Isolation of yeast and mammalian stress granule cores

Abstract

Stress granules are dynamic, conserved RNA-protein (RNP) assemblies that form when translation is limiting; and are related to pathological aggregates in degenerative disease. Mammalian stress granules are comprised of two structures - an unstable shell and more stable cores. Herein we describe methodology for isolation of stress granule cores from both yeast and mammalian cells. The protocol consists of first enriching for stress granule cores using centrifugation and then further purifying stress granule cores using immunoprecipitation. The stress granule core isolation protocol provides a starting point for assisting future endeavors aimed at discovering conserved RNA regulatory mechanisms and potential links between RNP aggregation and degenerative disease.

Keywords: Liquid-liquid phase separation; Purification; RNP granule; Stress granule.

Fig. 1

Quantification of percentage of G3BP and PABPC1 in granules. (A) Example of the quantification of percent of G3BP in granules taken from multiple U-2 OS cells expressing G3BP-GFP. Yellow line represents cytoplasm boundary. Red line represents boundary of the nucleus. Green lines represent stress granule boundary. Fraction of total intensity of GFP (G3BP) in stress granules was determined by comparing total intensity of all granules in image to total intensity within cell boundaries using ImageJ. (B) SIM image of the same granule imaged for G3BP (top) am PABPC1. Cytoplasm and stress granule (SG) are labeled. Graph shown alongside shows normalized quantification of intensity (left to right) along with white line shown in the image. Intensity is normalized to background subtracted average intensity in the cytoplasm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Isolation of yeast stress granule cores. Scheme for preparation of stress granule core enriched fraction from crude cell lysate from yeast cells. Image shows stress granule core enriched fraction from cells carrying Pab1-GFP.

Fig. 3

Isolation of mammalian stress granule cores. Scheme for preparation of stress granule core enriched fraction from crude cell lysate from U-2 OS cells expressing G3BP-GFP. Image shows stress granule core enriched fraction from cells carrying G3BP-GFP.

Fig. 4

Expected results for mammalian stress granule core isolation. A) Representative images of Dynabeads following immunoprecipitation of G3BP-GFP using anti-GFP antibody. B) SYPRO Ruby staining following affinity purification of G3BP-GFP stress granule cores. Quantification is normalized to input and corrected for background using ImageJ. C) Mass spectrometry results of G3BP-GFP stress granule cores. Scale bars represent 2 μm.