Browsing by Subject "Active Transport, Cell Nucleus"
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Item Characterization of the Beta-Karyopherin Protein Ipo9 During Germ Cell Differentiation(2019-12-04) Palacios, Victor Manuel; Krämer, Helmut; Buszczak, Michael; Carroll, Thomas J.; Mendell, Joshua T.Germ cells participate in the most fascinating process in nature, the fusion of two cells that ultimately give rise to an entire organism. Errors in germ cell development directly affect the fertility of the parent organism and/or the health of their offspring. A myriad of molecules such as transcription factors, signaling pathway effectors, chromatin modifiers and meiotic related proteins play fundamental roles during germ cell development. In order for these proteins to work they need to access the nucleus, through nuclear trafficking machinery. The karyopherin family of proteins is responsible for nuclear import and export. The contribution of nuclear trafficking during gametogenesis is not well understood. Here we demonstrated that the well conserved β-karyopherin Importin-9 (Ipo9) is essential in the germline for proper gametogenesis in female and male flies. We generated a molecular null allele of Ipo9 and showed that Ipo9 is required in females for chromosome segregation during meiosis and the accumulation of nuclear actin during egg chamber development. Additionally, Ipo9 is essential during spermatogenesis for spermatid elongation, proper elimination of histones during the transition from histone-based to protamine-based chromatin packaging, import of proteasome components and chromosome segregation during meiosis. Lastly, at the molecular level, we showed that the N-terminal domain, which is critical for nuclear import, is required for Ipo9 function during gametogenesis. Overall, this work advances our understanding of how nuclear trafficking regulates germ cell development.Item Chemical Intervention of Influenza Virus mRNA Nuclear Export(2020-07-15) Esparza, Matthew Aaron; Shay, Jerry W.; Fontoura, Beatriz; Grinnell, Frederick; Minna, John D.Influenza A viruses are human pathogens with limited therapeutic options, making it crucial to devise strategies for the identification of new classes of antiviral medications. The influenza A virus genome is constituted of 8 RNA segments. Two of these viral RNAs are transcribed into mRNAs that are alternatively spliced. The M1 mRNA encodes the M1 protein but is also alternatively spliced to yield the M2 mRNA during infection. M1 to M2 mRNA splicing occurs at nuclear speckles, and M1 and M2 mRNAs are exported to the cytoplasm for translation. M1 and M2 proteins are critical for viral trafficking, assembly, and budding. We show that influenza virus utilizes nuclear speckles to promote post-transcriptional splicing of its M1 mRNA. We assign previously unknown roles for the viral NS1 protein and cellular factors to an intranuclear trafficking pathway that targets the viral M1 mRNA to nuclear speckles, mediates splicing at these nuclear bodies, and exports the spliced M2 mRNA from the nucleus. In addition, gene knockout of the cellular protein NS1-BP, a constituent of the M mRNA speckle-export pathway, inhibits M mRNA nuclear export without significantly altering bulk cellular mRNA export, providing an avenue to preferentially target influenza virus. We performed a high-content, image-based chemical screen using single-molecule RNA-FISH to label viral M mRNAs followed by multistep quantitative approaches to assess cellular mRNA and cell toxicity. We identified inhibitors of viral mRNA biogenesis and nuclear export that exhibited no significant activity towards bulk cellular mRNA at non-cytotoxic concentrations. Among the hits is a small molecule that inhibits nuclear export of a subset of viral and cellular mRNAs via the mRNA export factor UAP56 without altering bulk cellular mRNA nuclear export. These findings underscore specific nuclear export requirements for viral mRNA nuclear export. This RNA export inhibitor also impaired replication of diverse influenza virus strains at non-toxic concentrations. Thus, this screening strategy yielded compounds that alone or in combination may serve as leads to new ways of treating influenza virus infection and are novel tools for studying viral RNA trafficking in the nucleus.Item Identification of Oncogenic KRAS-Associated Vulnerabilities in Non-Small Cell Lung Cancer(2016-05-26) Kim, Ji Mi; Lum, Lawrence; White, Michael A.; Fontoura, Beatriz; Cobb, Melanie H.Activating mutations in KRAS are frequently involved in the pathogenesis of non-small cell lung cancer (NSCLC), the disease responsible for the most cancer-related deaths in the US. Despite intensive efforts to develop drugs that directly interfere with KRAS activity over the past decade, no effective inhibitor has been developed. As an alternative, synthetic-lethal therapeutic opportunities are being pursued using large-scale, RNAi-based, functional genomics platforms. We first addressed two major challenges associated with RNAi-based primary synthetic-lethal screens; a prevalent miRNA-like behavior of siRNA and cell line-dependent phenotypic diversity within intra-lineage KRAS-driven cancer. In consideration of these, we performed a whole-genome synthetic-lethal siRNA screen, powered by 106 NSCLC lines and integrated with gene set enrichment analysis. This identified components of nuclear transport machinery as selectively essential for KRAS mutant NSCLC lines. We found that pharmacological inhibition of a key nuclear export receptor, XPO1 (a.k.a. CRM1), was sufficient to induce robust and selective apoptosis in KRAS mutant NSCLC cells in vitro and to cause significant impairment of KRAS mutant tumor growth in vivo. Mechanistically, XPO1-depedent nuclear export machinery was required to maintain NFκB-mediated survival signaling. We discovered that a subset of KRAS mutant NSCLC lines bypassed the addiction to XPO1-dependent nuclear export via YAP1 activation as a consequence of previously unappreciated co-occurring loss-of-function mutations in FSTL5 and mutations in Hippo pathway. The intrinsic resistance was reversed by coadministration of YAP1/TEAD inhibitor. Thus, our study suggests that XPO1 can be exploited for a promising therapeutic opportunity for