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Using Crispr To Generate Integrated Ssa4-Gfp Reporter Strains, Emma Norman, Rebecca Adams

Science University Research Symposium (SURS)

Using CRISPR to Generate Integrated Ssa4-GFP Reporter Strains

Emma Norman, Rebecca Adams, PhD

Proteins play critical roles in numerous cellular processes. In order to synthesize these important molecules in eukaryotes, DNA is first transcribed into an intermediate molecule, mRNA, in the nucleus. The export of mRNA from its origin in the nucleus to the site of protein production, the cytoplasm, is an integral step in protein synthesis. When a cell is subjected to stress, such as heat shock, most mRNA export is halted, as export proteins are inactivated, and cellular machinery is redirected towards recovery. However, particular transcripts, including the …

Uncovering Genes Involved In Non-Npc-Associated Dbp5 Function, Jonah Hooks, Rebecca Adams

Belmont University Research Symposium (BURS)

During an mRNA’s lifecycle, RNA-binding proteins (RBP) are added and removed in order to facilitate export from the nucleus to the cytoplasm through the nuclear pore complex (NPC). During the RNA export process, a class of proteins termed Dead-box proteins (Dbps) are strategically located at the cytoplasmic face of the NPC to remove mRNPs from the mRNA-protein complex, providing one way directionality for export in the process termed mRNA-protein (mRNP) remodeling. Specifically, Mex67 is an RBP which ferries the transcript through the nuclear pore complex and is removed by Dbp5, which is located at the cytoplasmic …

Analysis Of Ssa4 Reporter Expression By Q-Pcr, Susveen Sharanshi, Rebecca Adams

Belmont University Research Symposium (BURS)

The synthesis of genome-encoded proteins via mRNA translation is integral to cell survival. In eukaryotes, such as S. cerevisiae, the mRNA that is produced in the nucleus must be exported to the cytoplasm for translation to occur, and this process is highly regulated. Specifically, the export of mRNA occurs via travel through nuclear pore complexes (NPCs), which are selective doorways embedded in the nuclear envelope. During cellular stress, such as heat shock, the cell needs to regulate gene expression to permit survival, and mRNA export is one step at which this occurs. At these high temperatures, a cell’s proteins …

Neuropilin-2 Regulates Androgen-Receptor Transcriptional Activity In Advanced Prostate Cancer, Samikshan Dutta, Navatha Shree Polavaram, Ridwan Islam, Sreyashi Bhattacharya, Sanika Bodas, Thomas Mayr, Sohini Roy, Sophie Alvarez, Marieta I. Toma, Anza Darehshouri, Angelika Borkowetz, Stefanie Conrad, Susanne Fuessel, Manfred Wirth, Gustavo B. Baretton, Paramita Ghosh, Kenneth J. Pienta, David L. Klinkebiel, Surinder K. Batra, Michael H. Muders, Kaustubh Datta

Department of Agronomy and Horticulture: Faculty Publications

Aberrant transcriptional activity of androgen receptor (AR) is one of the dominant mechanisms for developing of castration-resistant prostate cancer (CRPC). Analyzing AR-transcriptional complex related to CRPC is therefore important towards understanding the mechanism of therapy-resistance. While studying its mechanism, we observed that a transmembrane protein called neuropilin-2 (NRP2) plays a contributory role in forming a novel AR-transcriptional complex containing nuclear pore proteins. Using immunogold electron microscopy, high-resolution confocal microscopy, chromatin immunoprecipitation, proteomics, and other biochemical techniques, we delineated the molecular mechanism of how a specific splice variant of NRP2 becomes sumoylated upon ligand stimulation and translocates to the inner nuclear …

Exploring The Functionality Of Putative Bop3 Post-Translational Modifications, Liliya Tkachuk

Honors Scholars Collaborative Projects

All eukaryotic cells require that transcribed mRNAs undergo export form the nucleus to the cytoplasm where they can be translated into proteins. This process requires a host of proteins which are conserved between the unicellular budding yeast, S. cerevisiae, and humans. During this process, Mex67 and other associated proteins facilitate the mRNA to travel across the nuclear pore complex (NPC), doorways embedded in the nuclear envelope. Upon the exit of mRNA, Mex67 is released and recycled back into the nucleus to facilitate the export of more mRNA. This occurs through the action of Dbp5, whose activity is regulated through …

Exploring The Functionality Of Putative Bop3 Post-Translational Modifications, Liliya Tkachuk, Rebecca Adams Phd

Belmont University Research Symposium (BURS)

All eukaryotic cells require that transcribed mRNAs undergo export form the nucleus to the cytoplasm where they can be translated into proteins. This process requires a host of proteins which are conserved between the unicellular budding yeast, S. cerevisiae, and humans. During this process, Mex67 and other associated proteins facilitate the mRNA to travel across the nuclear pore complex (NPC), doorways embedded in the nuclear envelope. Upon the exit of mRNA, Mex67 is released and recycled back into the nucleus to provide the export of more mRNA. This release occurs through the action of Dbp5, whose activity is regulated …

Interaction Of Influenza A Virus Ns2/Nep Protein With The Amino-Terminal Part Of Nup214, Burçak Şenbaş Akyazi, Ayşegül Pi̇ri̇nçal, Atsushi Kawaguchi, Kyosuke Nagata, Kadi̇r Turan

