Regulation of protein synthesis during cellular stress

Project Summary/Abstract Cells experience and rapidly respond to environmental stressors by modulating which mRNAs are selected for protein synthesis. A central factor in this process is the essential heterotrimeric translation initiation factor eIF2. eIF2 is a G-protein complex that, in its active GTP-bound form, delivers initiator methionyl tRNA (Met-tRNAiMet) to the ribosome. During cellular stress, eIF2 is phosphorylated on its α subunit (eIF2α-P) in a pathway known as the Integrated Stress Response (ISR). eIF2α-P inhibits the exchange of eIF2-GDP for eIF2-GTP, causing a decrease in active eIF2 levels and general protein synthesis. In addition, specific stress-responsive factors, including the bZIP transcription factors ATF4 and CHOP, are translationally induced when active eIF2 levels are low. The ATF4 and CHOP mechanisms of translation control involve upstream open reading frames (uORFs) in their mRNA 5’-leaders. Importantly, the majority of mammalian transcripts contain uORFs, so the presence of an uORF is not sufficient for selection of an mRNA for translation during the ISR. Rather, we and others have determined that unique combinations of uORF features dictate how a given mRNA will be translationally regulated when active eIF2 levels are low. Additionally, while emphasis has been placed on understanding the regulatory schemes that drive GTP and Met-tRNAiMet binding to eIF2, we lack a complete picture of eIF2 biogenesis itself. This is critical to note as the efficiency of eIF2 heterotrimer formation also dictates the overall level of active eIF2. Furthermore, impaired eIF2 heterotrimer formation, modulation of active eIF2 levels, and dysregulation of the ISR are implicated in multiple stress- related diseases, including neuropathologies, diabetes, and various cancers. Our broad objective is to understand how cells regulate protein synthesis to maintain homeostasis. We hypothesize that a decrease in active eIF2 levels, whether driven by impaired eIF2 complex formation or eIF2α-P, underlies gene expression programs that are specific to the ISR and determine cell fate. We are using innovative and holistic approaches that feature -omics methodologies coupled with biochemistry, molecular biology, and structure-function studies to address three key questions. 1) How is formation of the eIF2 complex coordinated? 2) What is an ISR translational signature, and what are the mechanisms by which eIF2 selects or disregards mRNAs for translation? 3) How does eIF2-mediated translational control modulate the proteome and cell signaling to determine cell fate? Completion of the proposed studies will provide fundamental insight into the regulation of eIF2 function and how cells modulate protein synthesis to maintain homeostasis, laying the foundation for advancements in the diagnosis and treatment of stress-related diseases.