Transcriptome-Wide Comparison of Stress Granules and P-Bodies Reveals that Translation Plays a Major Role in RNA Partitioning

Abstract

The eukaryotic cytosol contains multiple RNP granules, including P-bodies and stress granules. Three different methods have been used to describe the transcriptome of stress granules or P-bodies, but how these methods compare and how RNA partitioning occurs between P-bodies and stress granules have not been addressed. Here, we compare the analysis of the stress granule transcriptome based on differential centrifugation with and without subsequent stress granule immunopurification. We find that while differential centrifugation alone gives a first approximation of the stress granule transcriptome, this methodology contains nonspecific transcripts that play a confounding role in the interpretation of results. We also immunopurify and compare the RNAs in stress granules and P-bodies under arsenite stress and compare those results to those for the P-body transcriptome described under nonstress conditions. We find that the P-body transcriptome is dominated by poorly translated mRNAs under nonstress conditions, but during arsenite stress, when translation is globally repressed, the P-body transcriptome is very similar to the stress granule transcriptome. This suggests that translation is a dominant factor in targeting mRNAs into both P-bodies and stress granules, and during stress, when most mRNAs are untranslated, the composition of P-bodies reflects this broader translation repression.

Keywords: P-body; RNA; stress granule; transcriptome.

FIG 1

Unstressed RNA granule pellet transcriptomes are reproducible. (A) Three-dimensional (3D) scatterplot depicting the normalized read counts from RNA-seq libraries from unstressed RNA granule pellet triplicates. (B) 3D scatterplot depicting the normalized read counts from RNA-seq libraries from unstressed total RNA triplicates. (C) Table depicting pairwise Pearson correlation coefficients between RNA granule pellets and total RNA in unstressed cells. (D) Scatterplot of unstressed RG enrichment obtained from mouse fibroblasts versus unstressed RG enrichment in U-2 OS cells.

FIG 2

Characterization of the unstressed RNA granule pellet. (A) MA plot depicting the log₂ fold change values (unstressed RG pellet/unstressed total RNA) versus abundance (fragments per kilobase per million [FPKM]). Genes are color-coded by their significance. Significant genes (P < 0.01) are colored red, while nonsignificant (P > 0.01) genes are colored blue. (B) Gene ontology analysis for enriched and depleted transcripts. (C) Zoom image of scatterplot highlighting the position of mitochondrial transcripts. (D) Box plot depicting transcript length for RG-enriched and RG-depleted transcripts in both stressed and unstressed cells. (E) Box plot depicting translation efficiency values (18) for RG-enriched and RG-depleted transcripts in unstressed cells.

FIG 3

Stressed RNA granule pellet transcriptomes are reproducible. (A) 3D scatterplot depicting the normalized read counts from RNA-seq libraries from stressed RNA granule pellet triplicates. (B) 3D scatterplot depicting the normalized read counts from RNA-seq libraries from stressed total RNA triplicates. (C) Table depicting pairwise Pearson correlation coefficients between RNA granule pellets and total RNA in stressed cells. (D) MA plot depicting the log₂ fold change values (stressed RG pellet/stressed total RNA) versus abundance (FPKM). Genes are color-coded by their significance. Significant genes (P < 0.01) are colored red, while nonsignificant (P > 0.01) genes are colored blue. (E) Scatterplot of stressed RG enrichment obtained from mouse fibroblasts versus stressed RG enrichment in U-2 OS cells. (F) Gene ontology for enriched and depleted transcripts.

FIG 4

Characterization of the stressed RNA granule pellet. (A) Scatterplot depicting the correlation between stressed pellet enrichment versus unstressed pellet enrichment. (B) Box plots showing transcript lengths for pellet-enriched and pellet-depleted transcripts during arsenite stress and unstressed conditions. (C) Same as panel B but for translation efficiency (TE) values (obtained from Subtelny et al. [18]). (D) Same as panel B but color-coded by transcript length. (E) Same as panel B but color-coded by TE values.

