Abstract
The sequence of events leading to stress granule assembly in stressed cells remains elusive. We show here, using isotope labeling and ion microprobe, that proportionally more RNA than proteins are present in stress granules than in surrounding cytoplasm. We further demonstrate that the delivery of single strand polynucleotides, mRNA and ssDNA, to the cytoplasm can trigger stress granule assembly. On the other hand, increasing the cytoplasmic level of mRNA-binding proteins like YB-1 can directly prevent the aggregation of mRNA by forming isolated mRNPs, as evidenced by atomic force microscopy. Interestingly, we also discovered that enucleated cells do form stress granules, demonstrating that the translocation to the cytoplasm of nuclear prion-like RNA-binding proteins like TIA-1 is dispensable for stress granule assembly. The results lead to an alternative view on stress granule formation based on the following sequence of events: after the massive dissociation of polysomes during stress, mRNA-stabilizing proteins like YB-1 are outnumbered by the burst of nonpolysomal mRNA. mRNA freed of ribosomes thus becomes accessible to mRNA-binding aggregation-prone proteins or misfolded proteins, which induces stress granule formation. Within the frame of this model, the shuttling of nuclear mRNA-stabilizing proteins to the cytoplasm could dissociate stress granules or prevent their assembly.
Highlights
In eukaryotic cells, the mechanism leading to the formation of mRNA-containing granules remains a matter of debate [1,2]
We checked that the mRNA cytoplasmic level was not significantly reduced during short term transcription inhibition using in situ hybridization (Supplementary Figure S9A)
The stability of polysomes in the cytoplasm is reinforced by the clustering of ribosomes along the mRNA strand leaving little room for aggregation
Summary
The mechanism leading to the formation of mRNA-containing granules remains a matter of debate [1,2]. A remarkable aspect of RNA-containing granules is the absence of encapsulating membranes which leaves RNA and associated RNA-binding proteins free to shuttle in and out of granules in a dynamical equilibrium rendering such aggregates, by nature, unstable [8] For this reason, the isolation of RNA-containing granules from cells and their further characterization in vitro remain an issue. The precipitated proteins appeared in turn to provide a platform which can recruit and retain selectively RNA with long 3 UTR [9,10] From these results, it was proposed that the aggregation of LC domain-enriched proteins is a critical event in RNAgranule formation.
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