Nancy Kedersha

Stress granules and processing bodies are dynamically linked sites of mRNP remodeling

Stress granules (SGs) are cytoplasmic aggregates of stalled translational preinitiation complexes that accumulate during stress. GW bodies/processing bodies (PBs) are distinct cytoplasmic sites of mRNA degradation. In this study, we show that SGs and PBs are spatially, compositionally, and functionally linked. SGs and PBs are induced by stress, but SG assembly requires eIF2α phosphorylation, whereas PB assembly does not. They are also dispersed by inhibitors of translational elongation and share several protein components, including Fas-activated serine/threonine phosphoprotein, XRN1, eIF4E, and tristetraprolin (TTP). In contrast, eIF3, G3BP, eIF4G, and PABP-1 are restricted to SGs, whereas DCP1a and 2 are confined to PBs. SGs and PBs also can harbor the same species of mRNA and physically associate with one another in vivo, an interaction that is promoted by the related mRNA decay factors TTP and BRF1. We propose that mRNA released from disassembled polysomes is sorted and remodeled at SGs, from which selected transcripts are delivered to PBs for degradation.

Stress granules: the Tao of RNA triage

Cytoplasmic RNA structures such as stress granules (SGs) and processing bodies (PBs) are functional byproducts of mRNA metabolism, sharing substrate mRNA, dynamic properties and many proteins, but also housing separate components and performing independent functions. Each can exist independently, but when coordinately induced they are often tethered together in a cytosolic dance. Although both self-assemble in response to stress-induced perturbations in translation, several recent reports reveal novel proteins and RNAs that are components of these structures but also perform other cellular functions. Proteins that mediate splicing, transcription, adhesion, signaling and development are all integrated with SG and PB assembly. Thus, these ephemeral bodies represent more than just the dynamic sorting of mRNA between translation and decay.

RNA granules: Figure 1.

Cytoplasmic RNA granules in germ cells (polar and germinal granules), somatic cells (stress granules and processing bodies), and neurons (neuronal granules) have emerged as important players in the posttranscriptional regulation of gene expression. RNA granules contain various ribosomal subunits, translation factors, decay enzymes, helicases, scaffold proteins, and RNA-binding proteins, and they control the localization, stability, and translation of their RNA cargo. We review the relationship between different classes of these granules and discuss how spatial organization regulates messenger RNA translation/decay.

RNA granules: post-transcriptional and epigenetic modulators of gene expression

The composition of cytoplasmic messenger ribonucleoproteins (mRNPs) is determined by their nuclear and cytoplasmic histories and reflects past functions and future fates. The protein components of selected mRNP complexes promote their assembly into microscopically visible cytoplasmic RNA granules, including stress granules, processing bodies and germ cell (or polar) granules. We propose that RNA granules can be both a cause and a consequence of altered mRNA translation, decay or editing. In this capacity, RNA granules serve as key modulators of post-transcriptional and epigenetic gene expression.

TIA‐1 is a translational silencer that selectively regulates the expression of TNF‐α

TIA‐1 and TIAR are related proteins that bind to an AU‐rich element (ARE) in the 3′ untranslated region of tumor necrosis factor alpha (TNF‐α) transcripts. To determine the functional significance of this interaction, we used homologous recombination to produce mutant mice lacking TIA‐1. Although lipopolysaccharide (LPS)‐stimulated macrophages derived from wild‐type and TIA‐1−/− mice express similar amounts of TNF‐α transcripts, macrophages lacking TIA‐1 produce significantly more TNF‐α protein than wild‐type controls. The half‐life of TNF‐α transcripts is similar in wild‐type and TIA‐1−/− macrophages, indicating that TIA‐1 does not regulate transcript stability. Rather, the absence of TIA‐1 significantly increases the proportion of TNF‐α transcripts that associate with polysomes, suggesting that TIA‐1 normally functions as a translational silencer. TIA‐1 does not appear to regulate the production of interleukin 1β, granulocyte–macrophage colony‐stimulating factor or interferon γ, indicating that its effects are, at least partially, transcript specific. Mice lacking TIA‐1 are hypersensitive to the toxic effects of LPS, indicating that this translational control pathway may regulate the organismal response to microbial stress.

