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Principles and Properties of Disordered Regions in Post-Transcriptional Control

转录后控制中无序区域的原理和特性

基本信息

批准号：
10535908
负责人：
Joseph Heimlich Lobel
金额：
$ 6.76万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10535908
关键词：
Address Affect Affinity Chromatography Binding Biochemical Biological Biological Assay Biological Process Biophysics Catalogs Cell physiology Complement Couples Data Set Development Disease Elements Environment Equipment and supply inventories Fluorescence-Activated Cell Sorting Functional disorder Gene Expression Genetic Transcription Genetic Translation Immunity In Vitro Investigation Learning Length Libraries Machine Learning Mass Spectrum Analysis Measurement Measures Mediating Messenger RNA Methodology Methods Molecular Mutagenesis Mutate Mutation Pathway interactions Peptides Phenotype Post-Transcriptional Regulation Property Protein Biosynthesis Protein Region Proteins Proteome RNA-Binding Proteins RNA-Protein Interaction Regulatory Element Reporter Scanning Signal Transduction Structure Techniques Testing Transcript Translations Work Yeasts base biophysical properties cofactor deep sequencing experimental study genome-wide high throughput screening in vivo innovation mRNA Decay mRNA Expression mRNA Stability mutation screening programs reconstitution recruit

项目摘要

Project Summary/Abstract Intrinsically disordered regions of proteins interact with specific regulatory partners to control gene expression. Despite their biological importance, we do not understand how molecular features of disordered regions contribute to distinct regulatory programs because current methodologies cannot interrogate sequence- function relationships on a proteome-wide scale. This limitation is seen in post-transcriptional control, where traditional biochemical experiments have identified several disordered regions that recruit translation or mRNA decay machinery to target transcripts but have been unable to define general rules that govern their function. I propose to establish a mechanistic framework explaining how the biophysical properties of disordered regions influence post-transcription regulatory activity by building on an innovative high-throughput approach to uncover important functional features. Understanding the molecular principles describing how disordered regions control post-transcriptional gene expression requires systematically measuring the regulatory effects of many thousands of disordered sequences. To address this need, I have developed a high-throughput functional assay that couples post- transcriptional effects to a fluorescent signal and enables large-scale profiling of regulatory activity. This approach uses a heterologous RNA-protein interaction that tethers a query peptide to a reporter mRNA encoding a fluorescent protein, whose expression changes based on the regulatory activity of the tethering construct. I leveraged this assay to catalog functional post-transcriptional regulators in the yeast proteome by expressing a library of disordered peptides in a reporter strain and used fluorescence activated cell sorting to isolate active elements, which were identified by deep sequencing. I now propose to learn the molecular grammar of post- transcriptional regulatory disordered regions by measuring how changes in sequence composition affects activity using my high-throughput assay. First, I will perform scanning mutagenesis of functional disordered regions and use my tethering screen to define residues and motifs necessary for activity. Following discovery of regulatory motifs, I will embed these sequences in different physicochemical environments and examine how local context influences function. These high-throughput measurements will allow me to uncover correlations between sequence features and post-transcriptional activity using machine learning approaches. Finally, I will test how the biophysical properties of disordered regions affect regulatory mechanisms in their endogenous context through phenotypic analysis and biochemical reconstitution. Taken together, this proposal will establish the general principles of disordered regions involved in post-transcriptional control by developing and utilizing an innovative high-throughput functional profiling method.

项目总结/文摘