TIA-1 is an RNA binding protein that promotes the assembly of stress granules (SGs), discrete cytoplasmic inclusions into which stalled translation initiation complexes are dynamically recruited in ...cells subjected to environmental stress. The RNA recognition motifs of TIA-1 are linked to a glutamine-rich prion-related domain (PRD). Truncation mutants lacking the PRD domain do not induce spontaneous SGs and are not recruited to arsenite-induced SGs, whereas the PRD forms aggregates that are recruited to SGs in low-level-expressing cells but prevent SG assembly in high-level-expressing cells. The PRD of TIA-1 exhibits many characteristics of prions: concentration-dependent aggregation that is inhibited by the molecular chaperone heat shock protein (HSP)70; resistance to protease digestion; sequestration of HSP27, HSP40, and HSP70; and induction of HSP70, a feedback regulator of PRD disaggregation. Substitution of the PRD with the aggregation domain of a yeast prion, SUP35-NM, reconstitutes SG assembly, confirming that a prion domain can mediate the assembly of SGs. Mouse embryomic fibroblasts (MEFs) lacking TIA-1 exhibit impaired ability to form SGs, although they exhibit normal phosphorylation of eukaryotic initiation factor (eIF)2alpha in response to arsenite. Our results reveal that prion-like aggregation of TIA-1 regulates SG formation downstream of eIF2alpha phosphorylation in response to stress.
Exposure to arsenite inhibits protein synthesis and activates multiple stress signaling pathways. Although arsenite has diverse effects on cell metabolism, we demonstrated that phosphorylation of ...eukaryotic translation initiation factor 2 at Ser-51 on the α subunit was necessary to inhibit protein synthesis initiation in arsenite-treated cells and was essential for stress granule formation. Of the four protein kinases known to phosphorylate eukaryotic translation initiation factor 2α, only the heme-regulated inhibitor kinase (HRI) was required for the translational inhibition in response to arsenite treatment in mouse embryonic fibroblasts. In addition, HRI expression was required for stress granule formation and cellular survival after arsenite treatment. In vivo studies elucidated a fundamental requirement for HRI in murine survival upon acute arsenite exposure. The results demonstrated an essential role for HRI in mediating arsenite stress-induced phosphorylation of eukaryotic translation initiation factor 2α, inhibition of protein synthesis, stress granule formation, and survival.
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 polyA 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.
Environmental stress-induced phosphorylation of eIF2alpha inhibits protein translation by reducing the availability of eIF2-GTP-tRNA(i)Met, the ternary complex that joins initiator tRNA(Met) to the ...43S preinitiation complex. The resulting untranslated mRNA is dynamically routed to discrete cytoplasmic foci known as stress granules (SGs), a process requiring the related RNA-binding proteins TIA-1 and TIAR. SGs appear to be in equilibrium with polysomes, but the nature of this relationship is obscure. We now show that most components of the 48S preinitiation complex (i.e., small, but not large, ribosomal subunits, eIF3, eIF4E, eIF4G) are coordinately recruited to SGs in arsenite-stressed cells. In contrast, eIF2 is not a component of newly assembled SGs. Cells expressing a phosphomimetic mutant (S51D) of eIF2alpha assemble SGs of similar composition, confirming that the recruitment of these factors is a direct consequence of blocked translational initiation and not due to other effects of arsenite. Surprisingly, phospho-eIF2alpha is recruited to SGs that are disassembling in cells recovering from arsenite-induced stress. We discuss these results in the context of a translational checkpoint model wherein TIA and eIF2 are functional antagonists of translational initiation, and in which lack of ternary complex drives SG assembly.
The TIA-1-interacting protein Fas-activated serine/threonine phosphoprotein (FAST) is a component of a signaling cascade that is initiated by ligation of the Fas receptor. Immunofluorescence ...microscopy using affinity-purified antibodies raised against recombinant FAST reveals that the endogenous protein associates with mitochondria. Subcellular fractionation confirms that FAST is a component of mitochondria. FAST is tethered to mitochondria by a lysine/arginine-rich domain at its carboxyl terminus that is structurally similar to the mitochondrial tethering motifs of monoamine oxidase B and cytochrome
b5. At the mitochondrial membrane, FAST interacts with BCL-X
L. The BCL-X
L binding domain maps to a BCL-2-homology-3 (BH3)-related domain that is distinct from the mitochondrial-tethering domain (MTD). Although interactions between FAST and BCL-X
L require both the BH3-related domain and the MTD, the requirement for mitochondrial tethering can be conferred by a heterologous MTD. Our results suggest that FAST–BCL-X
L interactions are likely to regulate mitochondrial metabolism during Fas-induced apoptosis.
