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.     

'Unmasking' of stored maternal mRNAs and the activation of protein synthesis at fertilization in sea urchins

The isolation and in vitro assay of maternal mRNPs has led to differing conclusions as to whether maternal mRNAs in sea urchin eggs are in a repressed or 'masked' form. To circumvent the problems involved with in vitro approaches, we have used an in vivo assay to determine if the availability of mRNA and/or components of the translational machinery are limiting protein synthesis in the unfertilized egg. This assay involves the use of a protein synthesis elongation inhibitor to create a situation in the egg in which there is excess translational machinery available to bind mRNA. Eggs were fertilized and the rate of entry into polysomes of individual mRNAs was measured in inhibitor-treated and control embryos using 32P-labeled cDNA probes. The fraction of ribosomes in polysomes and the polysome size were also determined. The results from this in vivo approach provide strong evidence for the coactivation of both mRNAs and components of the translational machinery following fertilization. The average polysome size increases from 7.5 ribosomes per message in 15 min embryos to approximately 10.8 ribosomes in 2 h embryos. This result gives additional support to the idea that translational machinery, as well as mRNA, is activated following fertilization. We also found that individual mRNAs are recruited into polysomes with different kinetics, and that the fraction of an mRNA in polysomes in the unfertilized egg correlates with the rate at which that mRNA is recruited into polysomes following fertilization.     

Stress-Induced Translational Control in Potato Tubers May Be Mediated by Polysome-Associated Proteins

Potato tubers exhibit distinct responses to wounding and hypoxia that include selective translation of stress-induced mRNAs. Newly synthesized wound-response mRNAs are bound to polysomes, whereas preexisting mRNAs are displaced and degraded. mRNAs that are induced and translated during hypoxic conditions are bound to ribosomes as expected. However, preexisting wound-response mRNAs whose translation is inhibited during hypoxia remain bound to polysomes, indicating that there are at least two distinct mechanisms by which translation is regulated in response to stress conditions. A 32-kD phosphoprotein is associated with polyribosomes from wounded tubers. This protein remains polysome bound as long as wound-response mRNAs are present, even during hypoxia when these mRNAs are no longer translated. However, association of the 32-kD protein with polysomes is not elicited by hypoxic stress alone. The kinase that phosphorylates this protein is active only for the first 24 hr after wounding and is not active during periods of hypoxia. This protein may mediate recognition of the wound-response mRNAs by ribosomes.     

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.     

Translational regulation of the expression of ribosomal protein genes in Xenopus laevis

The mRNAs coding for ribosomal proteins (rp-mRNA) are subjected to translational control during Xenopus oogenesis and embryogenesis, and also during nutritional changes in Xenopus cultured cells. This regulation, which appears to respond to the cellular need for new ribosomes, operates by changing the fraction of rp-mRNA engaged on polysomes, each translated rp-mRNA molecule always remaining fully loaded with ribosomes. All rp-mRNAs analyzed up to now show this translational behavior, and also share some structural features in their untranslated portions. In particular they all have rather short 5' untranslated regions, similar to each other, and always start at the very 5' end with a stretch of several pyrimidines. Fusion to a reporter-coding sequence of the 5' untranslated region of r-protein S19 has shown that this is involved in the translational regulation.     

Isolation of a cDNA clone encoding S-adenosylmethionine decarboxylase. Expression of the gene in mitogen-activated lymphocytes

S-Adenosylmethionine decarboxylase was purified from bovine liver and digested with endopeptidase Lys-C; the resulting peptides were chromatographically separated. Peptides containing either methionine or tryptophan were subjected to sequence analysis. An oligonucleotide mixture of 48 sequences, which was 17 nucleotides in length, was synthesized based on one of these peptide sequences. This synthetic oligonucleotide mixture was labeled and used to screen a bovine cDNA library in phage lambda gt11. A clone was identified which contained a 1350-nucleotide insert. This insert contained nucleotide sequences coding for amino acid sequences of two of the peptides that were analyzed, thus proving that this cDNA clone codes for S-adenosylmethionine decarboxylase. A subcloned fragment from the coding region of the cDNA was used as a probe to analyze the expression of this gene in mitogen-activated lymphocytes. Northern blots revealed two message species of 2.4 and 3.6 kilobases in length. Both mRNAs were coordinately expressed and were present in polysomes. The levels of these mRNAs increased approximately 4-fold by 9 h after activation of the cells. The magnitude of the increase in these messages is to be compared with an 8- to 10-fold increase in the rate of synthesis of the protein. The apparent increase in translational efficiency of this message upon lymphocyte activation was confirmed by analyzing polysomes from these cells. In resting lymphocytes, the average size of polysomes containing mRNA coding for S-adenosylmethionine decarboxylase was 1.4 ribosomes per mRNA, and this value increased to 2.7 in stimulated cells. Thus, it appears that the increase in translational efficiency of this mRNA arises from an elevated rate of translational initiation, leading to more ribosomes per polysome encoding this particular message. This is not a general effect on the expression of all proteins, since there is no change in the translational efficiency of cytoplasmic actin upon activation of lymphocytes.     

