Polysomally protected viruses.

It is conceivable that an RNA virus could use a polysome, that is, a string of ribosomes covering the RNA strand, to protect the genetic material from degradation inside a host cell. This paper discusses how such a virus might operate, and how its presence might be detected by ribosome profiling. There are two possible forms for such apolysomally protected virus, depending upon whether just the forward strand or both the forward and complementary strands can be encased by ribosomes (these will be termed type 1 and type 2, respectively). It is argued that in the type 2 case the viral RNA would evolve anambigrammaticproperty, whereby the viral genes are free of stop codons in a reverse reading frame (with forward and reverse codons aligned). Recent observations of ribosome profiles of ambigrammatic narnavirus sequences are consistent with our predictions for the type 2 case.

The effects of polydisperse crowders on the compaction of the Escherichia coli nucleoid.

DNA binding proteins, supercoiling, macromolecular crowders, and transient DNA attachments to the cell membrane have all been implicated in the organization of the bacterial chromosome. However, it is unclear what role these factors play in compacting the bacterial DNA into a distinct organelle-like entity, the nucleoid. By analyzing the effects of osmotic shock and mechanical squeezing on Escherichia coli, we show that macromolecular crowders play a dominant role in the compaction of the DNA into the nucleoid. We find that a 30% increase in the crowder concentration from physiological levels leads to a three-fold decrease in the nucleoid's volume. The compaction is anisotropic, being higher along the long axes of the cell at low crowding levels. At higher crowding levels, the nucleoid becomes spherical, and its compressibility decreases significantly. Furthermore, we find that the compressibility of the nucleoid is not significantly affected by cell growth rates and by prior treatment with rifampicin. The latter results point out that in addition to poly ribosomes, soluble cytoplasmic proteins have a significant contribution in determining the size of the nucleoid. The contribution of poly ribosomes dominates at faster and soluble proteins at slower growth rates.

Proteomic analysis of polyribosomes identifies splicing factors as potential regulators of translation during mitosis.

Precise regulation of mRNA translation is critical for proper cell division, but little is known about the factors that mediate it. To identify mRNA-binding proteins that regulate translation during mitosis, we analyzed the composition of polysomes from interphase and mitotic cells using unbiased quantitative mass-spectrometry (LC-MS/MS). We found that mitotic polysomes are enriched with a subset of proteins involved in RNA processing, including alternative splicing and RNA export. To demonstrate that these may indeed be regulators of translation, we focused on heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a test case and confirmed that it is recruited to elongating ribosomes during mitosis. Then, using a combination of pulsed SILAC, metabolic labeling and ribosome profiling, we showed that knockdown of hnRNP C affects both global and transcript-specific translation rates and found that hnRNP C is specifically important for translation of mRNAs that encode ribosomal proteins and translation factors. Taken together, our results demonstrate how proteomic analysis of polysomes can provide insight into translation regulation under various cellular conditions of interest and suggest that hnRNP C facilitates production of translation machinery components during mitosis to provide daughter cells with the ability to efficiently synthesize proteins as they enter G1 phase.

Hybrid agent-based model for quantitative in-silico cell-free protein synthesis.

An advanced vision of the mRNA translation is presented through a hybrid modeling approach. The dynamics of the polysome formation was investigated by computer simulation that combined agent-based model and fine-grained Markov chain representation of the chemical kinetics. This approach allowed for the investigation of the polysome dynamics under non-steady-state and non-continuum conditions. The model is validated by the quantitative comparison of the simulation results and Luciferase protein production in cell-free system, as well as by testing of the hypothesis regarding the two possible mechanisms of the Edeine antibiotic. Calculation of the Hurst exponent demonstrated a relationship between the microscopic properties of amino acid elongation and the fractal dimension of the translation duration time series. The temporal properties of the amino acid elongation have indicated an anti-persistent behavior under low mRNA occupancy and evinced the appearance of long range interactions within the mRNA-ribosome system for high ribosome density. The dynamic and temporal characteristics of the polysomal system presented here can have a direct impact on the studies of the co-translation protein folding and provide a validated platform for cell-free system studies.

