Paula Upla

Integrative structure and functional anatomy of a nuclear pore complex

Nuclear pore complexes play central roles as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm. However, their large size and dynamic nature have impeded a full structural and functional elucidation. Here we determined the structure of the entire 552-protein nuclear pore complex of the yeast Saccharomyces cerevisiae at sub-nanometre precision by satisfying a wide range of data relating to the molecular arrangement of its constituents. The nuclear pore complex incorporates sturdy diagonal columns and connector cables attached to these columns, imbuing the structure with strength and flexibility. These cables also tie together all other elements of the nuclear pore complex, including membrane-interacting regions, outer rings and RNA-processing platforms. Inwardly directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized into distinct functional units. This integrative structure enables us to rationalize the architecture, transport mechanism and evolutionary origins of the nuclear pore complex.

Purification and analysis of endogenous human RNA exosome complexes

As a result of its importance in key RNA metabolic processes, the ribonucleolytic RNA exosome complex has been the focus of intense study for almost two decades. Research on exosome subunit assembly, cofactor and substrate interaction, enzymatic catalysis and structure have largely been conducted using complexes produced in the yeast Saccharomyces cerevisiae or in bacteria. Here, we examine different populations of endogenous exosomes from human embryonic kidney (HEK) 293 cells and test their enzymatic activity and structural integrity. We describe methods to prepare EXOSC10-containing, enzymatically active endogenous human exosomes at suitable yield and purity for in vitro biochemistry and negative stain transmission electron microscopy. This opens the door for assays designed to test the in vitro effects of putative cofactors on human exosome activity and will enable structural studies of preparations from endogenous sources.

Structure, Dynamics, Evolution and Function of a Major Scaffold Component in the Nuclear Pore Complex

The Nuclear Pore Complex, composed of proteins termed Nucleoporins (Nups), is responsible for the nucleo-cytoplasmic transport in eukaryotes. NPCs form an annular structure composed of the nuclear ring, cytoplasmic ring, a membrane ring, and two inner rings.Nup192 is a major component of the NPCs inner ring. We report the crystal structure of Saccharomyces cerevisiae Nup192 residues 2 to 960 [ScNup192(2-960)], which adopts an α-helical fold with three domains (i.e., D1, D2 and D3). SAXS and EM studies reveal that ScNup192(2-960) could undergo long-range transition between an “open” and “closed” conformations. We obtained a structural model of full-length ScNup192 based on EM, structure of ScNup192(2-960), and homology modeling.Evolutionary analyses using ScNup192(2-960) structure suggest that NPCs and vesicle coating complexes are descended from a common membrane-coating ancestral complex.We show that suppression of Nup192 expression leads to compromised nuclear transport and hypothesize a role for Nup192 in modulating the permeability of the NPC central channel.