Structure-Function Mapping of the Nuclear Pore Complex-Renewal

ABSTRACT Nuclear pore complexes (NPCs) are huge macromolecular assemblies that serve as the only conduit for bidirectional transport between the nucleus and cytoplasm. We have determined the constituents, architecture, and detailed high precision structure of the archetypal yeast NPC. However, despite our increasing structural information on NPCs, we still lack a fundamental understanding of the mechanics of numerous of its functions. With our detailed maps in hand, we are, for the first time, in a unique position to map and reveal the structural changes associated with functional states that throw light on mechanisms underlying critical aspects of NPC function. Our hypothesis is that, despite some overlap, discrete and distinct structural stages and states are associated with NPCs’ varied functions. We have therefore established a powerful pipeline for analyzing NPCs and their vicinal associated complexes both structurally and functionally in defined functional states onto which we map quantitative phenotypic information. This information will allow us to move from static models of NPCs to working models of the machine in action, breathing life into our NPC maps and dissecting out how particular functionalities are mechanistically supported at the structural level. We focus on two such functionalities that are central to nuclear function at two related levels: first, as a regulator of transport, NPCs control mRNA packaging and export to the cytoplasm to both mediate and regulate gene expression; and second, NPCs directly control genes by binding chromatin and its regulators to alter expression states epigenetically. Both processes are incompletely understood at the molecular level, and have profound effects on cellular function as evidenced by the fact that disruptions of NPC-associated proteins associated with these functions lead to many human diseases. For Aim 1, we will determine the molecular machinery of NPC-mediated mRNP export by studying NPCs effectively “frozen” in defined intermediate stages of mRNP export. For Aim 2, we will determine the molecular machinery of NPC-mediated chromatin organization, specifically focusing on subtelomeric gene silencing. Using our established pipeline, we will identify and structurally characterize these NPC stages and states and their vicinal interactomes. Realizing these Aims will generate NPC structure-function maps in unprecedented detail, which will be of great use to the field to understanding how the mRNP export and chromatin remodeling machineries act in concert with different parts of the NPC to enable their functionalities and will shed light on the nature of numerous disorders associated with dysfunction in these processes. The resulting structure-function NPC maps promise to set the stage for tapping the NPC’s tremendous potential as a drug target for many human conditions ranging from cancers to infectious diseases to developmental and neurological disorders. 1

ABSTRACT Nuclear pore complexes (NPCs) are huge macromolecular assemblies that serve as the only conduit for bidirectional transport between the nucleus and cytoplasm. We have determined the constituents, architecture, and detailed high precision structure of the archetypal yeast NPC. However, despite our increasing structural information on NPCs, we still lack a fundamental understanding of the mechanics of numerous of its functions. With our detailed maps in hand, we are, for the first time, in a unique position to map and reveal the structural changes associated with functional states that throw light on mechanisms underlying critical aspects of NPC function. Our hypothesis is that, despite some overlap, discrete and distinct structural stages and states are associated with NPCs’ varied functions. We have therefore established a powerful pipeline for analyzing NPCs and their vicinal associated complexes both structurally and functionally in defined functional states onto which we map quantitative phenotypic information. This information will allow us to move from static models of NPCs to working models of the machine in action, breathing life into our NPC maps and dissecting out how particular functionalities are mechanistically supported at the structural level. We focus on two such functionalities that are central to nuclear function at two related levels: first, as a regulator of transport, NPCs control mRNA packaging and export to the cytoplasm to both mediate and regulate gene expression; and second, NPCs directly control genes by binding chromatin and its regulators to alter expression states epigenetically. Both processes are incompletely understood at the molecular level, and have profound effects on cellular function as evidenced by the fact that disruptions of NPC-associated proteins associated with these functions lead to many human diseases. For Aim 1, we will determine the molecular machinery of NPC-mediated mRNP export by studying NPCs effectively “frozen” in defined intermediate stages of mRNP export. For Aim 2, we will determine the molecular machinery of NPC-mediated chromatin organization, specifically focusing on subtelomeric gene silencing. Using our established pipeline, we will identify and structurally characterize these NPC stages and states and their vicinal interactomes. Realizing these Aims will generate NPC structure-function maps in unprecedented detail, which will be of great use to the field to understanding how the mRNP export and chromatin remodeling machineries act in concert with different parts of the NPC to enable their functionalities and will shed light on the nature of numerous disorders associated with dysfunction in these processes. The resulting structure-function NPC maps promise to set the stage for tapping the NPC’s tremendous potential as a drug target for many human conditions ranging from cancers to infectious diseases to developmental and neurological disorders. 1