Investigating the role of the RNA helicase F57B9.3 in translational regulation under conditions of lipid depletion - The relationship between protein homeostasis and nutrient sensing is critical for cellular function and survival. It is well established that amino acid and glucose depletion results in global inhibition of protein translation. Yet, lipids are the most energy-rich macromolecule, and it is still unclear if and how lipid depletion affects translation rates. Utilizing genetic models of lipid depletion in C. elegans via RNA interference, my preliminary studies reveal that translational regulation upon lipid depletion is seemingly different than that of amino acid or glucose deprivation. Specifically, I found that lipid depletion increases translation rates and coincides with a ~35-fold transcriptional induction of a eukaryotic initiation factor, F57B9.3 (homolog of the RNA helicase, eIF4A2). To gain mechanistic insight into the role of F57B9.3 and its potential link with lipid homeostasis, this proposal outlines two main objectives: 1) determine the transcription factor necessary for induction of F57B9.3 upon lipid depletion and 2) understand how F57B9.3 activation impacts translation upon lipid depletion. My preliminary studies identified ELT-2 as the transcriptional regulator of F57B9.3. Thus, the experiments proposed in Aim 1 will use a combination of CRISPR/Cas-9 gene editing strategies to determine the genetic elements required for F57B9.3 transcriptional induction upon lipid depletion by removing the GATA motif within the F57B9.3 promoter. Additionally, I will employ ChIP-qPCR to investigate ELT-2 binding to F57B9.3 GATA motif. In Aim 2, I will determine if F57B9.3 is required for lipid-depletion induced translation through various protein pulse labeling techniques and biochemical assays such as polysome profiling. Additionally, I will define the mRNAs bound to F57B9.3 by performing CLIP-sequencing. The successful completion of these aims will broaden our current understanding of translational regulation under lipid depletion and provide a molecular mechanism underlying lipid depletion induced changes in translation. Furthermore, understanding the mechanism by which lipid homeostasis is restored through protein translation can provide insight into a myriad of metabolic disorders and characterizing a novel role for the eIF4A2 homolog in response to lipid imbalances has strong therapeutic potential.
