PROJECT SUMMARY Translational control plays a critical role in maintaining protein homeostasis under stress conditions as it allows immediate and selective changes in protein levels. A long-standing question in the field of translational control is the mechanism through which cellular mRNAs are able to undergo cap-independent translation. How cells orchestrate differential modes of mRNA translation upon stress remains poorly understood. The goal of this project is to investigate dynamic O-GlcNAcylation in response to stress and understand its role in cap- independent mRNA translation. O-GlcNAc has been proposed to regulate diverse cellular processes, including transcription and cell signaling pathways. Our preliminary results have indicated that O-GlcNAcylation switches the function of eIF4G1 from cap-dependent to cap-independent initiation. We further uncovered a mechanistic linkage between O-GlcNAcylation, ABCF1, and mRNA methylation in cap-independent mRNA translation. These findings led to the central hypothesis that stress-induced O-GlcNAc modification of eIF4G1 licenses cap-independent mRNA translation by remodeling pre-initiation complex formation, recognizing methylated mRNA, and facilitating cap-independent translation. To test this hypothesis, the following Aims are proposed: 1) Characterize the functional switch of eIF4G1 upon O-GlcNAcylation; 2) Define the role of ABCF1 in O- GlcNAc signaling; 3) Dissect the network between mRNA methylation and O-GlcNAc signaling. These Aims are independent of one another but unified in their central focus on O-GlcNAc signaling in translational control of stress response. By integrating innovative approaches into fundamental studies of translational regulation, the proposed studies will open up new avenues of research in the field of mRNA translation. The mechanistic insights we gain from this study will provide paradigms for better understanding of translational control in cellular homeostasis and stress adaptation.