Stable ribosome binding to the endoplasmic reticulum enables compartment-specific regulation of mRNA translation

In eukaryotic cells, protein synthesis is compartmentalized; mRNAs encoding secretory/membrane proteins are translated on endoplasmic reticulum (ER)-bound ribosomes, whereas mRNAs encoding cytosolic proteins are translated on free ribosomes. mRNA partitioning between the two compartments occurs via positive selection: free ribosomes engaged in the translation of signal sequence-encoding mRNAs are trafficked from the cytosol to the ER. After translation termination, ER-bound ribosomes are thought to dissociate, thereby completing a cycle of mRNA partitioning. At present, the physiological basis for termination-coupled ribosome release is unknown. To gain insight into this process, we examined ribosome and mRNA partitioning during the unfolded protein response, key elements of which include suppression of the initiation stage of protein synthesis and polyribosome breakdown. We report that unfolded protein response (UPR)-elicited polyribosome breakdown resulted in the continued association, rather than release, of ER-bound ribosomes. Under these conditions, mRNA translation in the cytosol was suppressed, whereas mRNA translation on the ER was sustained. Furthermore, mRNAs encoding key soluble stress proteins (XBP-1 and ATF-4) were translated primarily on ER-bound ribosomes. These studies demonstrate that ribosome release from the ER is termination independent and identify new and unexpected roles for the ER compartment in the translational response to induction of the unfolded protein response.     

Ribosomal slowdown mediates translational arrest during cellular division

Global mRNA translation is transiently inhibited during cellular division. We demonstrate that mitotic cells contain heavy polysomes, but these are significantly less translationally active than polysomes in cycling cells. Several observations indicate that mitotic translational attenuation occurs during the elongation stage: (i) in cycling nonsynchronized cultures, only mitotic cells fail to assemble stress granules when treated with agents that inhibit translational initiation; (ii) mitotic cells contain fewer free 80S complexes, which are less sensitive to high salt disassembly; (iii) mitotic polysomes are more resistant to enforced disassembly using puromycin; and (iv) ribosome transit time increases during mitosis. Elongation slowdown guarantees that polysomes are retained even if initiation is inhibited at the same time. Stalling translating ribosomes during mitosis may protect mRNAs and allow rapid resumption of translation immediately upon entry into the G(1) phase.     

Pioneer round of translation mediated by nuclear cap-binding proteins CBP80/20 occurs during prolonged hypoxia

Nonsense-mediated mRNA decay (NMD) is one of the mRNA surveillance mechanisms, which eliminates aberrant mRNAs harboring premature termination codons. NMD targets only mRNAs bound by the nuclear cap-binding protein complex CBP80/20 which directs the pioneer round of translation. Here we demonstrate that NMD occurs efficiently during prolonged hypoxia in which steady-state translation is drastically inhibited. Accordingly, CBP80 remains in the nucleus, and processing bodies are unaffected with regard to their abundance and number under prolonged hypoxic conditions. These results indicate that mRNAs enter the pioneer round of translation during prolonged hypoxia.     

Neuronal RNA granules: a link between RNA localization and stimulation-dependent translation.

RNA granules are a macromolecular structure observed in neurons, where they serve as motile units that translocate mRNAs. Isolated RNA granules are highly enriched in Staufen protein and ultrastructurally contain densely packed clusters of ribosomes. With depolarization, many mRNAs, including those involved in plasticity, rapidly shift from the RNA granule fraction to polysomes. Depolarization reorganizes granules and induces a less compact organization of their ribosomes. RNA granules are not translationally competent, as indicated by the failure to incorporate radioactive amino acids and the absence of eIF4E, 4G, and tRNAs. We concluded that RNA granules are a local storage compartment for mRNAs under translational arrest but are poised for release to actively translated pools. Local release of mRNAs and ribosomes from granules may serve as a macromolecular mechanism linking RNA localization to translation and synaptic plasticity.     

La protein is associated with terminal oligopyrimidine mRNAs in actively translating polysomes

La is an abundant, mostly nuclear, RNA-binding protein that interacts with regions rich in pyrimidines. In the nucleus it has a role in the metabolism of several small RNAs. A number of studies, however, indicate that La protein is also implicated in cytoplasmic functions such as translation. The association of La in vivo with endogenous mRNAs engaged with polysomes would support this role, but this point has never been addressed yet. Terminal oligopyrimidine (TOP) mRNAs, which code for ribosomal proteins and other components of the translational apparatus, bear a TOP stretch at the 5' end, which is necessary for the regulation of their translation. La protein can bind the TOP sequence in vitro and activates TOP mRNA translation in vivo. Here we have quantified La protein in the cytoplasm of Xenopus oocytes and embryo cells and have shown in embryo cells that it is associated with actively translating polysomes. Disruption of polysomes by EDTA treatment displaces La in messenger ribonucleoprotein complexes sedimenting at 40-60 S. The results of polysome treatment with either low concentrations of micrococcal nuclease or with high concentrations of salt indicate, respectively, that La association with polysomes is mediated by mRNA and that it is not an integral component of ribosomes. Moreover, the analysis of messenger ribonucleoprotein complexes dissociated from translating polysomes shows that La protein associates with TOP mRNAs in vivo when they are translated, in line with a positive role of La in the translation of this class of mRNAs previously observed in cultured cells.     

