Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.     

Cell-free translation of polyribosomes from detached pumpkin cotyledons: Effects of starvation and cytokinin

The effects of 6-benzylaminopurine (BAP, 5.10^?5M) treatment of pumpkin cotyledons and their starvation after excision upon polysome/monosome ratio and translational capacity of polysomes in cell-free system were studied. It has been found that starvation causes a progressive polysome degradation. Polysome translation in a wheat germ cell-free proteinsynthesizing system reveals that the translation capacity of polysome preparations decreases with the time after cotyledon excision much more sharply than polysome/monosome ratio. This indicates the starvation damage in elongation steps of protein synthesis. The decrease of postribosomal supernatants activity in the system of poly(U)-directed polyphenylalanine synthesis confirms this conclusion. BAP treatment brings about a very rapid monosome mobilization into polysomes and activation of cell-free translation of ribosome preparations which is however closely parallel to the polysome percentage in them. That means that during this initial period of BAP action only protein synthesis initiation is under BAP control. The experiments with aurintricarboxylic acid (ATA) support this idea.     

The cell cycle dependence of protein synthesis during Xenopus laevis development.

The regulation of early embryonic development in the amphibian Xenopus laevis depends largely upon translational and post-translational regulatory mechanisms to direct the complex cytodifferentiations that take place during early cleavage and blastula formation. The cell cycle dependence of protein synthesis was examined in developing Xenopus embryos as well as in cycling cell-free lysates from Xenopus eggs. In both cases M-phase and the activation of the M-phase kinase were found to be correlated with an inhibition of translation. Translation in both the rough endoplasmic reticulum and cytosolic-free ribosomes were affected by this inhibition. Since elongation was found to be unaffected by M-phase, shifts in the polysome profiles during M-phase indicated that the inhibition affected initiation processes. The activity of the M-phase kinase may inhibit initiation through the modification of initiation factors or some other component during this process. The cell cycle dependence of translation may affect developmental mechanisms controlled by the titration of regulatory proteins.     

Molecular Factors Affecting the Accumulation of Recombinant Proteins in the <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> Chloroplast

In an effort to develop microalgae as a robust system for the production of valuable proteins, we analyzed some of the factors affecting recombinant protein expression in the chloroplast of the green alga Chlamydomonas reinhardtii. We monitored mRNA accumulation, protein synthesis, and protein turnover for three codon-optimized transgenes including GFP, bacterial luciferase, and a large single chain antibody. GFP and luciferase proteins were quite stable, while the antibody was less so. Measurements of protein synthesis, in contrast, clearly showed that translation of the three chimeric mRNAs was greatly reduced when compared to endogenous mRNAs under control of the same atpA promoter/UTR. Only in a few conditions this could be explained by limited mRNA availability since, in most cases, recombinant mRNAs accumulated quite well when compared to the atpA mRNA. In vitro toeprint and in vivo polysome analyses suggest that reduced ribosome association might contribute to limited translational efficiency. However, when recombinant polysome levels and protein synthesis are analyzed as a whole, it becomes clear that other steps, such as inefficient protein elongation, are likely to have a considerable impact. Taken together, our results point to translation as the main step limiting the expression of heterologous proteins in the C. reinhardtii chloroplast.     

Quantitative polysome analysis identifies limitations in bacterial cell-free protein synthesis

Cell-free protein synthesis (CFPS) is becoming increasingly used for protein production as yields increase and costs decrease. CFPS optimization efforts have focused primarily on energy supply and small molecule metabolism, though little is known about the protein synthesis machinery or what limits protein synthesis rates. Here, quantitative polysome profile analysis was used to characterize cell-free translation, thereby elucidating many kinetic parameters. The ribosome concentration in Escherichia coli-based CFPS reactions was 1.6 +/- 0.1 microM, with 72 +/- 4% actively translating at maximal protein synthesis rate. A translation elongation rate of 1.5 +/- 0.2 amino acids per second per ribosome and an initiation rate of 8.2 x 10(-9) +/- 0.3 x 10(-9) M/s, which correlates to, on average, one initiation every 60 +/- 9 s per mRNA, were determined. The measured CFPS initiation and elongation rates are an order of magnitude lower than the in vivo rates and further analysis identified elongation as the major limitation. Adding purified elongation factors (EFs) to CFPS reactions increased the ribosome elongation rate and protein synthesis rates and yields, as well as the translation initiation rate, indicating a possible coupling between initiation and elongation. Further examination of translation initiation in the cell-free system showed that the first initiation on an mRNA is slower than subsequent initiations. Our results demonstrate that polysome analysis is a valid tool to characterize cell-free translation and to identify limiting steps, that dilution of translation factors is a limitation of CFPS, and that CFPS is a useful platform for making novel observations about translation.     

