CLEAVAGE, POLYADENYLATION, AND TRANSPORT OF MRNA

mRNA 的切割、聚腺苷酸化和运输

• 批准号: 2176352
• 负责人: Marvin P. Wickens
• 金额: $ 25.03万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-04-01 至 1995-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2176352
• 关键词: Anura; alternatives to animals in research; cell free system; cell nucleus; chemical cleavage; cytoplasm; developmental genetics; early embryonic stage; embryogenic cleavage; enzyme mechanism; eukaryote; fresh water environment; gene expression; genetic manipulation; messenger RNA; microinjections; molecular cloning; mutant; nucleic acid probes; nucleic acid sequence; oogenesis; polyadenylate; posttranscriptional RNA processing; protein biosynthesis; radiotracer; tissue /cell culture

Eukaryotic mRNAs generally are not replicas of the genes that encode them. Instead, mRNA is produced by a series of covalent modifications of a primary transcript including capping, splicing and polyadenylation. In principle, each maturation step provides a means of regulating mRNA formation. The ultimate objective of our work is to understand the molecular mechanism of mRNA synthesis and the means by which it is regulated. In this proposal, we focus on a form of regulated RNA processing that is critical in early development -- the addition and removal of poly (A) from maternal mRNAs. We propose to study these regulated RNA processing reactions in frog oocytes and embryos. We focus on the periods of oocyte maturation and fertilization, and use cyclin and c-mos mRNAs as models. In the last grant period, we laid the groundwork for the work proposed here. We identified sequences that regulate poly (A) addition and removal, and characterized the enzymes involved. We developed assays for poly (A) addition and removal, both in vivo, by microinjection, and in vitro, using extracts of frog eggs. In addition, we elucidated key features of the mechanism by which poly (A) is added in the nucleus of all cells; this knowledge provides an invaluable intellectual framework and critical reagents for the work we now propose In the next grant period, sequences that are critical for poly (A) addition and removal during development will be identified precisely. We will characterize and purify the enzymes involved, both by classical means and molecular cloning, and determine how those activities are regulated during development. We will test our hypothesis that changes in the length of the poly (A) tail of c-mos mRNA help control the embryonic cell cycle. By in vitro complementation tests, we will determine whether factors that direct cytoplasmic polyadenylation during development are functionally interchangeable with those that add poly (A) in the nucleus. The work proposed will elucidate fundamental mechanisms of gene expression, and therefore has important practical applications. Changes in poly (A) length occur throughout development, and in the embryos of all animal species so far examined. Our detailed investigations of the regulation of c-mos, a proto-oncogene normally expressed only in the germ line, bear on how cell division is controlled both in normal and abnormal growth.

真核mRNA通常不是编码它们的基因的复制品。 相反，mRNA是由一系列共价修改产生的 主要转录本，包括封盖，剪接和聚腺苷酸化。 在 原理，每个成熟步骤提供了调节mRNA的手段 形成。 我们工作的最终目标是了解分子机制 mRNA合成及其调节的手段。 在这个 提案，我们专注于一种受调节的RNA处理形式，这是至关重要的 在早期开发中 - 从母体中添加和去除poly（a） mrnas。 我们建议研究这些调节的RNA处理反应 青蛙卵母细胞和胚胎。 我们专注于卵母细胞成熟时期和 受精，并使用细胞周期蛋白和C-MOS mRNA作为模型。 在最后的赠款期间，我们为提议的工作奠定了基础 这里。 我们确定了调节聚（a）添加和去除的序列， 并表征了所涉及的酶。 我们开发了poly（a）的测定法 加法和去除，无论是在体内，通过微注射和体外，都使用 青蛙鸡蛋的提取物。 此外，我们阐明了 在所有细胞的核中添加聚（a）的机制；这 知识提供了一个宝贵的智力框架和批判性的 我们现在建议的工作的试剂 在下一个赠款期间，对poly（a）添加至关重要的序列 并且将精确地确定开发过程中的去除。 我们将 通过经典手段和 分子克隆，并确定如何调节这些活动 发展。 我们将测试我们的假设，即长度的变化 C-MOS mRNA的聚（A）尾部有助于控制胚胎细胞周期。 由In 体外互补测试，我们将确定指导的因素是否 发育过程中的细胞质聚腺苷酸化是功能 与在细胞核中添加poly（a）的互换。 提出的工作将阐明基因表达的基本机制， 因此具有重要的实际应用。 poly（a）的变化 长度在整个发育中以及所有动物的胚胎中发生 到目前为止检查的物种。 我们对监管的详细调查 C-MOS是一种通常仅在种系中表达的原始癌基因， 细胞分裂如何在正常和异常生长中受到控制。