Membrane-bound mRNAs are recruited from preinitiated ribonucleoprotein particles in injected Xenopus oocytes

Messenger RNA injected Xenopus oocytes exhibit a differential capacity for translation. mRNAs translated in the free cytoplasm are translated efficiently whereas mRNAs translated on the rough endoplasmic reticulum (RER) membrane are translated inefficiently. If mRNA injected oocytes are injected additionally with proteins isolated from the RER, enhanced translation of RER-bound mRNAs is observed. When examined by sucrose gradient centrifugation and RNA dot blots, most of the injected RER-bound mRNA sediments less than or equal to the 80 S monosome. The RER proteins recruit these preinitiated mRNAs onto polysomes as evidenced by a shift in sedimentation to the polysome region of a sucrose gradient. When examined by immunoblotting, the RER proteins are shown to contain a protein which reacts specifically with an antibody directed against docking protein (SRP-receptor protein). However, this putative docking protein does not appear to be the protein which actually recruits the preinitiated mRNAs onto polysomes.     

Actin Depolymerization Affects Stress-Induced Translational Activity of Potato Tuber Tissue

Changes in polymerized actin during stress conditions were correlated with potato (Solanum tuberosum L.) tuber protein synthesis. Fluorescence microscopy and immunoblot analyses indicated that filamentous actin was nearly undetectable in mature, quiescent aerobic tubers. Mechanical wounding of postharvest tubers resulted in a localized increase of polymerized actin, and microfilament bundles were visible in cells of the wounded periderm within 12 h after wounding. During this same period translational activity increased 8-fold. By contrast, low-oxygen stress caused rapid reduction of polymerized actin coincident with acute inhibition of protein synthesis. Treatment of aerobic tubers with cytochalasin D, an agent that disrupts actin filaments, reduced wound-induced protein synthesis in vivo. This effect was not observed when colchicine, an agent that depolymerizes microtubules, was used. Neither of these drugs had a significant effect in vitro on run-off translation of isolated polysomes. However, cytochalasin D did reduce translational competence in vitro of a crude cellular fraction containing both polysomes and cytoskeletal elements. These results demonstrate the dependence of wound-induced protein synthesis on the integrity of microfilaments and suggest that the dynamics of the actin cytoskeleton may affect translational activity during stress conditions.     

Translational control during early development

Early development in many animals is programmed by maternally inherited messenger RNAs. Many of these mRNAs are translationally dormant in immature oocytes, but are recruited onto polysomes during meiotic maturation, fertilization, or early embryogenesis. In contrast, other mRNAs that are translated in oocytes are released from polysomes during these later stages of development. Recent studies have begun to define the cis and trans elements that regulate both translational repression and translational induction of maternal mRNA. The inhibition of translation of some mRNAs during early development is controlled by discrete sequences residing in the 3' and 5' untranslated regions, respectively. The translation of other RNAs is due to polyadenylation which, at least in oocytes of the frog Xenopus laevis, is regulated by a U-rich cytoplasmic polyadenylation element (CPE). Although similar, the CPE sequences of various mRNAs are sufficiently different to be bound by different proteins. Two of these proteins and their interactions are described here.     

Characterization of hibernating ribosomes in mammalian cells

Protein synthesis across kingdoms involves the assembly of 70S (prokaryotes) or 80S (eukaryotes) ribosomes on the mRNAs to be translated. 70S ribosomes are protected from degradation in bacteria during stationary growth or stress conditions by forming dimers that migrate in polysome profiles as 100S complexes. Formation of ribosome dimers in Escherichia coli is mediated by proteins, namely the ribosome modulation factor (RMF), which is induced in the stationary phase of cell growth. It is reported here a similar ribosomal complex of 110S in eukaryotic cells, which forms during nutrient starvation. The dynamic nature of the 110S ribosomal complex (mammalian equivalent of the bacterial 100S) was supported by the rapid conversion into polysomes upon nutrient-refeeding via a mechanism sensitive to inhibitors of translation initiation. Several experiments were used to show that the 110S complex is a dimer of nontranslating ribosomes. Cryo-electron microscopy visualization of the 110S complex revealed that two 80S ribosomes are connected by a flexible, albeit localized, interaction. We conclude that, similarly to bacteria, rat cells contain stress-induced ribosomal dimers. The identification of ribosomal dimers in rat cells will bring new insights in our thinking of the ribosome structure and its function during the cellular response to stress conditions.Key words: ribosome, translation, stress, starvation, polysome