TIA-1 is an RNA binding protein that promotes the assembly of stress granules (SGs), discrete cytoplasmic inclusions into which stalled translation initiation complexes are dynamically recruited in ...cells subjected to environmental stress. The RNA recognition motifs of TIA-1 are linked to a glutamine-rich prion-related domain (PRD). Truncation mutants lacking the PRD domain do not induce spontaneous SGs and are not recruited to arsenite-induced SGs, whereas the PRD forms aggregates that are recruited to SGs in low-level–expressing cells but prevent SG assembly in high-level–expressing cells. The PRD of TIA-1 exhibits many characteristics of prions: concentration-dependent aggregation that is inhibited by the molecular chaperone heat shock protein (HSP)70; resistance to protease digestion; sequestration of HSP27, HSP40, and HSP70; and induction of HSP70, a feedback regulator of PRD disaggregation. Substitution of the PRD with the aggregation domain of a yeast prion, SUP35-NM, reconstitutes SG assembly, confirming that a prion domain can mediate the assembly of SGs. Mouse embryomic fibroblasts (MEFs) lacking TIA-1 exhibit impaired ability to form SGs, although they exhibit normal phosphorylation of eukaryotic initiation factor (eIF)2α in response to arsenite. Our results reveal that prion-like aggregation of TIA-1 regulates SG formation downstream of eIF2α phosphorylation in response to stress.
FAST is a BCL-X(L)-associated mitochondrial protein Li, Wei; Kedersha, Nancy; Chen, Samantha ...
Biochemical and biophysical research communications,
2004-May-21, 20040521, Volume:
318, Issue:
1
Journal Article
Peer reviewed
The TIA-1-interacting protein Fas-activated serine/threonine phosphoprotein (FAST) is a component of a signaling cascade that is initiated by ligation of the Fas receptor. Immunofluorescence ...microscopy using affinity-purified antibodies raised against recombinant FAST reveals that the endogenous protein associates with mitochondria. Subcellular fractionation confirms that FAST is a component of mitochondria. FAST is tethered to mitochondria by a lysine/arginine-rich domain at its carboxyl terminus that is structurally similar to the mitochondrial tethering motifs of monoamine oxidase B and cytochrome b5. At the mitochondrial membrane, FAST interacts with BCL-X(L). The BCL-X(L) binding domain maps to a BCL-2-homology-3 (BH3)-related domain that is distinct from the mitochondrial-tethering domain (MTD). Although interactions between FAST and BCL-X(L) require both the BH3-related domain and the MTD, the requirement for mitochondrial tethering can be conferred by a heterologous MTD. Our results suggest that FAST-BCL-X(L) interactions are likely to regulate mitochondrial metabolism during Fas-induced apoptosis.
Genetic information flows from DNA to RNA to proteins. Precise control of the many steps of mRNA metabolism is critical for the continuation of this informational flow. Key regulatory points within ...the mRNA metabolic lifecycle include splicing, nuclear export, surveillance in the form of nonsense-mediated decay (NMD), and regulation of translation initiation. Tissue-specific alternative splicing events are key sources of genetic diversity. To gain insights into the mechanism of tissue-specific splicing events, we characterized a novel, neuron-specific RNA-binding protein known as Fox-3. We showed that Fox-3 can act either as a splicing enhancer or a splicing suppressor, and can affect the splicing of several target genes with significant physiological relevance to human disease. Nuclear mRNA export is a key step in gene expression, and yet much was unknown about its mechanism, particularly in metazoan organisms. We performed a whole-genome RNAi screen in Drosophila cells, and identified seventy-two factors required for metazoan mRNA export. Further, by comparing the export requirements of particular spliced and unspliced transcripts, we identified export factors that are specific to the nuclear processing requirements of their target transcripts. We characterized a novel export factor identified in the screen, known as dmPCID2, and showed that in addition to its role in export dmPCID2 associates with actively translating polysomes in the cytoplasm. We further characterized the human homolog of this protein, PCID2, and found that PCID2 is required for efficient NMD in human cells. Appropriate metabolic responses to environmental stress are critical for cellular survival. Regulation of translation initiation is a key stress response mechanism. We demonstrate the dynamic formation of stress granules (SGs) in Drosophila cells in response to heat and oxidative stress. SGs are sites of mRNA triage during cellular stress, and their formation is regulated by inhibition of translation initiation. Further, we show that heat stress bypasses the normal mechanisms that regulate translational arrest. The culmination of these results reveals several new mechanisms for the metabolic regulation of mRNAs. The processes elucidated here all intersect with human health and disease, highlighting the important role of regulation of mRNA metabolism for cellular function.
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