Addressing Protein Synthesis Regulation within Small Numbers of Discrete Neurons

解决少量离散神经元内的蛋白质合成调控问题

• 批准号: 10586226
• 负责人: MICHAEL ROSBASH
• 金额: $ 40.63万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-30 至 2028-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10586226
• 关键词: Address; Adult; Affinity Chromatography; Animals; Area; Biochemical; Biochemistry; Biocompatible Materials; Biological; Biological Assay; Biology; Brain; Brain Diseases; Catalytic Domain; Cell Culture System; Cell Culture Techniques; Cell Line; Cells; Chimeric Proteins; Circadian Rhythms; Circadian gene expression; Communities; Complement; Computing Methodologies; Data; Development; Disease; Drosophila genus; Enzymes; Fractionation; Fragile X Syndrome; Future; Gene Expression; Gene Expression Regulation; Genetic; Genetic Transcription; Human; In Vitro; Knock-out; Labor Complications; Laboratory Study; Lateral; Malaria; Malignant Neoplasms; Mammalian Cell; Mammals; Medicine; Messenger RNA; Methods; Mus; Neurodegenerative Disorders; Neurons; Neurosciences; Noise; Organism; Pacemakers; Parasites; Persons; Pharmaceutical Preparations; Plants; Plasmids; Polyribosomes; Population; Post-Transcriptional Regulation; Protein Biosynthesis; Proteins; RNA; RNA Editing; RNA-Binding Proteins; Recombinant Proteins; Recombinants; Regulation; Research Personnel; Ribosomal Proteins; Ribosomes; Role; Side; Signal Transduction; Site; Solid Neoplasm; Specificity; System; Testing; Therapeutic Intervention; Tissues; Transcript; Transgenes; Transgenic Animals; Translating; Translational Regulation; Translations; Tumor Stem Cells; Work; biological systems; cancer stem cell; cell type; circadian; experimental study; fly; genetic regulatory protein; in vivo; knock-down; mRNA sequencing; nanobodies; novel strategies; overexpression; posttranscriptional; ribosome profiling; success; tool; transcriptome sequencing; translation assay

Project Summary/Abstract Transcription dominates the gene expression landscape of circadian rhythms and a number of neuroscience areas. Yet post-transcriptional regulation, including translational regulation and the role of RNA binding proteins (RBPs), has become increasingly recognized as important in recent years. Moreover, understanding the roles of RBPs in diverse cell types and diseases and ultimately therapeutic intervention requires identifying the RNA targets of RBPs. The ribosome can be considered a RBP, so this focus on post-transcriptional regulation and RBP targets includes identifying ribosome-associated transcripts, namely RNAs that change their translational status under defined circumstances. RBP identification is particularly challenging from small numbers of cells, e.g., cancer stem cells within a large, heterogeneous solid tumor or discrete neuronal subtypes. These settings preclude traditional biochemistry and therefore require new approaches. Two new methods recently appeared, TRIBE and STAMP, which exploit the RNA editing enzymes ADAR and APOBEC, respectively. Their ribosome versions, Ribo-STAMP and Ribo-TRIBE, are even more recent and fuse a ribosomal protein to the editing enzymes. This is so that the enzyme will be near RNAs that are being translated and will “mark” them by changing their sequence. These edits are identified by mRNA sequencing and straightforward computational methods, even from single cells. We propose to compare Ribo-STAMP and Ribo-TRIBE side-by-side in mammalian cell culture systems, to assess their efficacy and to determine the optimal configuration of ribosome-editing enzyme fusions. We also propose to develop Ribo-TRIBE for use in Drosophila; we recently discovered that STAMP does not work in this organism, which limits us to TRIBE. We will then extend these methods to the more biological context of neurons, from Drosophila as well as from mouse brains. To complement these efforts, we will develop an extension of the TRIBE/STAMP theme called Nanobody-editing. It consists of fusing the editing enzyme, ADAR or APOBEC, to a GFP-nanobody, which will then deliver the editing enzyme to any GFP-tagged RNA binding protein or GFP-tagged ribosome. The chimeric, recombinant protein will be used in vitro as a recombinant protein or expressed in vivo. Nanobody- editing will facilitate identifying RBP and ribosome targets, because already existing GFP-tagged RBP or ribosomes can serve as substrates. Moreover, in vitro editing will in many cases obviate the need to generate new transgenes and transgenic animals. Lastly, TRIBE and as well as Ribo-TRIBE will be used to characterize translational regulation “around the clock” within a few key Drosophila circadian neurons. These efforts will deepen our understanding of circadian gene expression regulation as well as provide the community with new tools with which to study translation in important but challenging biological systems.

项目摘要/摘要 转录主导昼夜节律的基因表达景观和许多神经科学 区域。然而，转录后调节，包括翻译调节和RNA结合的作用 近年来，蛋白质（RBP）已变得越来越重要。而且，理解 RBP在潜水细胞类型和疾病中的作用以及最终的治疗干预需要确定 RBP的RNA靶标。核糖体可以视为RBP，因此专注于转录后 调节和RBP目标包括识别与核糖体相关的转录本，即改变的RNA 他们在定义的情况下的翻译状态。 RBP识别尤其具有挑战性 细胞的数量，例如大型，异质实体瘤或离散神经元内的癌症干细胞的数量 亚型。这些设置排除了传统的生物化学，因此需要新的方法。两个新 最近出现的方法是利用RNA编辑酶Adar和Apobec的Tribe and Stamp， 分别。它们的核糖体版本，Ribo-Stamp和Ribo-Tribe，甚至是最近的融合 核糖体蛋白至编辑酶。这是这样，酶将在RNA附近 翻译并通过更改其顺序“标记”它们。这些编辑通过mRNA测序确定 和直接的计算方法，甚至来自单个单元格。我们建议比较Ribo-Stamp和 哺乳动物细胞培养系统中的肋骨部位并排，以评估其效率并确定 核糖体编辑酶融合的最佳配置。我们还建议开发Ribo-Tribe以供 果蝇；我们最近发现，邮票在这种生物体中不起作用，这将我们限制在部落中。我们 然后将这些方法扩展到来自果蝇的神经元的更生物学环境 鼠标大脑。为了完成这些努力，我们将开发一个部落/邮票主题的扩展名为 纳米机构编辑。它包括将编辑酶，ADAR或APOBEC融合到GFP-Nanobody中，这将 然后将编辑酶输送到任何GFP标记的RNA结合蛋白或GFP标记的核糖体中。 嵌合，重组蛋白将在体外用作重组蛋白或在体内表达。纳米机 编辑将有助于识别RBP和核糖体目标，因为已经存在GFP标签的RBP或 核糖体可以用作底物。此外，在许多情况下，体外编辑将消除生成的需求 新的转基因和转基因动物。最后，将使用部落和肋骨部落来表征 在几个关键的果蝇昼夜节律神经元中“遍布时钟”的翻译调节。这些努力会 加深我们对昼夜节律表达调节的理解，并为社区提供新的理解 在重要但挑战生物系统中研究翻译的工具。