DNA is abundant in yeast and mammalian stress granule cores.

(A) Abundance of histones (brown) and other representative DNA-binding proteins (gray) in the proteomes of yeast stress granule cores (strain JD1370 grown in synthetic defined (SD) media), normalized to that of the stress granule marker eIF4A [peptide spectrum matches (PSMs); means from n = 2 biological replicates ± s.d.; n.s., no significance; one-tailed Student’s t-test]. (B) Abundance of histones and other representative DNA-binding proteins in the proteomes of HEK293T cell stress granule cores, normalized to that of the stress granule marker G3BP1 (PSMs; means from n = 3 biological replicates ± s.d.; *, p < 0.05; one-tailed Student’s t-test. Stress granule markers, histones and non-histone DNA-binding proteins in green, yellow, and gray, respectively. (C) Negative-stain immuno-electron micrograph of stress granule cores from yeast strain YAG1021 carrying a chromosomally encoded FLAG-tagged histone H3. Anti-FLAG antibodies were conjugated to NanoGold beads (arrows; Methods). Magnification (× 23,000). (D) Native agarose gel electrophoresis (SYBR Gold stain) of yeast stress granule cores treated with DNase I. BP, bromophenol blue. (E) DNase I, psDNase and RNase H treatment (the first at two different concentrations) of total nucleic acids extracted from yeast stress granule cores analyzed as in (D). Bottom graph, integrated signal for the dashed box. (F) Comparison of nuclease susceptibility of total nucleic acids extracted from yeast and mammalian stress granule cores, analyzed as in (D). (G) Alkaline hydrolysis of total nucleic acids extracted from yeast stress granule cores (15, 10 and 5 ng) and rRNA (15 ng), analyzed by non-denaturing agarose gel electrophoresis (SYTOX Green stain). Samples marked (+) were incubated with 50 mM KOH for 15 minutes at 95 °C. Bottom graph, integrated signal for the dashed box.

Characteristics of eccDNAs from yeast and mammalian stress granule cores.

(A) Abundance and length distribution of high-confidence eccDNAs from yeast stress granule cores (unless noted, the data are from strain JD1370 cultured in SD medium). (B) Genomic coverage, per chromosome, of eccDNAs from yeast stress granule cores. The length of each chromosome is shown for comparison (overall correlation coefficient between relative eccDNA abundance and chromosome length is 0.95). (C) Abundance and length distribution of eccDNA from HEK293T cell stress granule cores. (D) Genomic coverage, per chromosome of eccDNA from HEK293T cell stress granule cores. The length of each chromosome is shown for comparison (overall correlation coefficient between relative eccDNA abundance and chromosome length is 0.95). (E) Relative abundance of gene elements within assemblies of eccDNAs from yeast and human stress granule cores. TSS, transcription start site; TTS, transcription termination site. (F) Repetitive elements within assemblies of eccDNAs of yeast stress granule cores. SINEs and LINEs, short and long interspersed nuclear elements, respectively. (G) Repetitive elements within eccDNAs from human stress granule cores.

Cytoplasmic eccDNAs colocalize with stress granules in human cells.

