The role of G3BP granules in mRNA translation regulation and cell adaptation to exogenous stress

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Project Summary/Abstract G3BP stress granules (SGs) are a component of the eukaryotic stress response. They are membrane-less organelles that form as a consequence of eIF2⍺ phosphorylation and global translation inhibition. The role of G3BP granules during cellular stress is not completely understood. They are composed of untranslated mRNAs and factors from the translational machinery leading to the model that G3BP SGs inhibit translation through the sequestration of macromolecules from the bulk cytoplasm. Furthermore, the biological function of G3BP granules may be regulated by their liquid-like properties. This feature may allow G3BP SGs to interact dynamically with the bulk cytoplasm and to reversibly dissociate when cells recover from stress. Moreover, it has been proposed that SG transitioning to solid-like structure during prolonged stress is detrimental to survival. However, detailed characterization of G3BP granules liquid stability and their role in cell survival during stress response is still lacking. I hypothesize that G3BP granules protect mammalian cells against stress by regulating translation of mRNAs and retaining a stable liquid-like phase in the bulk cytoplasm. To test the direct role of G3BP granules in translation inhibition, in my aim 1, I will characterize the protein and RNA composition of G3BP granules under exogenous stress in human cells through APEX2-proximity labeling coupled to RNA sequencing and Mass Spectrometry. Furthermore, to decouple the effects that stress induction may have on SG composition, I will characterize the protein/RNA molecules associated to SGs induced with optogenetics. Then, I will perform the Transcript Isoforms in Polysomes sequencing (TrIP-seq) technique, which characterizes the abundance of mRNAs associated to polysomes, to define the relationship between SG recruitment of mRNAs and their translation. To test the biological function of G3BP SG biophysical properties to cell survival, in my aim 2, I will study the liquid stability and reversibility of G3BP granule formation under acute and chronic stress with a microfluidics-based fluorescence microscopy approach currently developed in the Floor and Wittmann laboratories at UCSF. Then, I will evaluate transitions in the material properties of G3BP granules by performing fluorescence recovery after photobleaching (FRAP) experiments. Finally, I will determine the viability of cells through a propidium iodide-based assay to elucidate the role of liquid stability and kinetics of granule formation to survival from stress. In summary, this project will provide insights into the physiological role of G3BP granules to survival during the stress response and recovery and the biological function of their liquid stability in promoting cellular adaptability under exogenous stress.