Reactivation of stalled polyribosomes in synaptic plasticity.

Some forms of synaptic plasticity require rapid, local activation of protein synthesis. Although this is thought to reflect recruitment of mRNAs to free ribosomes, this would limit the speed and magnitude of translational activation. Here we provide compelling in situ evidence supporting an alternative model in which synaptic mRNAs are transported as stably paused polyribosomes. Remarkably, we show that metabotropic glutamate receptor activation allows the synthesis of proteins that lead to a functional long-term depression phenotype even when translation initiation has been greatly reduced. Thus, neurons evolved a unique mechanism to swiftly translate synaptic mRNAs into functional protein upon synaptic signaling using stalled polyribosomes to bypass the rate-limiting step of translation initiation. Because dysregulated plasticity is implicated in neurodevelopmental and psychiatric disorders such as fragile X syndrome, this work uncovers a unique translational target for therapies.

Polysome profiling shows extensive posttranscriptional regulation during human adipocyte stem cell differentiation into adipocytes.

Adipocyte stem cells (hASCs) can proliferate and self-renew and, due to their multipotent nature, they can differentiate into several tissue-specific lineages, making them ideal candidates for use in cell therapy. Most attempts to determine the mRNA profile of self-renewing or differentiating stem cells have made use of total RNA for gene expression analysis. Several lines of evidence suggest that self-renewal and differentiation are also dependent on the control of protein synthesis by posttranscriptional mechanisms. We used adipogenic differentiation as a model, to investigate the extent to which posttranscriptional regulation controlled gene expression in hASCs. We focused on the initial steps of differentiation and isolated both the total mRNA fraction and the subpopulation of mRNAs associated with translating ribosomes. We observed that adipogenesis is committed in the first days of induction and three days appears as the minimum time of induction necessary for efficient differentiation. RNA-seq analysis showed that a significant percentage of regulated mRNAs were posttranscriptionally controlled. Part of this regulation involves massive changes in transcript untranslated regions (UTR) length, with differential extension/reduction of the 3'UTR after induction. A slight correlation can be observed between the expression levels of differentially expressed genes and the 3'UTR length. When we considered association to polysomes, this correlation values increased. Changes in the half lives were related to the extension of the 3'UTR, with longer UTRs mainly stabilizing the transcripts. Thus, changes in the length of these extensions may be associated with changes in the ability to associate with polysomes or in half-life.

Fear learning increases the number of polyribosomes associated with excitatory and inhibitory synapses in the barrel cortex.

Associative fear learning, resulting from whisker stimulation paired with application of a mild electric shock to the tail in a classical conditioning paradigm, changes the motor behavior of mice and modifies the cortical functional representation of sensory receptors involved in the conditioning. It also induces the formation of new inhibitory synapses on double-synapse spines of the cognate barrel hollows. We studied density and distribution of polyribosomes, the putative structural markers of enhanced synaptic activation, following conditioning. By analyzing serial sections of the barrel cortex by electron microscopy and stereology, we found that the density of polyribosomes was significantly increased in dendrites of the barrel activated during conditioning. The results revealed fear learning-induced increase in the density of polyribosomes associated with both excitatory and inhibitory synapses located on dendritic spines (in both single- and double-synapse spines) and only with the inhibitory synapses located on dendritic shafts. This effect was accompanied by a significant increase in the postsynaptic density area of the excitatory synapses on single-synapse spines and of the inhibitory synapses on double-synapse spines containing polyribosomes. The present results show that associative fear learning not only induces inhibitory synaptogenesis, as demonstrated in the previous studies, but also stimulates local protein synthesis and produces modifications of the synapses that indicate their potentiation.

Fear and safety learning differentially affect synapse size and dendritic translation in the lateral amygdala.