(A) Partitioning of AHNAK DNA signal between cytoplasm (c), perinuclear (pn) and nuclear (n) volumes of a HEK293T cell. Nuclear envelope is labeled with Lamins A and C. (B-C) Localization of MYC and AHNAK DNA signals in relation to the stress granule marker G3BP and its activator protein Caprin1. (D-E) Concurrent detection of DNA and RNA targets within AHNAK and MYC genes in relation to the stress granule marker G3BP. As in (A-C), denaturing conditions at 75 °C were used to prioritize capturing DNA targets over mRNA targets (red arrows). (F-G) Detection of DNA by conventional anti-DNA IF combined with HCR-FISH against AHNAK and MAPT DNA targets. DNA signals (framed and numbered) proximal to translocase TOMM20 points to relationship with mitochondria. Cytoplasmic DNA without proximal TOMM20 label is marked by red arrows. (H) Colocalization of histone H4 with stress granules turnover protein VCP. Stress granules are demarcated by T30 signal (framed), which represents stress-induced condensation of polyadenylated nucleic acids. (I) Colocalization of histone H3 with the granule marker G3BP and control AHNAK mRNA. Colocalized signals for H3, G3BP, and mRNA are framed and numbered. Arrows point to some H3 and G3BP signals without AHNAK mRNA foci. In all panels, HEK293T cells underwent oxidative stress with 0.5 mM sodium arsenite for 1 hour and 20 minutes at 37 °C, and then were fixed with formaldehyde. Unless otherwise stated, detection of proteins and nucleic acids was carried out by HCR IF and HCR-FISH, respectively. All images represent one confocal Z-stack of 0.2 mm with nuclei stained by Hoechst 33342; proteins and DNA are pseudocolored for clarity. Scale bars 1 mm in (A) and (F-I), 2 mm in (B-E), and 0.5 mm in inserts of (B-C).

Cytoplasmic eccDNA is required for stress granule formation in yeast.

(A-C) Confocal microscopy of S. cerevisiae with endogenous PAB1-GFP (green) and transiently expressed (GAL promoter) cytoplasmic CRISPR machinery (Cas9NES) with gRNA (Ty1). CRISPR transformant variants are indicated together with treatment conditions. (D) Analysis of total DNA isolated from stress granule cores from wild-type (no transformation) and CRISPR-treated (variants indicated) cells after exposure to oxidative stress with 0.5% w/v sodium azide for 45 minutes (means from n = 2 biological replicates ± s.d.). (E) Depletion of 20-nt Ty1 (Ty12HDV) targets by enzymatically active cytoplasmic Cas9NES (3rd generation; Methods). Mann-Whitney U-test. (F) Effect of cytosolic CHD1NES on abundance of circular double stranded DNA from stress granules isolated from cells co-expressing active CRISPR machinery with gRNA as in (B) (means from n = 2 biological replicates ± s.d.). (G-H) Confocal microscopy of untreated and oxidatively stressed S. cerevisiae with an endogenous PAB1-GFP fusion (green) and transient (GAL promoter) co-expression of cytoplasmic active CRISPR machinery with cytosolic yeast CHD1NES or GCN5NES. In (D) and (F), one-tailed t-tests are used; the ordinates are the mean depth of coverage over sixteen chromosomes (mitochondrial genome is omitted); n.s., no significance; *, p < 0.05. All images represent one middle confocal Z-stack of 0.14 mm with nuclei stained by DAPI (blue). Scale bar 2 mm.

CRISPR-mediated suppression of stress granules compromises recovery from hypoxic stress.

(A) Recovery of CRISPR transformants after exposure to oxidative stress (45 minutes at 30 °C) applied in the early log phase (Methods). Spotting assay was carried out under moderate Cas9NES induction on SD(-URA) solid medium with D(+)-glucose (1.5% w/v) and D(+)-galactose (0.75% w/v) using indicated ten-fold serial dilutions. Stress-induced phenotypes of the CRISPR variants with controls are presented in Figures 4 and S5. (B) Quantitation of post-stress recovery efficiency as in (A) for indicated CRISPR transformants. Spotting assays were carried out on solid medium at 30 °C for 2.5 days as in (A) and quantified using the second (10-2) dilution. Data are mean for n = 3 ± s.d. and 44 technical replicates. Data were normalized to values of gRNAs alone; n.s., no significance; *, p < 0.05; **, p < 0.01; ****, p < 0.0001 (one-way ANOVA followed by Tukey’s test with a 95% confidence interval). (C) Representative time-course for post-stress recovery of CRISPR transformants in the SD(-URA) liquid medium as in (A) (n = 2 ± s.d.; technical replicates). The growth was initiated with the second (10-2) dilution.