’外核溶解的易感性来确定NMD是通过去烯化依赖性还是非依赖性脱核和5‘-3’外核溶解或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确定。我们将通过分析同步细胞培养的mRNA和遗传学方法来鉴定可能编码NMD mRNA靶点的调控基因，从而研究着丝粒和/或端粒相关基因可能被NMD协调调节的可能性。该项目为进一步了解NMD在调节野生型基因表达中的细胞作用以及最终了解NMD识别和降解特定野生型mrna的机制提供了重要的起点。总之，蛋白质分子是生物系统中几乎所有分子过程的功能组成部分。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一种化学信息中介提供，称为信使RNA （mRNA）。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是细胞调控其表达的重要机制。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的理解，以及识别特定野生型mRNA进行选择性降解的机制。

Nonsense-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.