Cytoplasmic circular dsDNA is a key constituent of stress granules

1. Problem Statement

Stress granules (SGs) are cytoplasmic, membraneless organelles formed in eukaryotic cells in response to environmental stress (e.g., oxidative stress, heat shock). They are critical for cell survival and post-stress recovery. While the composition of SGs is known to include proteins and RNA, the specific macromolecular interactions driving their assembly remain an active area of research.

• The Gap: Conventional wisdom holds that SGs are composed primarily of RNA and proteins. Previous scattered observations hinted at DNA presence, but a systematic biochemical characterization of the nucleic acid content of purified SG cores was lacking.
• The Question: What is the true nucleic acid composition of stress granule cores, and does DNA play a functional role in their assembly or the cellular stress response?

2. Methodology

The authors employed a multi-disciplinary approach combining biochemistry, genomics, cell biology, and CRISPR-based functional assays in both Saccharomyces cerevisiae (yeast) and HEK293T (human) cells.

• Biochemical Purification & Characterization:

  • Purified stress granule cores from yeast and HEK293T cells.
  • Proteomics: Mass spectrometry to identify DNA-binding proteins (e.g., histones, RPA, PARP1) within the cores.
  • Nuclease Sensitivity Assays: Treated purified cores and extracted nucleic acids with Proteinase K, RNases (A, T1, H), and DNases (DNase I, Plasmid-Safe DNase) to determine nucleic acid stability and structure.
  • Alkaline Hydrolysis: Used harsh alkaline conditions to degrade RNA while preserving DNA, quantifying the relative abundance of DNA vs. RNA.

• Genomic Analysis (Circle-Seq):

  • Adapted the Circle-Seq methodology to enrich for circular DNA.
  • Used hybrid sequencing (Illumina short-reads for accuracy + Oxford Nanopore long-reads for resolving complex/repetitive structures) to assemble extrachromosomal circular DNA (eccDNA) from SG cores.
  • Mapped sequences to reference genomes to determine genomic coverage and repeat content.

• Cellular Imaging (FISH & Immunofluorescence):

  • HCR-FISH (Hybridization Chain Reaction): Used to detect specific DNA sequences (e.g., AHNAK, MYC, MAPT) and RNA in fixed cells.
  • Colocalization: Visualized the overlap between cytoplasmic DNA signals and canonical SG markers (G3BP1, Caprin1, PAB1) and mitochondrial markers (TOMM20) to confirm cytoplasmic localization distinct from mitochondria.
  • Histone Staining: Confirmed the presence of histones (H3, H4) in SGs via immunofluorescence and electron microscopy.

• Functional CRISPR Assays (Yeast):

  • Cytoplasmic Targeting: Engineered a Cas9 variant fused to a Nuclear Export Signal (Cas9NES) to retain the nuclease in the cytoplasm.
  • Targeting: Designed gRNAs against abundant Ty1 retrotransposon sequences found in SG eccDNA.
  • Controls: Tested catalytically active Cas9NES, catalytically dead dCas9NES, and co-expression of chromatin-interacting proteins (CHD1, GCN5) to distinguish between DNA cleavage and DNA binding effects.
  • Phenotypic Assays: Monitored SG formation (via PAB1-GFP) and cell recovery from hypoxic/oxidative stress (sodium azide treatment).

3. Key Contributions & Results

A. Discovery of Abundant Circular dsDNA in SGs

• Composition: Biochemical analysis revealed that >50% of the nucleic acid content in purified SG cores is DNA.
• Structure: The DNA is predominantly double-stranded and circular (eccDNA).
  • Evidence: SG cores are resistant to RNase A/T1 but highly sensitive to DNase I. They are resistant to Plasmid-Safe DNase (which degrades linear dsDNA but spares circular dsDNA), confirming the circular nature.
  • Histones (H2B, H4 in humans; H3 in yeast) are abundant in SG cores, suggesting a chromatin-like organization.

B. Genomic Characterization of SG-eccDNA

• Diversity: SG-eccDNAs are polydisperse (ranging from kilobases to megabases) and cover all chromosomes.
• Sequence Content:
  • Yeast: Enriched in promoter regions, exons, and Ty1 retrotransposons (LTRs).
  • Human: Enriched in intergenic/intronic regions and SINEs (non-LTR retrotransposons).
• Conclusion: The eccDNA complement mirrors the nuclear genome but is distinct from mitochondrial DNA (confirmed by lack of colocalization with TOMM20).

C. Cytoplasmic Localization and Colocalization

• HCR-FISH confirmed that specific eccDNA sequences (e.g., AHNAK, MYC) are present in the cytoplasm of stressed human cells.
• These DNA foci colocalize with SG markers (G3BP1, Caprin1) and histones.
• Some SGs contain both DNA and RNA signals, suggesting the formation of DNA:RNA heteroduplexes (supported by RNase H sensitivity).

D. Functional Requirement of eccDNA for SG Assembly

• CRISPR Inhibition: Expressing cytoplasmic Cas9NES-gRNA targeting Ty1 sequences in yeast completely abolished stress granule formation upon oxidative stress.
• Mechanism of Inhibition:
  • Surprisingly, the catalytically dead dCas9NES-gRNA also blocked SG formation.
  • This indicates that the binding of the CRISPR complex to the DNA (sequestering it or blocking diffusion), rather than DNA cleavage, is sufficient to disrupt SG assembly.
• Rescue by Chromatin Factors: Co-expression of chromatin-interacting proteins (CHD1 or GCN5) restored SG formation in the presence of the CRISPR machinery. This suggests that eccDNAs in SGs exist in a chromatin-like state, and the CRISPR machinery disrupts the necessary protein-DNA interactions required for nucleation.

E. Physiological Consequence

• Yeast strains unable to form SGs due to cytoplasmic CRISPR targeting of eccDNA showed severely impaired recovery from oxidative/hypoxic stress compared to wild-type or control strains.
• This confirms that eccDNA is not just a passive component but is functionally required for the cytoprotective role of stress granules.

4. Significance

• Paradigm Shift: This study overturns the dogma that stress granules are exclusively RNA-protein condensates, identifying circular dsDNA as a major, functional structural component.
• Novel Mechanism of Assembly: It proposes that eccDNA acts as a "seed" or nucleation point for SG assembly, potentially through DNA:RNA heteroduplexes and chromatin-like packaging.
• Chromatin in the Cytoplasm: The findings suggest a conserved mechanism where nuclear chromatin components (histones, DNA circles) are exported to the cytoplasm to mediate stress responses, challenging the strict compartmentalization of chromatin biology.
• Disease Relevance: Given the link between SG dysregulation and neurodegeneration/cancer, and the known role of eccDNA in oncogene amplification and inflammation, this discovery opens new avenues for understanding disease mechanisms and potential therapeutic targets.
• Technical Innovation: The development of cytoplasmic CRISPR targeting (Cas9NES) as a tool to probe cytoplasmic DNA function provides a new methodological framework for future research.

In summary, Demeshkina and Ferré-D'Amaré demonstrate that cytoplasmic circular dsDNA (eccDNA), organized in a chromatin-like state, is a fundamental and required component for the formation and function of stress granules, linking the nuclear genome directly to the cytoplasmic stress response machinery.