Saturday, November 23, 2024
11/23/2024
Parent Award - Abstract Accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the Unfolded Protein Response (UPR) which aims at restoring a healthy cellular proteome. Dysregulation of the UPR is key to diseases, e.g. neurodegeneration. For this central role, the UPR forms a network of processes, involving complex transcription, translation, and RNA and protein degradation changes. For example, while the UPR shuts down global translation, it activates specific response genes, such as the transcription factor ATF4. Our goal is to investigate this response network. We have investigated this network from several angles: profiling the dynamics of the mammalian UPR, we identified regulatory signatures for hundreds of genes. We discovered a translation regulatory element in ATF4 whose role in translation induction of the gene under stress had been overlooked. The element consists of a start and stop codon and stalls ribosomes. We discovered start-stops in hundreds of genes enriched for signaling molecules. In addition, we profiled protein modifications, e.g. ubiquitination, in response to stress. Further, we began to compare the UPR in two closely related motor neurons with differential stress-sensitivity that is consistent with their role in Amyotrophic Lateral Sclerosis (ALS): stress-sensitive spinal motor neurons die early during ALS, while more stress-resistant cranial motor neurons survive until late stages of the disease. We identified molecular signatures, e.g. in the proteasome, that can explain the motor neurons’ differential stress sensitivity. Doing so, we created tools and resources for integrative analysis. In the next five years, we will address three major questions that arise from these findings: i) How does the cell, in general, induce stress response genes while general translation is halted? ii) What are the roles of protein modifications in the UPR? and iii) How do translation, protein modifications, and other pathways form an efficient and robust response network that restores proteome health upon stress? Specifically, we will investigate the role of start-stops and other elements in translation regulation of DROSHA and RAD23B, which function in the miRNA pathway and DNA damage repair, respectively, but also link to the ER stress response. Using a gain-of-function construct, we will deconvolute the mechanism of start-stop function and identify regulators of ribosome stalling that affect transcript localization, stability, and downstream re-initiation. We will complement these analyses with large-scale assessment of ribosome scanning and initiation, changes in transcript stability and in proteins bound to mRNAs in response to stress. A second research avenue will investigate protein ufmylation, a ubiquitin-like protein modification linked to the UPR and ER maintenance, but also to translation and the DNA damage response. We previously found that Ufl1, a key ufmylation gene, expresses different isoforms in cranial and spinal motor neurons, and our proposed work will investigate the impact of differential Ufl1 expression on the UPR in the two motor neurons. Complementing these analyses, we will attempt to identify novel and neuron type specific ufmylation targets and investigate their links to translation regulation, ribosome quality control, and DNA damage repair. The work will exploit our expertise in systems-scale and targeted analysis to understand new properties of the proteostasis network.
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