Free mRNA in excess upon polysome dissociation is a scaffold for protein multimerization to form stress granules

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.

An aberrant phase transition of stress granules triggered by misfolded protein and prevented by chaperone function

“…This might be caused by a shift from transient interactions to more stable interactions. It has been reported that free mRNA is required for SG formation and integrity, indicating the importance of RNA‐based interactions in SGs (Kedersha et al , ; Bounedjah et al , ). In agreement with this, we found that microinjection of RNase A into heat‐stressed cells expressing G3BP1‐mCherry leads to a dramatic reduction of SG size within seconds (data not shown).…”

Chemical Tools to Investigate Mechanisms Associated with HSP90 and HSP70 in Disease

“…It highlighted an unconventional role for HSP70 in the localization of a key regulatory protein at the leading edges of cancer cells and the important consequences that this holds for their ability to migrate (Boroughs et al, 2011). Others used VER155008 to investigate stress granule formation (Bounedjah et al, 2014), the pathogenicity and virulence of Entamoeba histolytica (Santos et al, 2015) to confirm a role for HSP70 in the infection cycle of baculoviruses. When VER-155008 was added early in infection, the synthesis of viral proteins, genome replication and the production of budded virions were markedly inhibited by the chemical tool indicating the dependence of virus reproduction on HSP70 (Lyupina et al, 2014).…”

RNA assemblages orchestrate complex cellular processes

“…Intriguingly, mRNAs in the hydrogels exhibited a preponderance of long 3 0 UTRs, which provide a platform for RBP interplay, and could mean that mRNAs in liquid droplets are likely to be regulated mRNA species. An emerging theme in mRNA studies is that extensive 3 0 UTRs act as scaffolds for post-transcriptional regulatory purposes, and that physiologic responses depend on competing and/or cooperating trans-acting factors [22][23][24]. P bodies and stress granules contain low-complexity RBPs P bodies and stress granules are cytoplasmic RNP bodies that exhibit droplet properties.…”