Fear learning is associated with changes in synapse strength in the lateral amygdala (LA). To examine changes in LA dendritic spine structure with learning, we used serial electron microscopy to re-construct dendrites after either fear or safety conditioning. The spine apparatus, a smooth endoplasmic reticulum (sER) specialization found in very large spines, appeared more frequently after fear conditioning. Fear conditioning was associated with larger synapses on spines that did not contain a spine apparatus, whereas safety conditioning resulted in smaller synapses on these spines. Synapses on spines with a spine apparatus were smaller after safety conditioning but unchanged with fear conditioning, suggesting a ceiling effect. There were more polyribosomes and multivesicular bodies throughout the dendrites from fear conditioned rats, indicating increases in both protein synthesis and degradation. Polyribosomes were associated with the spine apparatus under both training conditions. We conclude that LA synapse size changes bidirectionally with learning and that the spine apparatus has a central role in regulating synapse size and local translation.

Characterization of the Trypanosoma cruzi ortholog of the SBDS protein reveals an intrinsically disordered extended C-terminal region showing RNA-interacting activity.

The human SBDS gene and its yeast ortholog SDO1 encode essential proteins that are involved in ribosome biosynthesis. SDO1 has been implicated in recycling of the ribosomal biogenesis factor Tif6p from pre-66S particles as well as in translation activation of 60S ribosomes. The SBDS protein is highly conserved, containing approximately 250 amino acid residues in animals, fungi and Archaea, while SBDS orthologs of plants and a group of protists contain an extended C-terminal region. In this work, we describe the characterization of the Trypanosoma cruzi SBDS ortholog (TcSBDS). TcSBDS co-fractionates with polysomes in sucrose density gradients, which is consistent with a role in ribosome biosynthesis. We show that TcSBDS contains a C-terminal extension of 200 amino acids that displays the features of intrinsically disordered proteins as determined by proteolytic, circular dichroism and NMR analyses. Interestingly, the C-terminal extension is responsible for TcSBDS-RNA interaction activity in electrophoretic mobility shift assays. This finding suggests that Trypanosomatidae and possibly also other organisms containing SBDS with extended C-terminal regions have evolved an additional function for SBDS in ribosome biogenesis.

The discovery of polyribosomes.

By the early 1960s, evidence had accumulated that proteins were synthesized from special RNA copies of genes, named "messenger RNAs" (mRNAs), not directly from the stable RNAs found in the ribosomes of the cytoplasm. Yet, precisely how the protein chains were assembled along the RNA and, in particular, the relationship between the mRNAs and the ribosomes during protein synthesis, was obscure. In this account, I discuss how my laboratory found that multiple ribosomes traverse each mRNA, yielding the structures known as polysomes. This work led on to the first physical determination of the coding ratio, new insights into how protein chains are initiated, and an early suggestion that chloroplasts and mitochondria in eukaryotic cells might ultimately have been derived from symbiotic bacteria.

Phosphorylation of eIF4E by MNKs supports protein synthesis, cell cycle progression and proliferation in prostate cancer cells.

Deregulation of the phosphatidyl inositol trisphosphate kinase/AKT/mammalian target of rapamycin (mTOR) and RAS/mitogen-activated protein kinase (MAPK)/MNK pathways frequently occurs in human prostate carcinomas (PCas) and leads to aberrant modulation of messenger RNA (mRNA) translation. We have investigated the relative contribution of these pathways to translational regulation and proliferation of PCa cells. MNK-dependent phosphorylation of eIF4E is elevated in DU145 cells, which have low basal levels of AKT/mTOR activity due to the expression of the tumor suppressor PTEN. In contrast, eIF4E phosphorylation is low in PC3 and LNCaP cells with mutated PTEN and constitutively active AKT/mTOR pathway, but it can be strongly induced through inhibition of mTOR activity by rapamycin or serum depletion. Remarkably, we found that inhibition of MNKs strongly reduced the polysomal recruitment of terminal oligopyrimidine messenger RNAs (TOP mRNAs), which are known targets of mTOR-dependent translational control. Pull-down assays of the eIF4F complex indicated that translation initiation was differently affected by inhibition of MNKs and mTOR. In addition, concomitant treatment with MNK inhibitor and rapamycin exerted additive effects on polysomal recruitment of TOP mRNAs and protein synthesis. The MNK inhibitor was more effective than rapamycin in blocking proliferation of PTEN-expressing cells, whereas combination of the two inhibitors suppressed cell cycle progression in both cell lines. Microarray analysis showed that MNK affected translation of mRNAs involved in cell cycle progression. Thus, our results indicate that a balance between the activity of the AKT/mTOR and the MAPK/MNK pathway in PCa cells maintains a defined translational level of specific mRNAs required for ribosome biogenesis, cell proliferation and stress response and might confer to these cells the ability to overcome negative insults.

