Single-molecule resolution of RNA editing of mRNAs: visualizing GRIA2 editing in situ in ALS

mRNA RNA 编辑的单分子分辨率：在 ALS 中可视化 GRIA2 原位编辑

基本信息

批准号：
9488359
负责人：
Ian Alexander Mellis
金额：
$ 3.15万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-06-01 至 2020-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9488359
关键词：
Adenosine Affect Alleles Amyotrophic Lateral Sclerosis Animals Area Biological Biology Brain CRISPR/Cas technology Calcium Cell Line Cell Lineage Cell Nucleus Cells Chemicals Code Collection Complement Cultured Cells Cytosine DRADA2b protein Data Deaminase Deamination Defect Disease Enzymes Epilepsy Event Family Fluorescent in Situ Hybridization Functional disorder Future Genetic Transcription Glutamate Receptor Glutamates Grain Guanosine Higher Order Chromatin Structure Human In Situ Individual Inosine Introns Knock-out Knockout Mice Laboratories Lead Lesion Measures Mediating Messenger RNA Methods Modification Molecular Motivation Motor Neurons Nerve Degeneration Neurologic Dysfunctions Nuclear Nucleotides Open Reading Frames Pathogenesis Patients Pattern Phenotype Population Prevalence Process Proteins Protocols documentation RNA RNA Editing Rattus Regulation Regulator Genes Resolution Reverse Transcriptase Polymerase Chain Reaction Ribosomal RNA Sampling Site Small Interfering RNA Spinal Cord Structure Surveys Techniques Thymidine Tissue Engineering Tissue Sample Tissues Toxic effect Transcript Transfer RNA Untranslated RNA Variant Work adenosine deaminase analog base cell type design insight knock-down mouse model neuron loss novel relating to nervous system single molecule single-cell RNA sequencing tissue processing tool trafficking transcriptome transcriptome sequencing trend

项目摘要

Project Summary Much of the basic biology of mRNA editing remains unknown despite decades of study, largely due to the limitations of existing experimental methods. Adenosine to Inosine (A to I) editinga result of adenosine deamination catalyzed by deaminases such as ADAR enzymesis one of the most common forms of posttranscriptional chemical modification of RNA, generally known as RNA editing. A to I editing is essential for not only noncoding RNA function, such as rRNA and tRNA higherorder structure stabilization, but also for posttranscriptional mRNA regulation. For example, GRIA2, which encodes the GluR2 subunit of the AMPA glutamate receptor, has a highly conserved ADAR2targeted editing site in its coding sequence. Perturbation of the orthologous rat Gria2 editing event leads to neurological dysfunction. In humans with Amyotrophic Lateral Sclerosis (ALS), many diseaseaffected motor neurons appear to die as a result of glutamatergic calcium toxicity and have deficient GRIA2 editing, and Adar2 knockout mice demonstrate an ALSlike phenotype, which is rescued upon exogenous expression of edited Gria2. GRIA2 and several other mRNA A to I editing targets have been studied for more than twenty years, and over the last four years transcriptomewide surveys of A to I editing have provided evidence for many more mRNA targets. Despite this motivation, much basic information about the biology of this important mRNA regulatory process, including its subcellular localization, timing relative to transcription, and association with other regulatory factors, remains unknown largely due to the limits of experimental techniques. Recently, our laboratory described a fluorescence in situ hybridization method for visualizing and quantifying singlenucleotide variants (SNVs) in single cells using a novel probe hybridization strategy known as SNV FISH. We are leveraging inosine's structure as a guanosine analog to adapt SNV FISH to the analysis of A to I editing in a protocol we refer to as inosine FISH (iFISH). Our preliminary data show that we can use iFISH to specifically identify edited and unedited GRIA2 transcripts in situ in cultured cells. In this project, we are quantifying subsinglecellular trends in A to I editing of known targets, such as GRIA2 and mRNA targets newly identified in transcriptomewide screens. Specifically, we are using iFISH to measure singlecell editing rate distributions, to visualize subcellular localization patterns of edited mRNAs, and to measure editing timing relative to transcription. We are also studying the effect of ADAR2 on subcellular GRIA2 trafficking. Lastly, we are applying our novel method toward better characterizing perturbations of GRIA2 editing in individual motor neurons in ALS lesions by extending the use of iFISH to fixed sections of postmortem brain and spinal cord tissue samples from healthy and diseased donors.

项目摘要尽管研究了数十年，但mRNA编辑的许多基本生物学仍然未知，由于现有实验方法的局限性。腺苷到肌苷（a至i）腺苷的编辑结果脱氨酸是由脱氨酶（例如ADAR酶）催化的，是最常见的形式之一 RNA的转录后化学修饰，通常称为RNA编辑。我编辑是必不可少的不仅对于非编码RNA函数，例如RRNA和TRNA高阶结构稳定，还用于转录后mRNA调节。例如，编码AMPA的GLUR2亚基的Gria2 谷氨酸受体，在其编码序列中具有高度保守的ADAR2TARGETING编辑位点。扰动直系同源的大鼠gria2编辑事件导致神经功能障碍。在肌营养性侧面的人硬化症（ALS），许多受疾病的运动神经元因谷氨酸钙而死亡毒性且具有不足的Gria2编辑，而ADAR2敲除小鼠表现出Alslike的表型，该表型在编辑的gria2的外源表达后被救出。 gria2和其他几个mRNA A到我编辑目标已经研究了二十多年，在过去的四年中我编辑为更多的mRNA靶标提供了证据。尽管有这种动机，但许多基本信息关于这个重要的mRNA调节过程的生物学，包括其亚细胞定位，时机相对于转录以及与其他监管工厂的关联，在很大程度上仍然未知实验技术。最近，我们的实验室描述了一种可视化和的荧光原位杂交方法使用新型探针杂交策略已知的单个细胞中量化单核苷酸变体（SNV）已知作为SNV鱼。我们正在利用Innosine的结构作为鸟嘌呤类似物，以使SNV鱼适应分析我在协议中进行编辑的A中，我们称为Inosine Fish（Ifish）。我们的初步数据表明我们可以使用在培养的细胞中特异性地识别和未经编辑的gria2转录本。在这个项目中，我们正在量化A至I编辑已知靶标的下细胞趋势，例如GRIA2和mRNA靶标在整个转录组屏幕上新识别。特别是，我们正在使用IFISH测量单核编辑速率分布，可视化编辑mRNA的亚细胞定位模式，并测量编辑时间相对于转录。我们还研究了ADAR2对亚细胞格里亚2型运输的影响。最后，我们正在应用我们的新方法来更好地表征单个电动机中的gria2编辑扰动通过扩展使用IFISH到固定截面的固定截面的ALS病变中的神经元来自健康和患病供体的组织样品。