Identification of the core organizational principles within nuclear mRNAs

University Of California At Davis, Davis CA

Investigators

Abstract

PROJECT SUMMARY Eukaryotic cells require messenger (m)RNAs to be exported through a nuclear pore complex for translation. From the start of transcription, RNA binding proteins (RBPs) are needed to package, surveil, and ultimately export processed mRNAs. One third of all RBPs are associated with disease and an overwhelming majority are involved in RNA metabolism, including mRNA export. These perturbations cause gene expression changes that lead to developmental delays and disease. A major challenge to defining how mutations in these RBPs contribute to tissue-specific disease is that export events occur within seconds making observation and study difficult. Recent advancements in fractionation and isolation methods of nuclear mRNA-protein complexes at distinct stages now provide a means to capture these elusive complexes for study. The central hypothesis of the work proposed here is that mRNA structure is a critical factor that leads to efficient nuclear export and that disruptions in RNA structure cause inefficient export and nuclear retention. To address this hypothesis, I aim to combine RNA structure probing with optimized fractionation and isolation methods to determine how RBPs organize and package nuclear mRNAs for export in S. cerevisiae. Given the high sequence similarity and conserved functional pathways, budding yeast offers a powerful model system to address this hypothesis utilizing highly optimized methods in an innovative way. Specifically, this project will be the first to define RNA structure, with single nucleotide resolution, within nuclear mRNA-protein complexes prior to export and address the synergy within these complexes from the perspective of both macromolecules. RNA structure modeling will be used in Aim 1 to define the principles of nuclear mRNA structure that lead to efficient export or retention by sequentially probing RNA structure in cells, fractioning or isolating nuclear mRNA-protein complexes, and subjecting mRNAs to mutational profiling. The outcome of these efforts will define organizational principles of mRNAs within nuclear mRNA-protein complexes to provide knowledge of how RNA structure and RBP occupancy influence gene expression. In Aim 2, I will define the role of a highly stoichiometric protein within nuclear mRNA-protein complexes, Yra1 (ALY/REF), in packaging and folding mRNAs for export. In Aim 3, I will attempt to shift RNA structure of an inefficiently exported mRNA, through mutation or tethering Yra1, to recover mRNA export and reinstate gene expression. Completion of these aims are fundamental to unraveling the complex relationship between mutations in RNA binding proteins essential for mRNA nuclear export and the emergence of tissue- specific disease. Moreover, this knowledge can be used to design therapeutics aimed at altering mRNA structure and gene expression to replace the function of proteins mutated in disease and alleviate disease pathology.