The RNA composition of G3BP1-induced lysate granules resembles the RNA composition of stress granules in cells. (A) Granules induced by increasing G3BP1 concentration by 20 µM incorporated labeled oligo-dT RNA (top). The addition of RNase (100 µg/ml) prevented the formation of granules (bottom). Images were obtained 30 min following induction with purified G3BP1. Scale bars, 10 µm. (B) U2OS cells stably expressing G3BP1-GFP were treated with 200 µg/ml cycloheximide (CHX) for 3 h before or immediately following lysis and the induction of lysate granules by increasing the concentration of G3BP1 by 5 µM using purified G3BP1. Images were obtained 30 min following induction with G3BP1. Scale bars, 5 µm. (C) G3BP1 lysate granule Z-scores (granule vs. lysate, x axis) were plotted against stress granule Z-scores (granule vs. lysate, y axis) to compare similarity in differentially expressed genes between lysate and stress granules (Khong et al., 2017) and were found to be highly similar. Pearson’s correlation analysis (r = 0.662). (D) RNA composition of whole cell lysates (left), stress granules (middle), and lysate granules (right) analyzed by RNA-seq. Both stress granules and G3BP1-induced lysate granules primarily contain mRNA compared with the lysate from which they were derived. The RNA composition of G3BP1-induced lysate granules is highly similar to the published stress granule transcriptome (Khong et al., 2017). (E) Individual mRNAs identified in both the stress granule transcriptome (Khong et al., 2017) and G3BP1-induced lysate granules were color-coded from the most prevalent to the least prevalent within the granule. Log(TPM) was used for color coding such that mRNAs with log(TPM) ≤ 0.5 were assigned the lightest shade of green, whereas mRNAs with log(TPM) ≥ 2.50 were assigned the darkest shade of green. (F) mRNAs that were identified in both the stress granule transcriptome (Khong et al., 2017) and G3BP1-induced lysate granules were rank ordered from the most depleted (blue) to the most enriched (red) within the granule compared with their respective lysates. (G) Average mRNA transcript length is positively correlated with the level of granule enrichment or depletion in both stress granules and lysate granules. Graph represents mean ± SEM. n = 6,148, 5,604 (lysate granule, stress granule) for RNAs enriched >1.5-fold; n = 4,414, 2,701 (lysate granule, stress granule) for RNAs enriched 1–1.5-fold; n = 1,004, 2,047 (lysate granule, stress granule) for RNAs depleted 1–1.5-fold; and n = 889, 2,099 (lysate granule, stress granule) for RNAs depleted >1.5-fold.
Figure 3.

The RNA composition of G3BP1-induced lysate granules resembles the RNA composition of stress granules in cells. (A) Granules induced by increasing G3BP1 concentration by 20 µM incorporated labeled oligo-dT RNA (top). The addition of RNase (100 µg/ml) prevented the formation of granules (bottom). Images were obtained 30 min following induction with purified G3BP1. Scale bars, 10 µm. (B) U2OS cells stably expressing G3BP1-GFP were treated with 200 µg/ml cycloheximide (CHX) for 3 h before or immediately following lysis and the induction of lysate granules by increasing the concentration of G3BP1 by 5 µM using purified G3BP1. Images were obtained 30 min following induction with G3BP1. Scale bars, 5 µm. (C) G3BP1 lysate granule Z-scores (granule vs. lysate, x axis) were plotted against stress granule Z-scores (granule vs. lysate, y axis) to compare similarity in differentially expressed genes between lysate and stress granules (Khong et al., 2017) and were found to be highly similar. Pearson’s correlation analysis (r = 0.662). (D) RNA composition of whole cell lysates (left), stress granules (middle), and lysate granules (right) analyzed by RNA-seq. Both stress granules and G3BP1-induced lysate granules primarily contain mRNA compared with the lysate from which they were derived. The RNA composition of G3BP1-induced lysate granules is highly similar to the published stress granule transcriptome (Khong et al., 2017). (E) Individual mRNAs identified in both the stress granule transcriptome (Khong et al., 2017) and G3BP1-induced lysate granules were color-coded from the most prevalent to the least prevalent within the granule. Log(TPM) was used for color coding such that mRNAs with log(TPM) ≤ 0.5 were assigned the lightest shade of green, whereas mRNAs with log(TPM) ≥ 2.50 were assigned the darkest shade of green. (F) mRNAs that were identified in both the stress granule transcriptome (Khong et al., 2017) and G3BP1-induced lysate granules were rank ordered from the most depleted (blue) to the most enriched (red) within the granule compared with their respective lysates. (G) Average mRNA transcript length is positively correlated with the level of granule enrichment or depletion in both stress granules and lysate granules. Graph represents mean ± SEM. n = 6,148, 5,604 (lysate granule, stress granule) for RNAs enriched >1.5-fold; n = 4,414, 2,701 (lysate granule, stress granule) for RNAs enriched 1–1.5-fold; n = 1,004, 2,047 (lysate granule, stress granule) for RNAs depleted 1–1.5-fold; and n = 889, 2,099 (lysate granule, stress granule) for RNAs depleted >1.5-fold.

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