Turkish Journal of Biology

Influenza A viruses have a single-stranded RNA genome consisting of 8 segments. Each RNA segment associates with the nucleoprotein (NP) and viral RNA polymerase to and from a viral ribonucleoprotein (vRNP) particle. The viral mRNA synthesis is dependent on a capped primer derived from nascent host RNA transcripts. For these processes to take place, vRNPs must pass through the cell nuclear pore complex (NPC) to the nucleus. The influenza A virus NS2 protein, also called the nuclear export protein (NES), has an important role in the nucleocytoplasmic transport of vRNPs. This protein interacts with the host cellular nucleoporins during the …

Biochemical And Structural Analysis Of The Nucleoporin Nup214 And Its Involvement In Mrna Export, Johanna Napetschnig

Student Theses and Dissertations

In order to gain a deeper understanding of the role of nups in leukemogenesis, and to make sense of the architecture and regulation of the mRNA export machinery at the NPC, I set out to biochemically and structurally characterize Nup214. In this thesis, I present the crystal structure of the human Nup214 N-terminal domain at 1.65 Ã… resolution. The structure reveals a sevenbladed !-propeller fold followed by a 30-residue C-terminal extended peptide segment (CTE). The CTE folds back onto the !-propeller and binds to its bottom face. Conserved surface patches on the Nup214 NTD reveal putative proteininteraction sites, one of …

Identification Of A Nuclear Export Signal In The Catalytic Subunit Of Amp-Activated Protein Kinase, N. Kazgan, Tyisha Williams, L. J. Forsberg, J. E. Brenman

Biology Faculty Research

The metabolic regulator AMP-activated protein kinase (AMPK) maintains cellular homeostasis through regulation of proteins involved in energy-producing and -consuming pathways. Although AMPK phosphorylation targets include cytoplasmic and nuclear proteins, the precise mechanisms that regulate AMPK localization, and thus its access to these substrates, are unclear. We identify highly conserved carboxy-terminal hydrophobic amino acids that function as a leptomycin B–sensitive, CRM1-dependent nuclear export sequence (NES) in the AMPK catalytic subunit (AMPKα). When this sequence is modified AMPKα shows increased nuclear localization via a Ran-dependent import pathway. Cytoplasmic localization can be restored by substituting well-defined snurportin-1 or protein kinase A inhibitor (PKIA) …

Biophysical Characterization Of Structure And Dynamics Of Nuclear Pore Complex Components, Martin Kampmann

Student Theses and Dissertations

The Nuclear Pore Complex (NPC) mediates nucleo-cytoplasmic transport in all eukaryotes and is among the largest cellular assemblies of proteins, called nucleoporins (nups). The details of NPC architecture, dynamics, and mechanism are still unknown. NPCs can be dissected biochemically into distinct subcomplexes. One of the best-characterized subcomplexes, the Nup84 complex, consists of seven nups and was proposed to form a membrane-coating module of the NPC. I optimized the isolation of the heptameric complex from budding yeast and analyzed its structure by negative-stain electron microscopy (EM). My data confirm the previously reported flexible Y-shape. I solved the three-dimensional structures of two …

Architecture Of A Coat For The Nuclear Pore Membrane, Kuo-Chiang Hsia

Student Theses and Dissertations

Nuclear pore complexes (NPCs) reside in the nuclear membrane and mediate macromolecules exchange between the nucleus and cytoplasm. Although the protein components of NPCs, termed collectively nucleoporin, have been identified, how nucleoporins arrange in NPC, however, is still an enigma. NPC is a highly symmetric protein complex, which contains an eight-fold rotational symmetry across the center of the pore and a two-fold symmetry in the plane of the nuclear envelope. In addition, according to electron microscopic reconstruction model, NPC can also be considered schematically as a series of concentric cylinders. A peripheral cylinder coating the nuclear pore membrane contains a …

Proteomic, Bioinformatic And Functional Characterization Of The Nuclear Pore Complex Of The African Trypanosome, Jeffrey Allen Degrasse

Student Theses and Dissertations

The eukaryotic genome, and its associated proteins, is intricately packaged and sequestered within the boundary of a double membrane, known as the nuclear envelope (NE). Transport across the NE is mediated by large protein assemblages known as nuclear pore complexes (NPCs). Yeast and vertebrate NPCs are comprised of about 30 proteins, termed nucleoporins (Nups), which are present in multiple copies. The origins and evolution of the nucleus and NPC are not yet clear, although it seems likely that the nucleus arose only once in eukaryotic evolution. To further our understanding of the evolution of the NPC, we characterized the NPC …

Myosin-Like Proteins In S. Cerevisiae: Multifunctional, Structural Components Of The Nuclear Envelope, Mario Niepel

Student Theses and Dissertations

The nuclear envelope (NE) separates the genetic material from the rest of the cell, delimits and defines the nucleus, organizes the intranuclear architecture and serves as a regulator for multiple nuclear processes. In all eukaryotes, filamentous coiled-coil proteins are associated with the intranuclear surface of the NE and are integral to proper nuclear function. One such protein, called Tpr in vertebrates, attaches to the NPC and appears to form the nuclear basket structure, is conserved throughout all eukaryotes. The two yeast homologs of Tpr are termed Mlp1p and Mlp2p. The Mlp proteins also attach to the nuclear face of the …