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.     

Synthesis of two proteins in chloroplasts and mRNA distribution between thylakoids and stroma during the cell cycle of Chlamydomonas reinhardii

Chloroplasts contain thylakoid-bound and free ribosomes and polysomes. Whether binding of polysomes plays an immediate role in the regulation of chloroplast protein synthesis is not yet clear. In the present work, variations of protein synthesis and of mRNA content were measured not in greening, but in fully differentiated chloroplasts during the cell cycle of synchronized cultures of Chlamydomonas reinhardii. At different times of the vegetative cell cycle, the RNA was extracted from free and thylakoid-bound chloroplast polysomes and the partition of mRNAs between stroma and thylakoids was measured for two proteins, i.e. the 32-kDa herbicide-binding membrane protein and the soluble large subunit of the ribulose-1,5-bisphosphate carboxylase. At the same time the rates of synthesis of these two proteins were also determined. At 2 h after the onset of light, the content of both mRNAs in chloroplasts had doubled and 75-90% of each of these mRNAs were found to be bound to the thylakoids. The rate of protein synthesis, however, increased 10-fold, but reached its maximum only after about 6 h in the light. The differences in the time courses, in the stimulation of the rate of protein synthesis, and in the mRNA-binding to thylakoids point to a translational regulation of protein synthesis. Furthermore, since a very high proportion of polysomes were bound to thylakoids, containing mRNA for both a membrane and a soluble protein, this light-induced binding of polysomes to thylakoids seems to be an essential, but not the only, prerequisite for protein synthesis in chloroplasts.     

Regulation of initiation factors during translational repression caused by serum depletion. Abundance, synthesis, and turnover rates

During growth in unreplenished medium, the fraction of active, polysomal ribosomes progressively decreases about 3-fold from 80-90% to only 20-40% due to a reduced rate of initiation. To assess whether the abundance of initiation factors could be involved in this repression of translational activity. HeLa cell cytoplasmic lysates were resolved by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and spots corresponding to the initiation factor proteins were quantitated. The relative abundance of most of the initiation factor proteins only decreases by 10-40% and roughly parallels that of the ribosomes. Measurement of the rates of synthesis and turnover of the initiation factor proteins establishes that during periods of active growth, synthesis and degradation occur coordinately with total cell protein. As growth rate decreases, the synthesis of some initiation factor proteins, particularly eukaryotic initiation factor (eIF)-3 subunits, becomes depressed. Serum stimulation of serum-depleted cells recruits most inactive ribosomes and mRNAs into polysomes, but most initiation factor mRNAs are not selectively recruited. The principal exceptions are eIF-3p24 which exhibits 4-5 fold enhanced synthesis and eIF-3p44 and eIF-4A whose syntheses are moderately stimulated.     

Free ribosomes and growth stimulation in human peripheral lymphocytes: Activation of free ribosomes as an essential event in growth induction

During the initial ten hours of growth in lymphocytes stimulated by phytohemagglutinin, the cells are converted from a state in which over 70% of all ribosomes are inactive free ribosomes, to one in which over 80% of ribosomes are in polysomes or in native ribosomal subunits. In this initial period, there was a neglible increase in total ribosomal RNA due to increased RNA synthesis, and abolition of ribosomal RNA synthesis with low concentrations of actinomycin D did not interfere with polysome formation. Therefore, the conversion is accomplished by the activation of existing free ribosomes rather than by accumulation of newly synthesized particles. The large free ribosome pool of resting lymphocytes is thus an essential source of components for accelerated protein synthesis early in lymphocyte activation, before increased synthesis can provide a sufficient number of new ribosomes. Free ribosomes accumulate once more after 24 to 48 hours of growth, when RNA and DNA synthetic activity are maximal. This reaccumulation of inactive ribosomes at the peak of growth activity may represent preparation for a return to the resting state where cells are again susceptible to stimulation. Activation of free ribosomes to form polysomes appears to involve modification of at least two steps: (a) dissociation of free ribosomes with stabilization as native subunits, and (b) adjustment of a rate-limiting step at initiation.