KRAS mutant lung cancer and provides strategies for genomics-guided application of clinically available XPO1 inhibitors.Item Nuclear Export Receptor CRM1 Recognizes Nuclear Export Signals with Diverse Conformations(2017-04-13) Fung, Ho Yee Joyce; Nam, Yunsun; Chook, Yuh Min; Grishin, Nick V.; Yu, Hongtao; Zhang, XuewuThe Chromosome Region of Maintenance 1 or CRM1 protein facilitates export of hundreds of proteins and RNA molecules from eukaryotic cell nuclei. CRM1 recognizes its protein cargoes by their 8-15 residues-long nuclear export signals or NESs, which bind to a hydrophobic groove in CRM1. NESs are highly variable in sequence and structure. Their sequences are described by multiple sequence patterns of four variably-spaced hydrophobic residues, and three previous structures showed CRM1-bound NESs adopting either helix-strand or mostly extended conformations while the CRM1 groove remains unchanged. The plasticity of CRM1-NES interaction and the repertoire of NES conformations were unclear. Many NES sequences also seem incompatible with the asymmetric and seemingly structurally invariant NES-bound CRM1 groove. I developed a general strategy to crystallize CRM1 bound to NES peptides in order to study how diverse sequences bind CRM1. In the first study, I solved crystal structures of CRM1 bound to NESs with unusual sequences, which bound the CRM1 groove in the opposite orientation (minus) to that of previously studied NESs (plus). Comparison of minus and plus NESs identified structural and sequence determinants for NES orientation. The binding of NESs to CRM1 in both orientations results in a large expansion in NES consensus patterns and therefore a corresponding expansion of potential NESs in the proteome. In the second study, I solved eight additional structures of diverse NESs, which show peptide conformations ranging from mostly loop-like to all-helical NESs, occupying the CRM1 groove to different extents. Comparison of >13 structures show a total of 5-6 different NES conformations where the only conserved structural element is one turn of helix, which has dihedral angles that proceed from helical to β-strand. All NESs also participate in hydrogen bonds with the human CRM1 Lys568 side chain, which functions as a specificity filter that prevents binding of non-NES peptides. The large conformational range of NES backbones explains the lack of a fixed pattern for its 3-5 hydrophobic anchor residues, which in turn explains the large array of peptide sequences that can function as NESs. We now have comprehensive structural knowledge for NESs of most known patterns. The structural information obtained is now the foundation for a new peptide docking/modeling approach to improve the accuracy of NES prediction.Item Toxic PRn Poly-Dipeptides Encoded by the C9orf72 Repeat Expansion Block Nuclear Import and Export(2017-03-02) Shi, Kevin; Nijhawan, Deepak; McKnight, Steven L.; Rizo-Rey, José; Rosen, Michael K.Expansion of the (GGGGCC)n hexanucleotide repeat within the first intron of the C9orf72 gene is the mutation that leads to the most prevalent heritable form of amyotrophic lateral sclerosis (ALS). The expanded repeat is aberrantly transcribed from both sense and anti-sense strands relative to the C9orf72 gene, and both transcripts are translated in an ATG-independent manner to yield five distinct poly-dipeptides. Expression of either the glycine:arginine (GRn) or proline:arginine (PRn) poly-dipeptide in Drosophila leads to neurodegneration of the eye, and when introduced into culturing medium, synthetic forms of both peptides lead to the death of human cells. We recently identified PRn poly-dipeptide interacting partners in vivo using an unbiased proteomics approach, demonstrating that the nuclear pore complex (NPC) as a major binding target. The biological significance of the PRn peptide-NPC interaction became apparent when we observed major defects in both mature mRNA export to the cytoplasm and protein import into the nucleus after cells were treated with PRn. The functional nucleocytoplasmic transport defect caused by PRn peptide was due to binding of the peptide to the central channel of the NPC as visualized by super-resolution microscopy. The NPC central channel is comprised of phenylalanine-glycine (FG) domain nucleoporins, and these proteins are essential for the NPC's active transport and passive size-exclusion permeability barrier functions. Purified FG domains of Nup54 and Nup98, both identified in our proteomics study as PRn targets, polymerized into structurally labile, cross-β sheet fibers under physiological conditions. Several lines of evidence suggested that the polymerized state of FG domains is relevant to intact NPC. First, PRn peptides only bound to polymerized Nup54 and Nup98 and not to the soluble versions of either protein, potentially representing how PRn binds to the FG-rich central channel. Second, the aliphatic alcohol 1,6-hexanediol (HD) selectively disrupted the permeability barrier of the NPC, while 2,5-hexanediol had no effect. Similarly, 1,6-HD can effectively solubilize Nup FG polymers in vitro, while 2,5-HD had no effect on polymer stability. Finally, PRn binding to the NPC abrogated the disruption of the permeability barrier by 1,6-HD. Similarly, PRn also protected the Nup FG polymers from solubilization by 1,6-HD. Our study has elucidated a major mechanism by which the C9orf72 expansion associated PRn poly-dipeptide inhibits the transport of macromolecules in and out of the nucleus, leading to a major disruption of cellular physiology. Our results support a model in which the FG domains of the NPC exist in equilibrium between the polymerized and unpolymerized states. By binding to polymerized FG domains and stabilizing them, the PRn peptide is understood to shift the equilibrium toward the polymerized state, with consequent blockage of nuclear transport. The aliphatic alcohol 1,6-hexanediol has the opposite effect, shifting the equilibrium toward the unstructured state of FG domains and disrupting the permeability barrier. The effects of the PRn peptide and aliphatic alcohols on nuclear pore function represent extreme conditions. More subtle changes in the equilibrium between structured and unstructured FG domains may regulate nuclear pore function in living cells.