FIG 5

Polysomes likely account for the differences in the stressed RG transcriptome and the stress granule transcriptome. (A) MA plot depicting the log₂ fold change values (stress granule RNA/stressed total RNA) versus abundance (FPKM). Genes are color-coded by their significance. Significant genes are colored red (P < 0.01), while nonsignificant genes are colored blue (P > 0.01). (B) Scatterplot of unstressed pellet enrichment versus stress granule enrichment. (C) Scatterplot of stressed pellet enrichment versus stress granule enrichment. (D) Same as panel C but color coded by pellet enrichment under unstressed conditions. (E) R2 values from multiple linear regression analysis for stressed pellet enrichment versus SG enrichment or stressed pellet enrichment versus SG enrichment and unstressed pellet enrichment. (F) Same as panel C but color-coded by percent GC content. (G) Same as panel C but color-coded by the fraction of optimal codons per transcript. (H) TapeStation analysis of the RG pellet during stress and of SG RNA using additional immunopurification steps.

FIG 6

Comparison of the SG and P-body transcriptome. (A) Scatterplot depicting the enrichment of RNAs in the unstressed RG pellet versus P-bodies. (B) Scatterplot depicting the enrichment of RNAs in the stressed RG pellet versus P-bodies. (C) Scatterplot depicting the enrichment of RNAs in P-bodies versus stress granules. (D) Same as panel C but color-coded by GC content.

FIG 7

Comparison of the stressed and unstressed P-body transcriptome. (A) Table depicting pairwise r² values between PB RNA and total RNA in arsenite-stressed cells. (B) Same as panel A but for G3BP1/G3BP2 knockout (ko) cells. (C) TapeStation analysis for IgG pulldown control. (D) TapeStation analysis for EDC3 pulldown of P-body RNA. (E) Scatterplot depicting the correlation between wild-type P-body enrichment and G3BP1Δ/G3BP2Δ P-body enrichment during stress. (F) P-body enrichment during unstressed conditions versus P-body enrichment during arsenite stress. (G) Stress granule enrichment versus P-body enrichment during arsenite stress.

FIG 8

Interplay between translation and length define the P-body transcriptome. (A) Scatterplot showing the correlation between the fraction of optimal codons and P-body enrichment during unstressed conditions. (B) Same as panel A but during arsenite stress. (C) Box plot showing fraction of optimal codons for enriched and depleted transcripts in stressed and unstressed P-bodies. (D) Scatterplot showing the correlation between length and P-body enrichment during unstressed conditions. (E) Scatterplot showing the correlation between length and P-body enrichment during stressed conditions. (F) Box plot showing transcript length for enriched and depleted transcripts in stressed and unstressed P-bodies.

FIG 9

smFISH validation of the P-body transcriptome. (A) smFISH of SPEN and IF of RCK, a P-body marker, during unstressed conditions. (B) Same as panel A but during arsenite stress. (C) smFISH of AHNAK and IF of RCK, a P-body marker, during unstressed conditions. (D) Same as panel C but during arsenite stress. (E) smFISH of DYNC1H1and IF of RCK, a P-body marker, during unstressed conditions. (F) Same as panel E but during arsenite stress. (G) smFISH of GAPDH and IF of RCK, a P-body marker, during unstressed conditions. (H) Same as panel G but during arsenite stress. (I) Quantification of percentage of transcripts associated with PBs by smFISH for SPEN, AHNAK, DYNC1H1, and GAPDH during no stress and arsenite stress. (J) Scatterplot depicting the correlation between P-body enrichment assessed by smFISH versus P-body enrichment assessed by RNA-seq.

FIG 10

Translation determines the RNA composition of P-bodies. Model depicting the critical role of translation in defining the RNA composition of PBs. (Left) During unstressed conditions, only transcripts with poor translation metrics are available for P-body sequestration. (Right) During conditions of stress, in which translation is limited, the RNA composition of PBs shifts to include more RNAs and more closely resemble the SG transcriptome.