Principles and Properties of Disordered Regions in Post-Transcriptional Control

项目总结/摘要 蛋白质的内含子无序区与特定的调控配偶体相互作用以控制基因的表达 表情尽管它们在生物学上很重要，但我们并不了解无序蛋白质的分子特征是如何影响它们的生物学特性的。 区域有助于不同的监管程序，因为目前的方法不能询问序列， 在蛋白质组范围内的功能关系。这种限制见于转录后控制， 传统的生物化学实验已经确定了几个募集翻译或mRNA的无序区域 衰变机制针对转录本，但无法定义管理其功能的一般规则。我 我建议建立一个机制框架，解释无序区域的生物物理特性 通过建立创新高通量方法来影响转录后调节活性， 重要的功能特征。 理解描述无序区域如何控制转录后的分子原理 基因表达需要系统地测量成千上万的紊乱的调节作用， 序列的为了满足这一需求，我开发了一种高通量功能测定， 该技术可以将转录效应转化为荧光信号，并能够对调控活性进行大规模分析。这 一种方法使用异源RNA-蛋白质相互作用，将查询肽与报告基因mRNA编码结合， 荧光蛋白，其表达基于系链构建体的调节活性而改变。我 利用这种测定方法，通过表达一种 在报告菌株中的无序肽文库，并使用荧光激活细胞分选来分离活性肽。 这些元素是通过深度测序鉴定的。我现在建议学习后- 通过测量序列组成的变化如何影响活性， 用我的高通量分析首先，我将对功能性无序区域进行扫描诱变， 用我的束缚筛选来定义活性所需的残基和基序。在发现监管 基序，我将嵌入这些序列在不同的物理化学环境，并研究如何当地的背景 影响功能。这些高通量测量将使我能够揭示 序列特征和转录后活性。最后，我将测试如何 无序区域的生物物理特性影响其内源性环境中的调节机制 通过表型分析和生化重建。总的来说，这项建议将建立 通过开发和利用一种新的方法， 创新的高通量功能分析方法。

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.