Visualizing repetitive diffusion activity of double-strand RNA binding proteins by single molecule fluorescence assays

Stress granules (SGs) are cytosolic, nonmembranous RNA–protein complexes. In vitro experiments suggested that they are formed by liquid–liquid phase separation; however, their properties in mammalian cells remain unclear. We analyzed the distribution and dynamics of two paradigmatic RNA-binding proteins (RBPs), Ras GTPase-activating protein SH3-domain–binding protein (G3BP1) and insulin-like growth factor II mRNA-binding protein 1 (IMP1), with single-molecule resolution in living neuronal cells. Both RBPs exhibited different exchange kinetics between SGs. Within SGs, single-molecule localization microscopy revealed distributed hotspots of immobilized G3BP1 and IMP1 that reflect the presence of relatively immobile nanometer-sized nanocores. We demonstrate alternating binding in nanocores and anomalous diffusion in the liquid phase with similar characteristics for both RBPs. Reduction of low-complexity regions in G3BP1 resulted in less detectable mobile molecules in the liquid phase without change in binding in nanocores. The data provide direct support for liquid droplet behavior of SGs in living cells and reveal transient binding of RBPs in nanocores. Our study uncovers a surprising disconnect between SG partitioning and internal diffusion and interactions of RBPs.

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Single-molecule imaging reveals translation of mRNAs localized to stress granules

10.1101/2020.03.31.018093 ◽

2020 ◽

Author(s):

Daniel Mateju ◽

Bastian Eichenberger ◽

Jan Eglinger ◽

Gregory Roth ◽

Jeffrey A. Chao

Keyword(s):

Single Molecule ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Cellular Stress ◽

Mrna Translation ◽

Stress Granules ◽

Living Cells ◽

Single Molecule Imaging ◽

Direct Role

SUMMARYCellular stress leads to reprogramming of mRNA translation and formation of stress granules (SGs), membraneless organelles consisting of mRNA and RNA-binding proteins. Although the function of SGs remains largely unknown, it is widely assumed they contain exclusively nontranslating mRNA. Here we re-examine this hypothesis using single-molecule imaging of mRNA translation in living cells. While our data confirms that non-translating mRNAs are preferentially recruited to SGs, we find unequivocal evidence for translation of mRNA localized to SGs. Our data indicate that SG-associated translation is not rare and that the entire translation cycle (initiation, elongation and termination) can occur for these transcripts. Furthermore, translating mRNAs can be observed transitioning between the cytosol and SGs without changing their translational status. Together, these results argue against a direct role for SGs in inhibition of mRNA translation.

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Cohesin SA1 and SA2 are RNA binding proteins that localize to RNA containing regions on DNA

Nucleic Acids Research ◽

10.1093/nar/gkaa284 ◽

2020 ◽

Vol 48 (10) ◽

pp. 5639-5655 ◽

Cited By ~ 1

Author(s):

Hai Pan ◽

Miao Jin ◽

Ashwin Ghadiyaram ◽

Parminder Kaur ◽

Henry E Miller ◽

...

Keyword(s):

Single Molecule ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Strand Break ◽

Data Sets ◽

Cohesin Complex ◽

Replication Fork Progression ◽

Chromatin Immunoprecipitation Sequencing ◽

Rna Immunoprecipitation

Abstract Cohesin SA1 (STAG1) and SA2 (STAG2) are key components of the cohesin complex. Previous studies have highlighted the unique contributions by SA1 and SA2 to 3D chromatin organization, DNA replication fork progression, and DNA double-strand break (DSB) repair. Recently, we discovered that cohesin SA1 and SA2 are DNA binding proteins. Given the recently discovered link between SA2 and RNA-mediated biological pathways, we investigated whether or not SA1 and SA2 directly bind to RNA using a combination of bulk biochemical assays and single-molecule techniques, including atomic force microscopy (AFM) and the DNA tightrope assay. We discovered that both SA1 and SA2 bind to various RNA containing substrates, including ssRNA, dsRNA, RNA:DNA hybrids, and R-loops. Importantly, both SA1 and SA2 localize to regions on dsDNA that contain RNA. We directly compared the SA1/SA2 binding and R-loops sites extracted from Chromatin Immunoprecipitation sequencing (ChIP-seq) and DNA-RNA Immunoprecipitation sequencing (DRIP-Seq) data sets, respectively. This analysis revealed that SA1 and SA2 binding sites overlap significantly with R-loops. The majority of R-loop-localized SA1 and SA2 are also sites where other subunits of the cohesin complex bind. These results provide a new direction for future investigation of the diverse biological functions of SA1 and SA2.

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