Stress granules (SGs) are macromolecular assemblies that form under cellular stress. Formation of these condensates is driven by the condensation of RNA and RNA-binding proteins such as G3BPs. G3BPs condense into SGs following stress-induced translational arrest. Three G3BP paralogs (G3BP1, G3BP2A, and G3BP2B) have been identified in vertebrates. However, the contribution of different G3BP paralogs to stress granule formation and stress-induced gene expression changes is incompletely understood. Here, we identified key residues for G3BP condensation such as V11. This conserved amino acid is required for formation of the G3BP-Caprin-1 complex, hence promoting SG assembly. Total RNA sequencing and ribosome profiling revealed that disruption of G3BP condensation corresponds to changes in mRNA levels and ribosome engagement during the integrated stress response (ISR). Moreover, we found that G3BP2B preferentially condenses and promotes changes in mRNA expression under endoplasmic reticulum (ER) stress. Together, this work suggests that stress granule assembly promotes changes in gene expression under cellular stress, which is differentially regulated by G3BP paralogs.

When influenza A releases its genomic segments into the cell, these segments travel to the nucleus where they are subsequently transcribed. As newly translated viral polymerase and nucleoprotein reach high levels, the virus switches from transcription to replication. Disruption of this regulation could endanger viral output hampering infection of the host. In our attempt to generate a genome mimetic based fluorescent flu reporter, we discovered a viral self loading mechanism in which the virus may be preventing compromised infection. We observed that naked RNA is incorporated well in a minigenome assay, but actively excluded during viral infection. This exclusion could be serving as a viral policing mechanism in which the virus actively excludes host naked RNA such as tRNA, rRNA, and snRNA. As a result, the virus would be utilizing most of its proteins allowing for maximum viral output. Through a series of experiments pre-expressing a variety of viral proteins before viral infection, polymerase components cotransfection, or nucleoprotein transfection, we discovered that NEP is an active factor that prevents viral inclusion of naked RNA. However, further analysis is required to determine the exact mechanism by which NEP mitigates this naked RNA exclusion, and whether other viral active factors are causing naked RNA exclusion.

ABSTRACT OF THE DISSERTATION

Neutral and Anionic Carboranes as Electronically and Sterically Diverse Phosphine Substituents

by

Christopher Alexander Lugo

Doctor of Philosophy, Graduate Program in Chemistry

University of California, Riverside, June 2017

Dr. Vincent Lavallo, Chairperson

The chemistry of carboranes has expanded greatly since their discovery in the 1960’s. Displaying 3D aromaticity, superior thermal and chemical stability, and facile tunability, these molecules have seen a wide range of applications but little has been said for their potential as transition metal catalysts. We sought to remedy this rift by 1) constructing a library of ligands that integrate carboranes into phosphines as R-group substituents, 2) ligating the ligands to an array of transition metal centers, and 3) surveying the reactivity of the isolated metal complexes.

By substituting the inherently bulky ortho-carborane with a terphenyl moiety at the C-2 position, and a phosphine at the C-1 position, a massive ligand was created. The donor and steric properties were analyzed via a rhodium carbonyl complex. A nido version of the above terphenyl carboranyl phosphine was also synthesized and its behavior with iridium was investigated. Through preparation of phosphines of the anionic 12 and 10-vertex carboranes, rhodium carbonyl complexes were synthesized to quantitatively assess their donor properties. Through utilization of a carboranylphosphino iron complex, the ability to cyclically trimerize methylacrylate has been demonstrated through the isolation a phosphonium enolate species. Amongst the vast library of monoanionic carboranyl phosphines there are none containing a dianionic phosphine. Using PCl3 as a launch pad, a variety of monoanionic and dianionic ligands were systematically prepared with ranging electronic and steric profiles.