Abstract
ABSTRACTAlthough the regulation of stress granules has become an intensely studied topic, current investigations of stress granule assembly, disassembly and dynamics are mainly performed in cultured cells. Here, we report the establishment of a stress granule reporter to facilitate the real-time study of stress granules in vivo. Using CRISPR/Cas9, we fused a green fluorescence protein (GFP) to endogenous G3BP1 in zebrafish. The GFP–G3BP1 reporter faithfully and robustly responded to heat stress in zebrafish embryos and larvae. The induction of stress granules varied by brain regions under the same stress condition, with the midbrain cells showing the highest efficiency and dynamics. Furthermore, pre-conditioning using lower heat stress significantly limited stress granule formation during subsequent higher heat stress. More interestingly, stress granule formation was much more robust in zebrafish embryos than in larvae and coincided with significantly elevated levels of phosphorylated eIF2α and enhanced heat resilience. Therefore, these findings have generated new insights into stress response in zebrafish during early development and demonstrated that the GFP–G3BP1 knock-in zebrafish could be a valuable tool for the investigation of stress granule biology.This article has an associated First Person interview with the first author of the paper.
Highlights
Stress granules are cytoplasmic structures rich in mRNA and RNAbinding proteins
The expression of green fluorescence protein (GFP)-tagged G3BP1 was under the control of the endogenous g3bp1 gene promoter, meaning that stress granule formation would not be affected by G3BP1 overexpression
We performed whole-genome sequencing of the F1 10AA-GFP–G3BP1 knock-in fish to examine whether GFP was erroneously inserted in other genes, and we found that g3bp1 was the only gene tagged, indicating that the GFP reporter was unique to G3BP1
Summary
Stress granules are cytoplasmic structures rich in mRNA and RNAbinding proteins. They are usually formed when translation initiation is inhibited. As the name suggests, stress granules are induced upon various stress insults, such as heat shock, viral. The formation of stress granules is considered to be a protective cellular mechanism for resource conservation and survival under unfavorable conditions, and is characterized by the translation inhibition of most house-keeping genes and the preferential translation of prosurvival stress-responsive genes (Anderson and Kedersha, 2002; Kedersha et al, 2013; McCormick and Khaperskyy, 2017)
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