CELLULAR ROLE AND STRUCTURE OF STRESS RECEPTORS FROM THE KINASE FAMILY: RNASE L AND GCN2

Abstract

Cells often experience external and internal stress. During evolutionary adaptation, all cells evolved dedicated stress response pathways which recognize stressful conditions and act to re- establish homeostasis and ensure survival. Understanding the molecular machinery of stress responses provides an important strategy for understanding human diseases, which always activate at least some stress responses. This thesis focuses on two phylogenetically and mechanistically related, yet biologically distinct, pathways of eukaryotic stress response. Specifically, I set out to determine 1) mechanisms of biogenesis of human endogenous double- stranded RNA (endo-dsRNA), which activates strong viral-like responses in uninfected human cells, and 2) the structural mechanism of the key Integrated Stress Response (ISR) kinase GCN2.The first part of this dissertation describes the identification of phosphorothioate DNAs (PS- DNAs) as triggers of endo-dsRNAs. PS-DNAs inhibit the decay of nuclear RNAs and induce endo- dsRNA via accumulation of high levels of intronic and intergenic inverted retroelements (IIIR). IIIRs do not turn on transcriptional RIG-I/MDA5/IFN signaling, but they trigger the dsRNA sensing pathways of OAS3/RNase L and PKR. Thus, nuclear RNA decay and nuclear-cytosolic RNA sorting protect the cell from activating an innate immune response. Our data suggest that the OAS3/RNase L and PKR arms of innate immunity diverge from antiviral IFN responses and monitor nuclear RNA decay by sensing cytosolic escape of IIIRs. The second part of this dissertation focuses on the ISR kinase, GCN2. GCN2 is unique within the protein kinome as it contains a histidyl tRNA synthetase-like (HRSL) domain. The HRSL domain presumably regulates GCN2 signaling; however, its precise function is unknown. Here, we report a 4.2-Å resolution single particle cryo-EM structure of the GCN2 HRSL domain. This structure reveals a symmetrical homodimer similar to the tRNA synthetase HisRS but bearing crossed alpha-helices that form a compact structure at the junction between the HRSL and kinase domains. We show that our structure represents the active state of GCN2 receptor. Based on our data, we propose that HRSL forces the GCN2 kinase domain into active dimers, while the remaining domains oppose the action of HRSL to prevent uncontrolled GCN2 activity and couple regulation to stress.