Saturday, November 23, 2024
11/23/2024
TITLE Molecular basis of mRNA export ABSTRACT In eukaryotes, the genetic information encoded in the chromatin is stored within the nucleus. For genetic programs to be executed, folded proteins, ribonucleic acids, and assembled macromolecular complexes must be transported across the nuclear envelope. Nuclear pore complexes (NPCs) are exclusive gateways for the regulated bidirectional exchange of macromolecules between the nucleus and cytoplasm. In humans, NPCs are assembled from multiple copies of 35 different proteins known as nucleoporins (nups) that give rise to a ~1,000-protein cylindrical structure spanning the nuclear envelope with a molecular mass of ~120 million Daltons. Due to its size and flexibility, obtaining an atomic structure of the NPC has been a formidable task that we have overcome over the last two decades with a divide-and-conquer approach. This approach relies on biochemical reconstitution and nup-nup interaction mapping, along with high-resolution structure determination of nups and nup complexes, combined with lower resolution cryo-electron tomography (cryoET) and in vivo validation. These efforts have resulted in a near-atomic composite structure accounting for ~90% of the human NPC’s mass. However, critical aspects of both its structure and function remain unexplored. The NPC’s cytoplasmic face harbors nups that mediate interactions between mobile transport factors and their protein or ribonucleic acid cargoes, as well as the machinery that dismantles messenger ribonucleoprotein particles (mRNPs) as they emerge from the NPC’s central transport channel, irreversibly releasing mRNA into the cytoplasm. Notably, genetic variations in these nups are prominently associated with various human diseases, including neurodegenerative and neoplastic disorders, and heightened susceptibility to viral infections. We have previously reconstituted nup complexes of fungal NPCs’ cytoplasmic faces, enabling the systematic dissection of their nup-nup interactome. We now propose to perform an equivalent reconstitution of the human NPC’s cytoplasmic face using recombinantly expressed and highly purified proteins. Our second goal is to expand our high-resolution structural characterization to still unresolved parts of the NPC. Third, we will functionally characterize previously identified RNA-binding events involving cytoplasmic nups and the mobile RNA-transporting machinery by determining high-resolution structures and assessing their functional relevance using in vitro activity assays and cell-based assays for nucleocytoplasmic cargo transport. Finally, we will investigate the mechanism of action of the ORF6 virulence factor from SARS-CoV-2 and related sarbecoviruses. We will employ a multidisciplinary approach to gain atomic-level insights into ORF6's interactions with the NPC and the nucleocytoplasmic transport machinery, as well as the effects these interactions have on nuclear transport, mRNA export, and NPC integrity. Overall, our research will contribute to understanding the NPC’s role in mRNA export and the viral interference mechanisms targeting this process, providing atomic- level detail toward addressing this fundamental cell biological question and the molecular basis for the development of antiviral treatments.
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