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. 2018 Mar 13;115(11):2734-2739.
doi: 10.1073/pnas.1800038115. Epub 2018 Feb 26.

RNA self-assembly contributes to stress granule formation and defining the stress granule transcriptome

Briana Van Treeck  1 , David S W Protter  1 , Tyler Matheny  1 , Anthony Khong  1   2 , Christopher D Link  3 , Roy Parker  4   2
Affiliations

RNA self-assembly contributes to stress granule formation and defining the stress granule transcriptome

Briana Van Treeck et al. Proc Natl Acad Sci U S A. .

Abstract

Stress granules are higher order assemblies of nontranslating mRNAs and proteins that form when translation initiation is inhibited. Stress granules are thought to form by protein-protein interactions of RNA-binding proteins. We demonstrate RNA homopolymers or purified cellular RNA forms assemblies in vitro analogous to stress granules. Remarkably, under conditions representative of an intracellular stress response, the mRNAs enriched in assemblies from total yeast RNA largely recapitulate the stress granule transcriptome. We suggest stress granules are formed by a summation of protein-protein and RNA-RNA interactions, with RNA self-assembly likely to contribute to other RNP assemblies wherever there is a high local concentration of RNA. RNA assembly in vitro is also increased by GR and PR dipeptide repeats, which are known to increase stress granule formation in cells. Since GR and PR dipeptides are involved in neurodegenerative diseases, this suggests that perturbations increasing RNA-RNA assembly in cells could lead to disease.

Keywords: RNA self-assembly; RNP granules; dipeptides; stress granules.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
RNA–RNA base-pairing influences RNA localization. All ncRNAs (excluding antisense) (A) and antisense ncRNAs (B) with lengths <3,000 nt and significant sequencing reads were plotted in a histogram showing their log2(fold change) enrichment in stress granule/total RNA. A red bracket highlights the second peak for RNAs enriched in stress granules. (C) Pie chart illustrating the proportion of all mammalian mRNAs enriched (red), depleted (blue), or neither (gray) in stress granules. Stress granule localization of binding partners to enriched (D) and depleted (E) antisense ncRNAs is shown. *One enriched antisense ncRNA transcript has a secondary binding partner to a depleted mRNA. (F) Examples of enriched antisense ncRNAs and their binding partners. SG, stress granule.
Fig. 2.
Fig. 2.
Various RNAs self-assemble in vitro. (A) Phase diagram of RNA assembly morphology under varying PEG and NaCl concentrations. Images correspond to labeled positions in the phase diagram: Droplets formed at 0 mM NaCl, 10% PEG (a); droplet/tangles formed at 300 mM NaCl, 7.5% PEG (b); tangles formed at 750 mM NaCl, 10% PEG (c); and no assemblies at 300 mM NaCl, 2.5% PEG (d). All conditions contain 1 mM MgCl2 and 150 μg/mL yeast total RNA. (B) Total yeast RNA in 150 mM NaCl and 1 mM MgCl2 with and without physiologically relevant conditions of spermine (223 μM) and spermidine (1,339 μM). (C) polyU, polyC, polyA, and polyG self-partition in vitro at 500 μg/mL (respective) homopolymers, 10% PEG, and 750 mM NaCl.
Fig. 3.
Fig. 3.
RNAs in self-assemblies in vitro largely recapitulate the stress granule transcriptome. (A) RNAs from in vitro assemblies formed at 150 mM NaCl identified as significantly (P < 0.01) and twofold enriched (1,488 RNAs, red dots) and depleted (1,456 RNAs, blue dots) compared to total yeast RNA. The box plots show the correlation between transcript length and RNA localization to in vitro assemblies (B) and yeast stress granules (C) (28). ***P < 0.001 between any three box plots. (D) In vitro-enriched mRNAs significantly overlap with mRNAs identified in the yeast stress granule transcriptome and exhibit significant lack of overlap with RNAs depleted from stress granules. n.s., not significant. (E) In vitro-depleted RNAs significantly overlap with RNAs depleted from stress granules and exhibit a significant lack of overlap with RNAs identified in the yeast stress granule transcriptome. (F) Degree of enrichment and depletion between in vitro assemblies and stress granules correlates (Pearson’s correlation, R = 0.53). RNAs that are more enriched in vitro than in vivo (Upper Left) tend to have greater proportions of optimal codons (orange).
Fig. 4.
Fig. 4.
Four-phase model of stress granule assembly. Model illustrating how assemblies may be RNA-dominated (Bottom Right) or protein-dominated (Top Left); however, a combination of interactions is often responsible for assembly within cells (Top Right). Arrows denote examples from the literature that lead to stress granule formation or dissolution. The yellow arrow shows the formation of stress granules through a large influx of nontranslating RNAs. The red arrow signifies a lack of stress granules when key proteins are deleted. The black arrow denotes the formation of stress granules through overexpression of certain RNA-binding proteins.
Fig. 5.
Fig. 5.
Pathogenic dipeptides increase RNA assembly. (A) Dipeptides (GR)10 and (PR)10 promote assembly, while (GP)10 does not. Fluorescent dipeptides (green) and RNA (SYTO 17, red) are both enriched in assemblies. (B) Phase diagram illustrating the assembly of (GR)10 and RNA. The squares signify lack of assembly. +, sparse and small assembly; ++, moderate assemblies (constituting either frequent but smaller assemblies or larger assemblies that were more sparse); +++, robust assembly. Green indicates protein-only assembly.
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