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Systematic characterization of trans regulation of A-to-I RNA editing in neurons

神经元中 A-to-I RNA 编辑反式调节的系统表征

基本信息

批准号：
9423930
负责人：
Jin Billy Li
金额：
$ 45.5万
依托单位：
STANFORD UNIVERSITY
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：
2017-09-15 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9423930
关键词：
ADAR1 Adenosine Affect Alzheimer&apos s Disease Amino Acids Amyotrophic Lateral Sclerosis Autistic Disorder Biochemical Biochemical Genetics Biological Assay Biotinylation Brain CRISPR screen Candidate Disease Gene Cells Circadian Rhythms Clustered Regularly Interspaced Short Palindromic Repeats Co-Immunoprecipitations Code Coupled Cytidine Deaminase DRADA2b protein Depression and Suicide Development Double-Stranded RNA Drosophila genus Enhancers Environment Enzymes Family Frequencies Genes Genetic Genetic Screening Genetic Transcription Goals Guanosine Human Immunofluorescence Microscopy In Situ In Vitro Inosine K562 Cells Knock-out Knowledge Lead Libraries Link Locomotion Malignant Glioma Mus Mutagenesis Mutation Nervous system structure Neurologic Neurons Phenotype Play Point Mutation Process Proteins Protocols documentation RNA RNA Editing Regulation Reporter Role Secondary to Seizures Site Structure System Technology Testing Tissues Variant Work Yeasts adenosine deaminase base crosslinking and immunoprecipitation sequencing follow-up genetic approach genome-wide human disease improved in vivo mutant nervous system disorder novel novel strategies protein function public health relevance spatiotemporal transcriptome transcriptomics

项目摘要

Project Summary/Abstract RNA editing is a critical process for generating spatiotemporal transcriptomic diversity that is particularly important in the brain. A-to-I (adenosine to inosine, which is recognized as guanosine) editing is the most common form of RNA editing in metazoans and is catalyzed by a family of enzymes called adenosine deaminases acting on RNA (ADARs). A-to-I editing occurs co-transcriptionally when double-stranded RNA (dsRNA) is bound and edited by ADAR enzymes, which occurs at high frequency in the nervous system. Alteration of RNA editing levels is implicated in a number of neurological disorders. Loss of ADAR can lead to neurological phenotypes such as seizure, altered locomotion and circadian rhythm. Previous work studying mutations in ADAR demonstrates the importance of a few amino acids critical for proper editing activity, including a handful known to cause human diseases. However, we still lack a comprehensive understanding of ADAR protein function. We know even less about other trans regulators of RNA editing despite the evidence suggesting their existence. In this work, we aim to develop systematic approaches to deciphering the trans regulation of A-to-I RNA editing. First, we will identify functional mutants of ADAR1 and ADAR2 in human cells. Using a CRISPR-based technology we recently developed, we will perform saturation mutagenesis of ADAR1 and ADAR2 to introduce point mutations in human cells. We will identify functional ADAR mutants with decreased or increased editing activity and further characterize how these mutations affect ADAR editing activity in vivo. Second, we will identify novel regulators of RNA editing through biochemical and genetic screens. We will identify ADAR-interacting proteins in induced human neurons. We will also carry out a genome-wide CRISPR/Cas9 screen in induced human neurons to find candidates that alter editing levels. Top candidate genes are subject to secondary CRISPR/Cas9 screening in mouse primary neurons as well as double knockout in pairwise combinations to analyze their genetic interactions. Third, we will determine mechanistically how editing regulators alter the transcriptome-wide landscape of RNA editing. We will perturb the regulators to examine how they affect editing levels transcriptome-wide in human cells, mouse primary neurons, and Drosophila brains. We will test whether the regulators physically interact with ADAR1/2 or each other, and if and how they interact with ADAR RNA substrates. This work will provide an unprecedented understanding of trans regulation of A-to-I RNA editing in neurons, revealing novel mechanisms underlying this largely unexplored machinery.

项目总结/文摘