Analysis of SG components using biotin-isoxazole fractionation

Text and figures adapted from (Panas et al., 2015a)

Although the biochemical isolation of SGs has proven difficult, a technique was developed which allows the cell-free formation of SG-associated proteins and RNA precipitates. Isoxazole is a small molecule known to induce the differentiation of stem cells (Sadek et al., 2008), and a biotinylated version (B-isox) was recently shown to precipitate a subset of RNA-binding proteins and RNAs in a temperature-dependent manner from cell lysates (Han et al., 2012; Kato et al., 2012). These precipitated proteins were highly enriched in SG-associated proteins and protein components of other types of RNA granules (Kato et al., 2012). Most B-isox precipitating proteins contain low-complexity (LC), unstructured regions of unknown function, and these LC regions are necessary and sufficient for their precipitation by B-isox. These studies led the McKnight laboratory (Kato et al., 2012) to propose that RNA granule assembly is mediated by a concentration and temperature dependent liquid/liquid phase transition. Although the mechanism of B-isox precipitation is not well understood, using B-isox precipitation to selectively precipitate SG-competent proteins can serve as a surrogate method for crude purification of SG components, essentially mimicking stress in vitro. The assay involves the simple addition of the B-isox to cell lysates, incubation with agitation at 4°C, followed by centrifugation to separate the precipitate and supernatant fractions and analysis by SDS-PAGE.

Reagents:

B-isox: 6-(5-(Thiophen-2-yl) isoxazole-3-carboxamido)hexyl 5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate,  (Sigma catalog number T51161-1MG). Vial contains 1 mg; dilute contents in 192 μL DMSO to obtain a 10 mM (100X) stock solution. Store at -20°C.

Protocol:

Lyse cells in EE buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 0.1% NP40, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, 1 μM DTT or 20 mM β-mercaptoethanol) supplemented with protease, phosphatase, and RNase inhibitors (optional). Extract with tumbling at 4°C for 20 min, then centrifuge for 15 min at maximum speed in a refrigerated eppendorf microcentrifuge. Remove supernatant and save a small fraction of for SDS-PAGE analysis as “input”.

Divide the remaining cleared lysate in half.  Add 1/100 dilution of 10 mM b-isox in DMSO to final concentration of 100 μM. Add 1/100 dilution of DMSO to the mock control. Optional: add RNase A at 20 μg/mL and tumble lysate in cold for 1h prior to addition of B-isox.

Tumble in cold room 90 minutes

Spin 10000 g for 10 min at 4°C.

Harvest the supernatant from both B-isox and mock-treated samples, and add equal volume of 2X reducing SDS-PAGE loading buffer. These samples will be used to compare the percent of individual proteins removed by B-isox precipitation.

Wash pellet twice by suspending in ice-cold EE buffer, vortexing, and incubation for 10 min on ice, followed by centrifugation at 10000 g for 10 min at 4°C.

Resuspend  B-isox and mock pellets in reducing SDS-PAGE loading buffer and analyze supernatant and pellet fractions by SDS-PAGE and immunoblotting.

Quantification of the band intensities can be performed using Image J.

Optional: Stain gel to see bands. Note that silver stain will detect both proteins and nucleic acid, whereas Coomassie or Ponceau will detect only proteins.

We used this technique to determine the B-isox solubility of G3BP1 and its binding partners in cells expressing EGFP-fusion proteins carrying the G3BP-binding motifs from SFV nsP3, described in (Panas et al., 2012). U2OS cell lines stably expressing low and high levels of EGFP-31-wt (clones 1 and 8, respectively) or a non-G3BP-binding version (EGFP-31-F3A) were lysed and subjected to B-isox precipitation in the presence or absence of RNase and analyzed using SDS-PAGE. Gels containing lysates, supernatants and B-isox precipitates were silver stained to confirm equal protein loading and to demonstrate that precipitation only occurred in the presence of B-isox (Fig A, B). Replicate samples were resolved on SDS-PAGE and transferred to nitrocellulose for immunoblotting (Fig 3C). The general profile of proteins precipitating with B-isox was similar to expected (Kato et al., 2012), and did not change appreciably with expression of either EGFP-31-wt or –F3A. RNase pretreatment of the lysates removed the high molecular mass bands (identified as RNA by their failure to stain with protein stains, data not shown), and resulted in the precipitation of a number of smaller bands identified as ribosomal proteins (data not shown). When pelleted material was analyzed by immunoblotting for SG proteins, we observed that CAPRIN-1, eIF3b and TIAR all precipitated equally well from cells expressing low or high levels of EGFP-31-wt or EGFP-31-F3A (Fig C). However, G3BP-1 and -2 precipitation was inhibited in cells expressing high levels of EGFP-31-wt (clone 8), indicating that the EGFP-31-wt/G3BP interaction alters it such that it is no longer “SG competent” by this criterion. This result is in agreement with our previous work showing that overexpression of EGFP-31-wt but not EGFP-31-F3A blocks the formation of SA-induced SGs (Panas et al., 2015b).

References

Han, T.W., M. Kato, S. Xie, L.C. Wu, H. Mirzaei, J. Pei, M. Chen, Y. Xie, J. Allen, G. Xiao, and S.L. McKnight. 2012. Cell-free formation of RNA granules: bound RNAs identify features and components of cellular assemblies. Cell. 149:768-779.

Kato, M., T.W. Han, S. Xie, K. Shi, X. Du, L.C. Wu, H. Mirzaei, E.J. Goldsmith, J. Longgood, J. Pei, N.V. Grishin, D.E. Frantz, J.W. Schneider, S. Chen, L. Li, M.R. Sawaya, D. Eisenberg, R. Tycko, and S.L. McKnight. 2012. Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels. Cell. 149:753-767.

Panas, M.D., N. Kedersha, and G.M. McInerney. 2015a. Methods for the characterization of stress granules in virus infected cells. Methods. 90:57-64.

Panas, M.D., T. Schulte, B. Thaa, T. Sandalova, N. Kedersha, A. Achour, and G.M. McInerney. 2015b. Viral and Cellular Proteins Containing FGDF Motifs Bind G3BP to Block Stress Granule Formation. PLoS Pathog. 11:e1004659.

Panas, M.D., M. Varjak, A. Lulla, K.E. Eng, A. Merits, G.B. Karlsson Hedestam, and G.M. McInerney. 2012. Sequestration of G3BP coupled with efficient translation inhibits stress granules in Semliki Forest virus infection. Mol Biol Cell. 23:4701-4712.

Sadek, H., B. Hannack, E. Choe, J. Wang, S. Latif, M.G. Garry, D.J. Garry, J. Longgood, D.E. Frantz, E.N. Olson, J. Hsieh, and J.W. Schneider. 2008. Cardiogenic small molecules that enhance myocardial repair by stem cells. Proc Natl Acad Sci U S A. 105:6063-6068.