Translational activation of 5'-TOP mRNA in pressure overload myocardium.

The present study was conducted to determine the magnitude and duration of ribosomal protein translation in response to pressure overload and determine if additional, paracrine events associated with mechanical transduction, such as integrin activation using a bioactive peptide ligand, RGD or endothelin stimulation lead to ribosomal protein translation. Polysome analysis of ventricular tissue samples obtained from an in vivo model of right-ventricular pressure overload (RVPO) showed a significant shift in the proportion of a 5'-terminal oligopyrimidine (5'-TOP) mRNA, rpL32, associated with the polysomal fraction when compared with non-5'-TOP mRNAs, beta-actin and beta-myosin heavy chain (beta-MHC), in the early stages of the hypertrophic response (24-48 h). Furthermore, this increase in polysome-bound rpL32 mRNA was accompanied by the phosphorylation of mammalian target of rapamycin (mTOR), p70 S6 kinase (S6K1), and S6 ribosomal protein. In our in vitro studies, treatment of primary cultures of adult feline cardiomyocytes (cardiocytes) with 100 nM endothelin, 9 mM RGD, 100 nM insulin, or 100 nM TPA activated mTOR via distinct signaling pathways and resulted in an increased proportion of polysome-bound rpL32 mRNA. Pre-treatment of cardiocytes with the mTOR inhibitor rapamycin blocked the agonist-induced rpL32 mRNA mobilization to polysomes. These results show that mechanisms that regulate ribosomal biogenesis in the myocardium are dynamically sensitive to pressure overload. Furthermore, our in vitro studies indicate that distinct pathways are operational during the early course of hypertrophic growth and converge to activate mTOR resulting in the translational activation of 5'-TOP mRNA.

Yeast phenotypic assays on translational control.

This chapter describes phenotypic assays on specific and general aspects of translation using yeast Saccharomyces cerevisiae as a model eukaryote. To study the effect on start codon selection stringency, a his4(-) or his4-lacZ allele altering the first AUG to AUU is employed. Mutations relaxing the stringent selection confer the His(+) phenotype in the his4(-) strain background or increase expression from his4-lacZ compared to that from wild-type HIS4-lacZ (Sui(-) phenotype). Translation of the Gcn4p transcription activator is strictly regulated by amino acid availability depending on upstream ORF (uORF) elements in the GCN4 mRNA leader. Mutations reducing the eIF2/GTP/Met-tRNA(i)(Met) complex level or the rate of its binding to the 40S subunit derepress GCN4 translation by allowing ribosomes to bypass inhibitory uORFs in the absence of the starvation signal (Gcd(-) phenotype). Mutations impairing scanning or AUG recognition generally impair translational GCN4 induction during amino acid starvation (Gcn(-) phenotype). Different amino acid analogs or amino acid enzyme inhibitors are used to study Gcd(-) or Gcn(-) phenotypes. The method of polysome profiling is also described to gain an ultimate "phenotypic" proof for translation defects.

In vivo stabilization of preinitiation complexes by formaldehyde cross-linking.

