POST-TRANSCRIPTIONAL REGULATION OF R-PROTEIN SYNTHESIS

R 蛋白合成的转录后调控

• 批准号: 3295213
• 负责人: Allan S Jacobson
• 金额: $ 13.92万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3295213
• 关键词: Dictyostelium; fungal genetics; fusion gene; genetic manipulation; genetic translation; in situ hybridization; messenger RNA; nuclease; nucleic acid sequence; plasmids; posttranslational modifications; protein biosynthesis; ribonucleoproteins; ribosomes

These experiments are designed to characterize features of mRNAs which are determinants of translational regulation and mRNA stability. We will study the r-protein mRNAs of Dictyostelium which we have already shown to be selectively excluded from polysomes at the start of development and to undergo an apparent decrease in stability later in development. 1) Translational regulation: The relative translational efficiency of r-protein mRNAs will be assessed in in vitro systems programmed with increasing amounts of total poly(A)+ RNA, with either initiation or elongation made rate limiting by the addition of inhibitors. Experiments to identify sequences which are responsible for translational discrimination in vivo will utilize recombinant plasmids in which regions of genes which encode mRNAs with different translational efficiencies in development have been exchanged. Translation of the chimeric mRNAs expressed from these plasmids will be examined in transformed, developing cells. In addition, expression of r-protein mRNAs from a promoter active in development will allow us to ask whether post-transcriptional modifications which may change with mRNA "age" are important in determining translatability in developing cells. The cellular location of untranslated, but stable, r-protein mRNAS will be examined by in situ hybridization to developing cells. 2) mRNA stability: The time and magnitude of the developmental change in r-protein mRNA stability will be determined by measurement of mRNA transcription (in nuclear run-off assays) and mRNA half-life (in pulse-chase experiments). Events known to affect mRNA stability in Dictyostelium will be correlated with the decrease in relative abundance of r-protein mRNAs. The importance of specific sequences and of efficient translation during development will be examined using chimeric transcripts from plasmids introduced by transformation. We will examine by in situ hybridization whether changes in stability are correlated with changes in subcellular location. Finally, we will try to identify a nuclease activity responsible for stage specific degradation of r-protein mRNAS in developmental cell lysates.

这些实验旨在表征 mRNA 是翻译调控的决定因素 mRNA 稳定性。 我们将研究 r 蛋白 mRNA 我们已经证明盘基网柄菌具有选择性 在发育开始时就被排除在多核糖体之外 在开发后期稳定性会明显下降。 1） 翻译调节：相对翻译效率 r-蛋白 mRNA 将在体外编程系统中进行评估 随着总 Poly(A)+ RNA 数量的增加， 通过添加以下物质来限制引发或伸长速率 抑制剂。 鉴定序列的实验 负责体内翻译歧视将利用 重组质粒，其中编码基因的区域 发育过程中具有不同翻译效率的 mRNA 已交换。 嵌合 mRNA 的翻译 从这些质粒表达的将在转化中进行检查， 发育中的细胞。 此外，r 蛋白 mRNA 的表达 积极参与开发的发起人将使我们能够问是否 可能随 mRNA 改变的转录后修饰 “年龄”对于确定开发中的可译性很重要 细胞。 未翻译但稳定的 r 蛋白的细胞位置 mRNAS将通过原位杂交进行检查以开发 细胞。 2) mRNA稳定性：稳定性的时间和强度 r 蛋白 mRNA 稳定性的发育变化将是 通过测量 mRNA 转录（在核中）来确定 径流测定）和 mRNA 半衰期（在脉冲追踪实验中）。 已知影响盘基网柄菌 mRNA 稳定性的事件将是 与 r 蛋白相对丰度的降低相关 mRNA。 特定顺序和高效的重要性 开发期间的翻译将使用嵌合体进行检查 通过转化引入的质粒的转录本。 我们将 通过原位杂交检查稳定性的变化是否 与亚细胞位置的变化相关。 最后，我们将 尝试确定负责特定阶段的核酸酶活性 发育细胞裂解物中 r 蛋白 mRNAS 的降解。