MK2‐induced tristetraprolin:14‐3‐3 complexes prevent stress granule association and ARE‐mRNA decay

Stress granules (SGs) are dynamic cytoplasmic foci at which stalled translation initiation complexes accumulate in cells subjected to environmental stress. SG‐associated proteins such as TIA‐1, TIAR and HuR bind to AU‐rich element (ARE)‐containing mRNAs and control their translation and stability. Here we show that tristetraprolin (TTP), an ARE‐binding protein that destabilizes ARE‐mRNAs, is recruited to SGs that are assembled in response to FCCP‐induced energy deprivation, but not arsenite‐induced oxidative stress. Exclusion of TTP from arsenite‐induced SGs is a consequence of MAPKAP kinase‐2 (MK2)‐induced phosphorylation at serines 52 and 178, which promotes the assembly of TTP:14‐3‐3 complexes. 14‐3‐3 binding excludes TTP from SGs and inhibits TTP‐dependent degradation of ARE‐containing transcripts. In activated RAW 264.7 macrophages, endogenous TTP:14‐3‐3 complexes bind to ARE‐RNA. Our data reveal the mechanism by which the p38‐MAPK/MK2 kinase cascade inhibits TTP‐mediated degradation of ARE‐containing transcripts and thereby contributes to lipopolysaccharide‐induced TNFα expression.

The hPLIC Proteins May Provide a Link between the Ubiquitination Machinery and the Proteasome

Although there is a binding site on the proteasome for the polyubiquitin chains attached to degradation substrates by the ubiquitination machinery, it is currently unclear whether in vivo the activities of the ubiquitination machinery and the proteasome are coupled. Here we show that two human homologs of the yeast ubiquitin-like Dsk2 protein, hPLIC-1 and hPLIC-2, physically associate with both proteasomes and ubiquitin ligases in large complexes. Overexpression of hPLIC proteins interferes with the in vivo degradation of two unrelated ubiquitin-dependent proteasome substrates, p53 and IkappaBalpha, but not a ubiquitin-independent substrate. Our findings raise the possibility that the hPLIC proteins, and possibly related ubiquitin-like family members, may functionally link the ubiquitination machinery to the proteasome to affect in vivo protein degradation.

The LAR transmembrane protein tyrosine phosphatase and a coiled-coil LAR-interacting protein co-localize at focal adhesions.

Focal adhesions are sites of cell‐extracellular matrix interactions that function in anchoring stress fibers to the plasma membrane and in adhesion‐mediated signal transduction. Both focal adhesion structure and signaling ability involve protein tyrosine phosphorylation. LAR is a broadly expressed transmembrane protein tyrosine phosphatase comprised of a cell adhesion‐like ectodomain and two intracellular protein tyrosine phosphatase domains. We have identified a novel cytoplasmic 160 kDa phosphoserine protein termed LAR‐interacting protein 1 (LIP.1), which binds to the LAR membrane‐distal D2 protein tyrosine phosphatase domain and appears to localize LAR to focal adhesions. Both LAR and LIP.1 decorate the ends of focal adhesions most proximal to the cell nucleus and are excluded from the distal ends of focal adhesions, thus localizing to regions of focal adhesions presumably undergoing disassembly. We propose that LAR and LIP.1 may regulate the disassembly of focal adhesions and thus help orchestrate cell‐matrix interactions.

Heme-regulated Inhibitor Kinase-mediated Phosphorylation of Eukaryotic Translation Initiation Factor 2 Inhibits Translation, Induces Stress Granule Formation, and Mediates Survival upon Arsenite Exposure

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.

A functional RNAi screen links O-GlcNAc modification of ribosomal proteins to stress granule and processing body assembly

Stress granules (SGs) and processing bodies (PBs) are microscopically visible ribonucleoprotein granules that cooperatively regulate the translation and decay of messenger RNA. Using an RNA-mediated interference-based screen, we identify 101 human genes required for SG assembly, 39 genes required for PB assembly, and 31 genes required for coordinate SG and PB assembly. Although 51 genes encode proteins involved in mRNA translation, splicing and transcription, most are not obviously associated with RNA metabolism. We find that several components of the hexosamine biosynthetic pathway, which reversibly modifies proteins with O-linked N-acetylglucosamine (O-GlcNAc) in response to stress, are required for SG and PB assembly. O-GlcNAc-modified proteins are prominent components of SGs but not PBs, and include RACK1 (receptor for activated C kinase 1), prohibitin-2, glyceraldehyde-3-phosphate dehydrogenase and numerous ribosomal proteins. Our results suggest that O-GlcNAc modification of the translational machinery is required for aggregation of untranslated messenger ribonucleoproteins into SGs. The lack of enzymes of the hexosamine biosynthetic pathway in budding yeast may contribute to differences between mammalian SGs and related yeast EGP (eIF4E, 4G and Pab1 containing) bodies.