Dynamic Shuttling of Tia-1 Accompanies the Recruitment of mRNA to Mammalian Stress Granules

Abstract

Mammalian stress granules (SGs) harbor untranslated mRNAs that accumulate in cells exposed to environmental stress. Drugs that stabilize polysomes (emetine) inhibit the assembly of SGs, whereas drugs that destabilize polysomes (puromycin) promote the assembly of SGs. Moreover, emetine dissolves preformed SGs as it promotes the assembly of polysomes, suggesting that these mRNP species (i.e., SGs and polysomes) exist in equilibrium. We used green flourescent protein–tagged SG-associated RNA-binding proteins (specifically, TIA-1 and poly[A] binding protein [PABP-I]) to monitor SG assembly, disassembly, and turnover in live cells. Fluorescence recovery after photobleaching shows that both TIA-1 and PABP-I rapidly and continuously shuttle in and out of SGs, indicating that the assembly of SGs is a highly dynamic process. This unexpected result leads us to propose that mammalian SGs are sites at which untranslated mRNAs are sorted and processed for either reinitiation, degradation, or packaging into stable nonpolysomal mRNP complexes. A truncation mutant of TIA-1 (TIA-1ΔRRM), which acts as a transdominant inhibitor of SG assembly, promotes the expression of cotransfected reporter genes in COS transfectants, suggesting that this process of mRNA triage might, directly or indirectly, influence protein expression.

Evidence That Ternary Complex (eIF2-GTP-tRNAiMet)–Deficient Preinitiation Complexes Are Core Constituents of Mammalian Stress Granules

“…Previous work from our lab and others (Nover et al, 1989;Kedersha et al, 1999Kedersha et al, , 2000 has established that SGs are highly dynamic cytoplasmic foci at which poly(A) ϩ RNA, TIA-1, TIAR, and PABP-I transiently accumulate in response to stress-induced phosphorylation of eIF2␣. Both the RNA and protein components of SGs are in equilibrium with polysomes, as evinced by the dispersal of SGs upon treatment of cells with drugs that stabilize polysomes (e.g., emetine and cycloheximide), and by the enhanced assembly of SGs in cells treated with drugs that destabilize polysomes (e.g., puromycin).…”

“…Stress granules (SGs) are discrete cytoplasmic foci at which untranslated mRNAs accumulate in cells subjected to environmental stress (Nover et al, 1983(Nover et al, , 1989Scharf et al, 1998;Kedersha et al, 1999Kedersha et al, , 2000. In mammalian cells, the assembly of SGs is initiated by the phosphorylation of eIF2␣ (Kedersha et al, 1999), a translation initiation factor that loads the initiator tRNA onto the small ribosomal subunit (reviewed by Berlanga et al, 1998;Gray and Wickens, 1998).…”

mRNP granules

“…For example, non-translating mRNA is an essential component for assembly of all mRNP granules. Supporting this, P-bodies and stress granules cannot form in the presence of cycloheximide or emetine, 19,20 which traps mRNAs in polysomes. In Figure 1.…”

“…Translation typically shows an inverse relationship with stress granule and P-body numbers. 19,22 For instance, during cellular stress, bulk translation is inhibited, leading to increased granules, whereas during stress recovery, translation levels increase as granule numbers fall. This later phenomenon also holds true for specific mRNAs, which exit stress granules and enter translation during the recovery phase.…”

Ataxin-2 Interacts with the DEAD/H-Box RNA Helicase DDX6 and Interferes with P-Bodies and Stress Granules

“…Because an association of DDX6 and SGs has been demonstrated recently (Wilczynska et al, 2005), we further investigated whether DDX6 and ATXN2, which itself is a component of SGs (Ralser et al, 2005a), colocalize under stress conditions. We primarily used the cell line DU145, because DU145 cells were previously used for studying the assembly of SGs (Kedersha et al, 1999(Kedersha et al, , 2000. To induce SGs, cells were treated with NaAsO 2 or heat-shocked and then fixed.…”