Free mRNA in excess upon polysome dissociation is a scaffold for protein multimerization to form stress granules.
Bottom Line: We further demonstrate that the delivery of single strand polynucleotides, mRNA and ssDNA, to the cytoplasm can trigger stress granule assembly.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.Within the frame of this model, the shuttling of nuclear mRNA-stabilizing proteins to the cytoplasm could dissociate stress granules or prevent their assembly.
Affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM), UMR829; Université Evry-Val d'Essonne, Evry 91025, France.Show MeSH
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Mentions: We first controlled whether YB-1 in our conditions behaves as a negative regulator of stress granule assembly, as previously described (40). We found that, while low expression levels of GFP-YB-1 do not interfere with arsenite-induced stress granules, above a threshold, stress granule assembly is significantly inhibited (Figure 4). In addition, arguing against a role of HSP70 (40), we observed that high level of YB-1 similarly leads to the inhibition of stress granule assembly when both the HSP70 activity and its expression were inhibited by VER-155008 plus puromycin treatment (Supplementary Figure S5B). The inhibitory effect of YB-1 expression on stress granule assembly could then rather result from the direct binding of YB-1 to mRNA. In order to address this issue, we analyzed the effect of YB-1 on mRNA:TIA-1 aggregates in vitro by AFM. This technique allows the visualization at a nanometric resolution of protein/nucleic acid complexes (‘Materials and Methods’ section, (41)). By mixing 2Luc mRNA and YB-1, we detected the presence of isolated mRNPs with a beads-on-a-string structure, as previously found by AFM (Figure 5A, (28)). They were well dispersed and isolated from each other. In contrast, TIA-1 in the presence of 2Luc mRNA leads to the formation of mRNP aggregates (Figure 5A and B), as expected from its prion-like self-attracting domain. Upon the addition of YB-1 to the mRNA:TIA-1 aggregates, we then clearly observed their dissociation into isolated mRNPs. YB-1 is then able to directly dissociate mRNA granules in vitro and, by a similar mechanism, potentially inhibits stress granule assembly when overexpressed in cells.
Affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM), UMR829; Université Evry-Val d'Essonne, Evry 91025, France.