Nup98-mediated Regulation of Gene Expression and RNA Metabolism

• Author / Creator

  Capitanio, Juliana S.

• The nucleus is a hallmark of eukaryotic cells. It segregates the genetic material from the cytoplasm. The nucleus is encapsulated by the nuclear envelope which is perforated with nuclear pore complexes (NPCs), macromolecular structures formed by proteins termed nucleoporins (Nups). NPCs and nuclear transport factors regulate nucleocytoplasmic transport. Many Nups also function away from NPCs, within the nucleoplasm, where they associate with chromatin and other nuclear factors to regulate gene expression and RNA metabolism. One such Nup, Nup98, has been shown to interact with chromatin to regulate gene expression in metazoan cells. To gain further insight into how Nup98 contributes to gene expression regulation in human cells, we began by identifying novel Nup98 binding partners and focusing on those with known roles in gene expression. Here, we report the identification of DHX9, a DExH/D-box helicase, as an intranuclear Nup98 binding partner. Our results show that the N-terminal domain of Nup98, containing FG/GLFG amino-acid repeats, binds to DHX9 in an RNA-facilitated manner. DHX9 interacts with Nup98 via its N- and C-terminal domains, which bind double-stranded and single-stranded RNAs, respectively. Importantly, the RNA-dependent ATPase activity of DHX9 is stimulated by its interaction with Nup98, resulting in increased DHX9-stimulated transcription of a reporter gene. Consistent with these observations, Nup98 and DHX9 bind interdependently to similar gene loci and their transcripts, regulating gene expression and RNA splicing. Based on our results, we propose that Nup98 affects gene expression and RNA metabolism by functioning as a cofactor that regulates DHX9 and, potentially, other RNA helicases in the cell. Several viruses exploit Nups and RNA helicases during infection. Nup98 and DHX9 are among these host factors hijacked by viruses to participate in their life cycle. Thus, we explored how the Nup98-DHX9 complex, characterized as a regulator of gene expression and RNA metabolism in the host cell, can be exploited by positive-strand RNA viruses of the Flaviviridae family (i.e. the Hepatitis C virus and Zika virus). We also examined the importance of other host RNA-binding nuclear factors and nuclear transport factors (NTFs) in the life cycle of these cytoplasmic RNA viruses. Despite the cytoplasmic replication cycle of the Hepatitis C virus (HCV) and Zika virus (ZIKV), their positive-strand RNA genomes (+vRNA) are present in the nuclei of infected cells. NTFs dynamically transport this nuclear pool of +vRNA, and altering nucleocytoplasmic transport can affect the nuclear localization of the viral genome in infected cells. Disrupting the nuclear localization of +vRNA changes its interaction with several RNA-binding host nuclear factors (including Nup98 and DHX9), consequently affecting the viral life cycle. These data point towards a previously unknown role for the nuclear environment in the life cycle of viruses that undergo replication in the cytoplasm of the host cells.

• Subjects / Keywords

  • DHX9
  • gene expression
  • Hepatitis C Virus
  • Zika Virus
  • NPC
  • mRNA metabolism
  • Nup98
  • nucleocytoplasmic transport

• Graduation date

  Fall 2017

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3JM23Z12

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.