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节问题

基本信息

批准号：
10091418
负责人：
MICHAEL ROSBASH
金额：
$ 32.72万
依托单位：
BRANDEIS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-30 至 2023-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10091418
关键词：
Address Behavior Binding Biochemical Biological Biological Assay Biology Brain Catalytic Domain Cell physiology Cells Circadian Rhythms Clock protein Clustered Regularly Interspaced Short Palindromic Repeats Dimerization Drosophila genus Enzymes Event Fusion Protein Expression Goals Grant Health Human In Vitro Intention Knock-in Light Mammals Measurement Mediating Mental disorders Messenger RNA Methods Molecular Biology Motor Activity Nerve Degeneration Neurons Neurosciences Noise Orthologous Gene Paper Pharmaceutical Preparations Poly(A)-Binding Proteins Post-Transcriptional Regulation Protein Biosynthesis Proteins Publications RNA RNA Editing RNA Recognition Motif RNA Sequences RNA-Binding Proteins Regulation Ribosomal Proteins Ribosomes Signal Transduction Source Stroke Substance Addiction Substance abuse problem System Techniques Time Transcript Translating Translational Regulation Translations Tribes Variant Work Yeasts brain cell circadian fly improved in vivo interest novel novel strategies overexpression programs protein TDP-43 response tissue/cell culture transcriptome sequencing translation factor

项目摘要

Project Summary/Abstract Recent publications indicate that post-transcriptional regulation makes an important contribution to circadian rhythms, in flies and in mammals. This evidence is now quite extensive and includes translational regulation, which impacts the synthesis of key clock proteins within the mammalian SCN as well as within the small number of central brain neurons that govern Drosophila circadian locomotor activity rhythms. These neurons pose a significant challenge – and opportunity – for circadian biology and for molecular biology more generally: how can one assess biochemical events within a few neurons? For almost all measurements, there is not enough signal and/or a problematic signal:noise ratio when an extract is made and assayed starting with only a few cells/brain. Even methods that tag brain neurons are plagued with this problem if the fraction of tagged neurons/brain is very low. To address this issue in the context of RNA binding proteins and their targets, my lab developed a new technique, which we call TRIBE (Targets of RNA Binding proteins Identified by Editing). It fuses the catalytic domain of the RNA editing enzyme ADAR to RNA binding proteins. TRIBE takes advantage of the fact that one can make clean RNA and sequence it even from a single cell despite the inability to work with an extract from the same source material. We have used TRIBE with three different RBPs and also showed that it can work within the “small number of specific neurons” context. We have recently validated TRIBE and improved its efficacy, so that it has many fewer false negatives, i.e., it recognizes a much greater fraction of RBP- associated mRNAs. We will extend the method in two new translation-centric directions: to identify specific targets of the important translation factor eIF4E-BP and to identify ribosome-associated transcripts. We also propose to address some remaining issues, and extend TRIBE in new directions, with CRISPR-mediated knock-ins as well as with dimerization approaches. The latter will bring the editing moiety to the RNA on command, i.e., in response to a drug- or light-mediated dimerization signal. In all cases, assaying successfully small numbers of discrete neurons is the key biological focus. It is an important goal relevant to many human health problems like mental illness, neurodegeneration, stroke, substance abuse and addiction.

项目摘要/摘要最近的出版物表明，转录后法规为昼夜节律，苍蝇和哺乳动物。这些证据现在非常广泛，包括翻译调节，这会影响哺乳动物SCN中关键时钟蛋白的合成以及在控制果蝇昼夜运动运动的少数中央脑神经元中活动节奏。这些神经元对昼夜节律生物学和机会构成了重大挑战和机会对于分子生物学，更普遍：一个人如何评估几个神经元内的生化事件？为了几乎所有测量值，都没有足够的信号和/或问题的信号：噪声比提取物是从仅几个细胞/大脑开始的。甚至标记脑神经元的方法如果标记的神经元/大脑的比例非常低，则困扰着这个问题。在 RNA结合蛋白及其靶标的背景，我的实验室开发了一种新技术，我们称之为部落（通过编辑确定的RNA结合蛋白的靶标）。它融合了RNA的催化结构域将酶ADAR编辑为RNA结合蛋白。部落利用一个人可以做出的事实清洁RNA并从单个单元目的地对其进行排序，无法与提取物一起工作相同的原始材料。我们使用了三个不同的RBP的部落，还表明它可以起作用在“少数特定神经元”环境中。我们最近验证了部落并改善了它的效率，使其具有较少的错误负面因素，即它认识到RBP-的一部分更大的一部分相关的mRNA。我们将以两个新的以译本为中心的方向扩展该方法：识别重要翻译因子EIF4E-BP的特定目标，并识别与核糖体相关的成绩单。我们还建议解决剩余的一些问题，并将部落扩展到新的方向，随着CRISPR介导的敲击以及二聚化方法。后来将带来编辑 RNA指挥的部分，即响应药物或光介导的二聚信号。总的来说案例，分析成功数量的离散神经元是关键的生物学重点。这是一个与许多人类健康问题有关的重要目标，例如精神疾病，神经退行性，中风，药物滥用和成瘾。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Comparison of TRIBE and STAMP for identifying targets of RNA binding proteins in human and Drosophila cells.

DOI：
10.1261/rna.079608.123
发表时间：
2023-08
期刊：
RNA (New York, N.Y.)
影响因子：
0
作者：
通讯作者：

TRIBE: Hijacking an RNA-Editing Enzyme to Identify Cell-Specific Targets of RNA-Binding Proteins.

DOI：
10.1016/j.cell.2016.03.007
发表时间：
2016-04-21
期刊：
CELL
影响因子：
64.5
作者：
McMahon, Aoife C.;Rahman, Reazur;Jin, Hua;Shen, James L.;Fieldsend, Allegra;Luo, Weifei;Rosbash, Michael
通讯作者：
Rosbash, Michael

Protocol for using TRIBE to study RNA-protein interactions and nuclear organization in mammalian cells.