FETAL DEVELOPMENT: THE EFFECTS OF MATURATION ON IN VITRO PROTEIN SYNTHESIS BY MOUSE BRAIN TISSUE

—The elucidation of the translational regulatory events which function during the critical fetal and neonatal period is an important prerequisite to our understanding of normal, as well as abnormal, brain growth and differentiation. Brain cell suspensions and cell-free homogenates were employed to study the protein synthetic activity during the maturation of fetal- neural tissue. The results clearly demonstrated that while neural tissue from 1-day postnatal mice was 10 times more active in protein synthesis than brain tissue from adult mice, the former was many fold less active in translational events than fetal neural tissue from 13-day post-zygotic mice. Fetal polypeptide synthetic activity was found to decrease from the 13th day to the 19th day post-zygotic. This decrement in the translational activity was not due to amino acid availability or pools, or to differences, quantitatively or qualitatively, in polysome concentrations. The enhanced rate of protein synthetic activity measured with neural tissue from 13-day post-zygotic mice was shown to be due to an increase in rate of protein synthesis and not to an enhanced rate of protein degradation.     

Effect of energy source on the efficiency of translational termination during cell-free protein synthesis

We studied how the fidelity of translation termination is affected by the method of ATP regeneration during cell-free protein synthesis. During the in vivo expression of hEPO, whose termination is directed by the UGA codon, we found that substantial proportions of the translational products showed a larger molecular weight than expected. Similar results were obtained in a cell-free synthesis reaction using phosphoenol pyruvate (PEP) or 3-phosphoglycerate (3PG) for ATP regeneration. However, when the energy source was switched to creatine phosphate (CP), the readthrough of the UGA codon was completely repressed and only the target protein of the correct size was expressed in a high yield. To the best of our knowledge, this is the first report describing the relationship between the regeneration of nucleotide triphosphates and protein readthrough, and we also believe that the discovery would pave the way to the selective and efficient expression of target proteins in cell-free protein synthesis systems.     

A model for protein translation: polysome self-organization leads to maximum protein synthesis rates

The genetic information in DNA is transcribed to mRNA and then translated to proteins, which form the building blocks of life. Translation, or protein synthesis, is hence a central cellular process. We have developed a gene-sequence-specific mechanistic model for the translation machinery, which accounts for all the elementary steps of the translation mechanism. We performed a sensitivity analysis to determine the effects of kinetic parameters and concentrations of the translational components on protein synthesis rate. Utilizing our mathematical framework and sensitivity analysis, we investigated the translational kinetic properties of a single mRNA species in Escherichia coli. We propose that translation rate at a given polysome size depends on the complex interplay between ribosomal occupancy of elongation phase intermediate states and ribosome distributions with respect to codon position along the length of the mRNA, and this interplay leads to polysome self-organization that drives translation rate to maximum levels.     

Translational control by influenza virus. Selective and cap-dependent translation of viral mRNAs in infected cells.

In cells infected by influenza virus type A, host protein synthesis undergoes a rapid and dramatic shutoff. To define the molecular mechanisms underlying this selective translation, a transfection/infection protocol was developed utilizing viral and cellular cDNA clones. When COS-1 cells were transfected with cDNAs encoding nonviral genes and subsequently infected with influenza virus, protein expression from the exogenous genes was diminished, similar to the endogenous cellular genes. However, when cells were transfected with a truncated influenza viral nucleocapsid protein (NP-S) gene, the NP-S protein was made as efficiently in influenza virus infected cells as in uninfected cells, showing that the NP-S mRNA, although expressed independently of the influenza virus replication machinery, was still recognized as a viral and not a cellular mRNA. Northern blot analysis demonstrated that the selective blocks to nonviral protein synthesis were at the level of translation. Moreover, polysome experiments revealed that the translational blocks occurred at both the initiation and elongation stages of cellular protein synthesis. Finally, we utilized this transfection/infection system as well as double infection experiments to demonstrate that the translation of influenza viral mRNAs probably occurred in a cap-dependent manner as poliovirus infection inhibited influenza viral mRNA translation.     

