Structural and Functional Characterization of the S. cerevisiae mRNA export platform - Project Abstract: The nuclear pore complex (NPC) is an ancient molecular machine embedded in all eukaryotic nuclear membranes that acts as a physical gatekeeper for macromolecules that enter or leave the nucleus. mRNA is bound by RNA-binding proteins to form messenger ribonucleoprotein particles (mRNPs) that aid in transit across the NPC so that they may exit into the cytoplasm to be translated into proteins. The NPC mediates mRNP export along a pathway that is distinct from transport of proteinaceous or ribosomal cargoes that culminates at a highly conserved cytoplasmic export location termed the mRNA export platform. At the mRNA export platform, mRNPs are remodeled, undergoing conformational and compositional changes, to facilitate release of packaged mRNA for translation into the cytoplasm. Mutations in or mis-regulation of proteins in the mRNA export pathway and particularly at this critical export complex are implicated in developmental defects, cancer, and in particular, aging. Previous studies that combined crosslinking mass-spectrometry and negative-stain electron microscopy with integrative modeling strategies yielded a low-resolution map of the architecture of the complex, but a high- resolution map of the entire mRNA export platform that yields molecular insights into the assembly of the complex and identifies key points of regulation has eluded us. Additionally, recent work has provided substantial evidence that the NPC has a functional role in post-transcriptional gene regulation, with preliminary data indicating that dysfunction in the mRNA export platform leads to disruption in this regulatory role. The goal of this proposal is to understand the molecular determinants of mRNA export platform assembly and elucidate the mechanisms by which aging-associated mutations affect platform assembly and lead to aberrant gene regulation, therefore manifesting in the onset of premature aging. Aim 1: To leverage single particle cryo-EM of the core Nup82 complex, as well as reconstitution biochemistry, crosslinking mass-spectrometry, and negative stain EM to generate a high-resolution integrative model of the mRNA export platform, onto which aging-associated mutations can be mapped to understand how these mutations disrupt platform assembly. Aim 2: To generate yeast strains carrying these aging-associated mutations that can be functionally characterized to uncover the mechanisms by which the mRNA export platform regulates gene expression and elucidate how these mutations affect this critical function and give rise to aging phenotypes. This work would give us an unprecedented understanding of post-transcriptional gene regulation at this critical mRNA export and remodeling site. Furthermore, this study would reveal new structural and molecular insights into the highly documented but poorly understood role of NPC dysfunction in the onset of aging and disease.