Translation initiation starts with the formation of the 43S preinitiation complex (PIC) consisting of several soluble factors, including the ternary complex (TC; elF2-GTP-Met-tRNA(i)(Met)), which associate with the small ribosomal subunit. In the next step, mRNA is recruited to form the 48S PIC and the entire machinery starts scanning the 5' untranslated region of the mRNA until the AUG start codon is encountered. The most widely used method to separate 40S and 60S ribosomal subunits from soluble factors, monosomes and polysomes, is sucrose density centrifugation (SDC). Since PICs are intrinsically unstable complexes that cannot withstand the forces imposed by SDC, a stabilization agent must be employed to detect the association of factors with the 40S subunit after SDC. This was initially achieved by adding heparin (a highly sulfated glycosaminoglycan) directly to the breaking buffer of cells treated with cycloheximide (a translation elongation inhibitor). However, the mechanism of stabilization is not understood and, moreover, there are indications that the use of heparin may lead to artifactual factor associations that do not reflect the factor occupancy of the 43S/48S PICs in the cell at the time of lysis. Therefore, we developed an alternative method for PIC stabilization using formaldehyde (HCHO) to cross-link factors associated with 40S ribosomal subunits in vivo before the disruption of the yeast cells. Results obtained using HCHO stabilization strongly indicate that the factors detected on the 43S/48S PIC after SDC approximate a real-time in vivo "snapshot" of the 43S/48S PIC composition. In this chapter, we will present the protocol for HCHO cross-linking in detail and demonstrate the difference between heparin and HCHO stabilization procedures. In addition, different conditions for displaying the polysome profile or PIC analysis by SDC, used to address different questions, will be outlined.

Molecular genetic structure-function analysis of translation initiation factor eIF5B.

Recently, significant progress has been made in obtaining three-dimensional (3-D) structures of the factors that promote translation initiation, elongation, and termination. These structures, when interpreted in light of previous biochemical characterizations of the factors, provide significant insight into the function of the factors and the molecular mechanism of specific steps in the translation process. In addition, genetic analyses in yeast have helped elucidate the in vivo roles of the factors in various steps of the translation pathway. We have combined these two approaches and use molecular genetic studies to define the structure-function properties of translation initiation factors in the yeast Saccharomyces cerevisiae. In this chapter, we describe our multistep approach in which we first characterize a site-directed mutant of the factor of interest using in vivo and in vitro assays of protein synthesis. Next, we subject the mutant gene to random mutagenesis and screen for second-site mutations that restore the factor's function in vivo. Following biochemical and in vivo characterization of the suppressor mutant, we interpret the results in light of the 3-D structure of the factor to define the structure-function properties of the factor and to provide new molecular insights into the mechanism of translation.

Cardiovirus 2A protein associates with 40S but not 80S ribosome subunits during infection.

Host translation shutoff induced in picornavirus-infected cells is a well-known phenomenon. The mechanisms by which separate genera of the picornavirus family achieve this shutoff differ. This study examined alterations in the cellular translational components in HeLa cells infected with encephalomyocarditis virus (EMCV), a cardiovirus. In agreement with previous reports, EMCV induced a marked decrease in host mRNA translation. The inhibition correlated with the appearance of a significantly enhanced 80S peak in cells and a concomitant decrease in polysome abundance. Characterization of the 80S material revealed that these ribosomes were virtually devoid of mRNA. Viral protein 2A was tightly associated with some of the free 40S ribosome subunits, but it was not present in the 80S pool which accumulated after infection. Expression of 2A protein in cells in the absence infection was able to modulate the cellular translational environment to increase the ratio of internal ribosome entry site-dependent translation to cap-dependent translation of a reporter construct. The results provide further evidence for a role of 2A protein in the mechanism of cardiovirus-induced host translational shutoff.

On the functions of the h subunit of eukaryotic initiation factor 3 in late stages of translation initiation.

