UCSF

The Integrated Stress Response (ISR) is a highly conserved cellular signaling network that regulates homeostasis by phosphorylating eukaryotic initiation factor 2 (eIF2), thereby reducing Ternary Complex (TC) availability and broadly arresting translation. Paradoxically, this reduction in TC upregulates essential stress-response genes like Activating Transcription Factor 4 (ATF4) through a delayed reinitiation mechanism governed by upstream open reading frames (uORFs). While the basic model of ATF4 translation is established, the exact molecular mechanisms facilitating post-termination small ribosomal subunit (40S) behavior remain obscure. Through systematic CRISPRi screening and the deployment of a rapid endogenous degron system in K562 cells, this study identifies a previously uncharacterized, non-canonical role for the h subunit of eIF3 in ATF4 translation. Systematic mutation of the ATF4 5’ UTR combined with fluorescent reporter assays demonstrate that eIF3h does not function as a general reinitiation or scanning factor. Instead, it is specifically required to enable the post-termination, TC-empty 40S ribosome to bypass the inhibitory uORF2 start codon. Complementary single-molecule FRET assays confirm that, while eIF3h plays no role in canonical translation, the TC-empty 40S exhibits profound vulnerabilities, including an inability to scan and a significantly destabilized half-life on the mRNA. Furthermore, by varying the interORF distance and measuring fluorescence accumulation, this work develops a first-principles mathematical model based on constant scanning speed and first-order kinetics to calculate relative intracellular TC concentrations. This novel, non-invasive proxy for real-time TC availability provides a critical new tool for interrogating ISR activation. Ultimately, this dissertation challenges current paradigms of start codon recognition by introducing the existence of a TC-independent uORF detection mechanism, offering broad implications for the study of translational reprogramming in health and neurodegenerative disease.