Principles and Properties of Disordered Regions in Post-Transcriptional Control

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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.