Document text
Principal Investigator: Andre Hoelz
Organization: CALIFORNIA INSTITUTE OF TECHNOLOGY
Fiscal Year: 2024
Award: $376,883
Funding agency: National Institute of General Medical Sciences
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.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><Accounting><Address><Anatomic Sites><Anatomic structures><Anatomy><Anti-viral Response><Architecture><Assay><Binding><Bioassay><Biochemical><Biological><Biological Assay><Blind Spots><COVID-19 virus><COVID19 virus><Cancers><Cell Body><Cell Function><Cell Nucleus><Cell Nucleus Active Transport><Cell Physiology><Cell Process><Cells><Cellular Function><Cellular Physiology><Cellular Process><Characteristics><Chromatin><CoV-2><CoV2><Complex><Cryo-electron Microscopy><Cryo-electron tomography><Cryoelectron Microscopy><Cytoplasm><Cytoplasmic Filaments><Defect><Degenerative Neurologic Disorders><Diffusion><Disease><Disorder><Dissection><Docking><Dysfunction><Electron Cryomicroscopy><Engineering / Architecture><Eukaryota><Eukaryote><Eukaryotic Cell><Event><Face><Functional disorder><Future><Genetic><Genetic Alteration><Genetic Change><Genetic Diversity><Genetic Variation><Genetic defect><Genome><Goals><Head><Health><Human><Human Genome><In Vitro><In vivo analysis><Knowledge><Link><Location><Macromolecular Complexes><Malignant Neoplasms><Malignant Tumor><Maps><Mediating><Membrane><Messenger RNA><Modern Man><Molecular><Molecular Interaction><Mutation><NPC><Nerve Degeneration><Nervous System Degenerative Diseases><Neural Degenerative Diseases><Neural degenerative Disorders><Neurodegenerative Diseases><Neurodegenerative Disorders><Neurologic Degenerative Conditions><Neuron Degeneration><Non-Polyadenylated RNA><Nuclear><Nuclear Envelope><Nuclear Membrane><Nuclear Pore Complex><Nuclear Pore Complex Proteins><Nuclear Structure><Nuclear Transport><Nucleocytoplasmic Shuttling><Nucleoporin Gene><Nucleoporins><Nucleus><Nup Protein><Pathogenicity Factors><Physiologic><Physiological><Physiopathology><Predisposition><Process><Proteins><Protomer><RNA><RNA Binding><RNA Gene Products><RNA Transport><RNA and protein interaction><RNA bound><RNA-Protein Interaction><Receptor Protein><Recombinants><Reporter><Research><Resolution><Rest><Retinal blind spot><Ribonucleic Acid><Ribonucleic Acid Transport><Ribonucleoproteins><Role><SARS corona virus 2><SARS-CO-V2><SARS-COVID-2><SARS-CoV-2><SARS-CoV2><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Sarbecovirus><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome related corona virus 2><Structure><Subcellular Process><Susceptibility><Tail><Translations><Validation><Viral><Viral Diseases><Viral Interference><Viral Pathogenesis><Virulence Factors><Virus><Virus Diseases><Work><Wuhan coronavirus><anti-viral development><anti-viral drug development><anti-viral therapeutic development><anti-viral therapy development><antiviral development><antiviral drug development><antiviral therapeutic development><antiviral therapy development><biologic><coronavirus disease 2019 virus><coronavirus disease-19 virus><cryo-EM><cryo-EM tomography><cryoEM><cryoEM tomography><cryoelectron tomography><cryogenic electron microscopy><dalton><degenerative diseases of motor and sensory neurons><degenerative neurological diseases><density><developing anti-viral agent><developing anti-viral drug><developing anti-viral therapeutic><developing anti-viral therapy><developing antiviral agent><developing antiviral drug><developing antiviral therapeutic><developing antiviral therapy><diffused><diffuses><diffusing><diffusions><electron cryo-tomography><faces><facial><flexibility><flexible><genetic information><genome mutation><hCoV19><human disease><human whole genome><in vitro activity><in vivo><in vivo evaluation><in vivo testing><insight><interdisciplinary approach><interfering virus><mRNA><mRNA Export><mRNP><macromolecular assembly><macromolecule><malignancy><membrane structure><messenger ribonucleoprotein><molecular mass><multidisciplinary approach><nCoV2><neoplasm/cancer><neoplastic><neural degeneration><neurodegeneration><neurodegenerative><neurodegenerative illness><neurological degeneration><neuronal degeneration><nucleocytoplasmic transport><particle><pathophysiology><programs><protein folding><protein protein interaction><protein purification><receptor><reconstitute><reconstitution><reconstruction><recruit><resolutions><segregation><social role><success><translation><validations><viral infection><virus infection><virus pathogenesis><virus-induced disease>