BACKGROUND: The eukaryotic translation initiation factor 3 (eIF3) has multiple roles during the initiation of translation of cytoplasmic mRNAs. How individual subunits of eIF3 contribute to the translation of specific mRNAs remains poorly understood, however. This is true in particular for those subunits that are not conserved in budding yeast, such as eIF3h. RESULTS: Working with stable reporter transgenes in Arabidopsis thaliana mutants, it was demonstrated that the h subunit of eIF3 contributes to the efficient translation initiation of mRNAs harboring upstream open reading frames (uORFs) in their 5' leader sequence. uORFs, which can function as devices for translational regulation, are present in over 30% of Arabidopsis mRNAs, and are enriched among mRNAs for transcriptional regulators and protein modifying enzymes. Microarray comparisons of polysome loading in wild-type and eif3h mutant seedlings revealed that eIF3h generally helps to maintain efficient polysome loading of mRNAs harboring multiple uORFs. In addition, however, eIF3h also boosted the polysome loading of mRNAs with long leaders or coding sequences. Moreover, the relative polysome loading of certain functional groups of mRNAs, including ribosomal proteins, was actually increased in the eif3h mutant, suggesting that regulons of translational control can be revealed by mutations in generic translation initiation factors. CONCLUSION: The intact eIF3h protein contributes to efficient translation initiation on 5' leader sequences harboring multiple uORFs, although mRNA features independent of uORFs are also implicated.

Human let-7a miRNA blocks protein production on actively translating polyribosomes.

MicroRNAs (miRNAs) regulate gene expression at a post-transcriptional level through base-pairing to 3' untranslated regions (UTRs) of messenger RNAs. The mechanism by which human let-7a miRNA regulates mRNA translation was examined in HeLa cells expressing reporter mRNAs containing the Caenorhabditis elegans lin-41 3' UTR. let-7a miRNA strongly repressed translation, yet the majority of control and lin-41-bearing RNAs sedimented with polyribosomes in sucrose gradients; these polyribosomes, together with let-7a miRNA and the miRISC protein AGO, were released from those structures by puromycin. RNA containing the lin-41 3' UTR and an iron response element in the 5' UTR sedimented with polysomes when cells were incubated with iron, but showed ribosome run-off when the iron was chelated. These data indicate that let-7a miRNA inhibits actively translating polyribosomes. Nascent polypeptide coimmunoprecipitation experiments further suggest that let-7a miRNA interferes with the accumulation of growing polypeptides.

Visualization of mRNA translation in living cells.

The role of mRNA localization is presumably to effect cell asymmetry by synthesizing proteins in specific cellular compartments. However, protein synthesis has never been directly demonstrated at the sites of mRNA localization. To address this, we developed a live cell method for imaging translation of beta-actin mRNA. Constructs coding for beta-actin, containing tetracysteine motifs, were transfected into C2C12 cells, and sites of nascent polypeptide chains were detected using the biarsenial dyes FlAsH and ReAsH, a technique we call translation site imaging. These sites colocalized with beta-actin mRNA at the leading edge of motile myoblasts, confirming that they were translating. beta-Actin mRNA lacking the sequence (zipcode) that localizes the mRNA to the cell periphery, eliminated the translation there. A pulse-chase experiment on living cells showed that the recently synthesized protein correlated spatially with the sites of its translation. Additionally, localization of beta-actin mRNA and translation activity was enhanced at cell contacts and facilitated the formation of intercellular junctions.

Involvement of DnaK3, one of the three DnaK proteins of cyanobacterium Synechococcus sp. PCC7942, in translational process on the surface of the thylakoid membrane.

The Synechococcus sp. PCC7942 strain carrying a missense mutation in the peptide-binding domain of DnaK3, one of the essential dnaK gene products, revealed temperature-sensitive growth. We also isolated suppressor mutants of this strain. One of the suppressors was mapped in the ribosomal protein gene rpl24 (syc1876), which encodes the 50S ribosomal protein L24. Subcellular localization of three DnaK proteins was determined, and the results indicated that a quantity of DnaK3 was dislocated from membrane-bound polysomes when dnaK3 temperature-sensitive mutant was incubated at non-permissive temperatures. Furthermore, we examined the photosystem II reaction center protein D1 and detected a translational intermediate polypeptide in membrane-bound polysome fractions prepared from dnaK3 temperature-sensitive cells grown at high temperature. These characteristic features of DnaK3 localizations and detection of D1 protein intermediate were not observed in the suppressor mutant even at high temperatures.