Mutations in the Escherichia coli23S rRNA Increase the Rate of Peptidyl-tRNA Dissociation from the Ribosome

We have studied in vivothe phenotypes of 23S rRNA mutations G2582A, G2582U, G2583C, and U2584C, which are located at the A site of Escherichia coli50S ribosomal subunit. All mutant rRNAs incorporated into 50S ribosomal subunits. Upon sucrose gradient fractionation of cell lysates, 23S rRNAs mutated at G2582 to A and G2583 to C accumulated in the 50S and 70S fractions and were underrepresented in the polysome fraction. Induction of 23S rRNAs mutated at G2582 and G2583 lead to a drastic reduction in cell growth. In addition, mutations G2582A and G2583C reduced to one-third the total protein synthesis but not the RNA synthesis. Finally, we show that 23S rRNA mutations G2582A, G2582U, and G2583C cause a significant increase in peptidyl-tRNA drop-off from ribosomes, thereby reducing translational processivity. The results clearly show that tRNA–23S rRNA interaction has an essential role in maintaining the processivity of translation.     

Alu RNA regulates the cellular pool of active ribosomes by targeted delivery of SRP9/14 to 40S subunits

The human genome contains about 1.5 million Alu elements, which are transcribed into Alu RNAs by RNA polymerase III. Their expression is upregulated following stress and viral infection, and they associate with the SRP9/14 protein dimer in the cytoplasm forming Alu RNPs. Using cell-free translation, we have previously shown that Alu RNPs inhibit polysome formation. Here, we describe the mechanism of Alu RNP-mediated inhibition of translation initiation and demonstrate its effect on translation of cellular and viral RNAs. Both cap-dependent and IRES-mediated initiation is inhibited. Inhibition involves direct binding of SRP9/14 to 40S ribosomal subunits and requires Alu RNA as an assembly factor but its continuous association with 40S subunits is not required for inhibition. Binding of SRP9/14 to 40S prevents 48S complex formation by interfering with the recruitment of mRNA to 40S subunits. In cells, overexpression of Alu RNA decreases translation of reporter mRNAs and this effect is alleviated with a mutation that reduces its affinity for SRP9/14. Alu RNPs also inhibit the translation of cellular mRNAs resuming translation after stress and of viral mRNAs suggesting a role of Alu RNPs in adapting the translational output in response to stress and viral infection.     

The regulation of hepatic protein synthesis during fasting in the rat

We have studied translational control in the model of 48 h of fasting in the rat. Our initial observations showed a paradoxical increase in ribosomal protein S6 (rpS6) phosphorylation and a decrease in eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation. These effects, which would favor an increase in protein synthesis, could be attributed to increased circulating concentrations of branched-chain amino acids in fasting. To determine what mechanisms might account for decreased hepatic translation in fasting, we examined the cap binding complex. eIF4E-bound 4E-BP1 did not increase. However, eIF4E-bound eIF4G and total cellular eIF4G were profoundly decreased in fasted liver. eIF4G mRNA levels were not lower after fasting. Based on the hypothesis that decreased eIF4G translation might account for the reduced eIF4G content, we fractionated ribosomes by sucrose density centrifugation. Immunoblotting for rpS6 showed modest polysomal disaggregation upon fasting. PCR analysis of polysome profiles revealed that a spectrum of mRNAs undergo different translational regulation in the fasted state. In particular, eIF4G was minimally affected by fasting. This indicated that reduced eIF4G abundance in fasting may be a function of its stability, whereas its recovery upon refeeding is necessarily independent of its own involvement in the cap binding complex. Western immunoblotting of polysome fractions showed that phosphorylated rpS6 was disproportionately present in translating polysomes in fed and fasted animals, consistent with a role in translational control. However, the translation of rpS8, an mRNA with a 5'-oligopyrimidine tract, did not coincide with rpS6 phosphorylation, thus dissociating rpS6 phosphorylation from the translational control of this subset of mRNAs.