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Target specificity of human RNA-induced silencing complex

人RNA诱导的沉默复合物的靶标特异性

基本信息

批准号：
10687141
负责人：
Kotaro Nakanishi
金额：
$ 31.66万
依托单位：
OHIO STATE 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 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10687141
关键词：
5&apos Untranslated Regions Acylation Algorithms Amino Acids Antiviral Response Binding Binding Proteins Binding Sites Biological Assay Biological Process Cells Chimera organism Complex Cryoelectron Microscopy Designer Drugs Development Disease Dissociation Drug Design Equipment and supply inventories Eukaryotic Cell Event Foundations Funding Gene Expression Gene Silencing Goals Guide RNA HIV Human Hydroxyl Radical Immunoprecipitation Individual Internal Ribosome Entry Site Investigation Knowledge Malignant Neoplasms Mass Spectrum Analysis Measures Mediating Messenger RNA Methods MicroRNAs Molecular Molecular Conformation N-methylisatoic anhydride Nucleotides Outcome Outcome Study Primer Extension Property Proteins RNA RNA Binding RNA Interference RNA purification RNA-Induced Silencing Complex Resolution Role Shapes Site Solid Specificity Structure Techniques Testing Therapeutic Translational Repression Translations Visualization Work X-Ray Crystallography base design experimental study flexibility hydroxyl group improved insight mutant neurodevelopment novel novel therapeutic intervention prediction algorithm prevent scaffold transcriptome sequencing

项目摘要

PROJECT SUMMARY In eukaryotic cells, gene expression is regulated at multiple levels, including post-transcriptional gene silencing, where microRNAs (miRNAs) bind to complementary target RNAs and cause translational repression. Argonaute (AGO) proteins and miRNAs form RNA-induced silencing complexes (RISCs), the core players in gene silencing. Humans have four AGO proteins, AGO1-4, which share a high sequence identity, and the majority of miRNAs bound are common across all AGOs. Therefore, it has been thought that the four AGOs work redundantly. Nevertheless, an increasing number of studies have found that each AGO has its unique roles in various biological processes and diseases in addition to gene silencing. Although the interaction of all four AGOs with miRNAs has been well characterized, little is known about how each RISC recognizes its target RNAs. Elucidation of this recognition will provide insight into the unique roles of each AGO. Meanwhile, characterization of RISC and target interactions will facilitate target prediction accuracy by improving prediction algorithms, which will take account of not only the complementarity between guide and target but also the type of AGO and target interaction. In this proposed study, we will pursue the following specific aims. In Aim 1, we will use cryo-electron microscopy and X-ray crystallography to determine the structures of all four homogenously purified RISCs with the same guide and target RNAs, which will provide insight into the differences in target recognition by the four AGOs. In Aim 2, to clarify these differences, we recently developed a novel SHAPE-based technique which allows us to visualize the conformational dynamics of target RNA bound to RISC. The method will enable us to characterize this interaction within the RISC binding channel and its periphery at a single-nucleotide resolution and can be expanded to understand how RISCs recognize guide- binding sites buried within highly structured target RNAs. In Aim 3, we will first use mass spectrometry to identify the unique protein binding partners of each AGO and their specific sites of interaction. Then, we will use tandem immunoprecipitation, followed by RNA sequencing, to determine how the binding of these proteins influences the target specificity of each AGO and directs their functionality towards alternative cellular events. The outcome from this study will provide a solid foundation for fields beyond gene silencing and enable the development of new strategies for higher accuracy guide-RNA drug design in therapeutic applications.

项目总结