mRNA Translation Regulation by the Gly-Ala Repeat of Epstein-Barr Virus Nuclear Antigen 1

The glycine-alanine repeat (GAr) sequence of the Epstein-Barr virus-encoded EBNA-1 prevents presentation of antigenic peptides to major histocompatibility complex class I molecules. This has been attributed to its capacity to suppress mRNA translation in cis. However, the underlying mechanism of this function remains largely unknown. Here, we have further investigated the effect of the GAr as a regulator of mRNA translation. Introduction of silent mutations in each codon of a 30-amino-acid GAr sequence does not significantly affect the translation-inhibitory capacity, whereas minimal alterations in the amino acid composition have strong effects, which underscores the observation that the amino acid sequence and not the mRNA sequence mediates GAr-dependent translation suppression. The capacity of the GAr to repress translation is dose and position dependent and leads to a relative accumulation of preinitiation complexes on the mRNA. Taken together with the surprising observation that fusion of the 5′ untranslated region (UTR) of the c-myc mRNA to the 5′ UTR of GAr-carrying mRNAs specifically inactivates the effect of the GAr, these results indicate that the GAr targets components of the translation initiation process. We propose a model in which the nascent GAr peptide delays the assembly of the initiation complex on its own mRNA.Epstein-Barr Virus (EBV) nuclear antigen 1 (EBNA-1) and latency-associated nuclear antigen 1 (LANA-1), from Kaposi''s sarcoma-associated herpesvirus (KSHV), are major latency proteins of these two gammaherpesviruses that are essential for maintaining viral episomes in infected cells (21, 22). Independent studies suggest that both proteins have evolved mechanisms to remain largely invisible to the immune system, which could otherwise eliminate latently infected cells (8, 9, 19, 25). These mechanisms act in cis and are mediated via an internal repeat region. In the case of EBNA-1 this region consists of an N-terminal glycine-alanine repeat (GAr), and for LANA-1 the region consists of a glutamine-glutamate-aspartate central repeat (QED-CR). Although the two domains do not share amino acid homology, both retard their own synthesis to reduce the production of defective ribosomal products that can be processed for the major histocompatibility complex (MHC) class I-restricted antigen presentation pathway (23, 24), highlighting the importance of translation control in regulating MHC class I-restricted antigen presentation. To compensate for their low rates of synthesis, both proteins also have slow turnover rates (4, 8).Regulation of translation for most prokaryotic and eukaryotic mRNAs occurs at the level of initiation, but there are examples where regulation of protein synthesis depends on the elongation stage (17). The two main types of translation initiation are the classic cap-dependent and the less frequent cap-independent translation mechanisms (5, 7, 11, 14, 16). In the former, the preinitiation complex is formed around the cap structure in the 5′ untranslated region (UTR) of the message, whereas in the latter the 40S subunit is directed toward the mRNA via an internal ribosome entry site (IRES). The mechanism of GAr- and LANA-1-mediated control of translation seems different from other types of viral regulation in several aspects. The EBNA-1 GAr is 60 to 300 amino acids long, depending on virus isolate, and is positioned in the N-terminal part of the protein. The GAr message is GC rich but does not activate protein kinase R and eukaryotic initiation factor 2α phosphorylation (25). The fact that the GAr has to be encoded to suppress translation, coupled with the restricted use of GGG and GGA codons to express Gly and of GCA to express Ala in the GAr (GAT, GAG, and CAG for aspartic acid, glutamic acid, and glutamine, respectively, in the LANA sequence), could suggest that codon exhaustion might explain the effect of these repeats. However, manipulations of sequence order, orientation, and composition of the QED-CR and GAr domains and the observation that antibodies directed toward the GAr can stimulate translation in vitro instead favor a direct role for the amino acid sequence (8, 25).Here, we have studied GAr-mediated regulation of translation in vitro and in vivo. The results presented suggest that, once synthesized, the nascent GAr peptide sequence prevents the assembly of the following upstream ribosomes. This knowledge should further understanding of how amino acid repeat sequences can affect mRNA translation in cis and should shed light on a novel type of viral control of mRNA translation and its implications in regulating MHC class I-restricted antigen presentation.     

Mammalian end binding proteins control persistent microtubule growth

End binding proteins (EBs) are highly conserved core components of microtubule plus-end tracking protein networks. Here we investigated the roles of the three mammalian EBs in controlling microtubule dynamics and analyzed the domains involved. Protein depletion and rescue experiments showed that EB1 and EB3, but not EB2, promote persistent microtubule growth by suppressing catastrophes. Furthermore, we demonstrated in vitro and in cells that the EB plus-end tracking behavior depends on the calponin homology domain but does not require dimer formation. In contrast, dimerization is necessary for the EB anti-catastrophe activity in cells; this explains why the EB1 dimerization domain, which disrupts native EB dimers, exhibits a dominant-negative effect. When microtubule dynamics is reconstituted with purified tubulin, EBs promote rather than inhibit catastrophes, suggesting that in cells EBs prevent catastrophes by counteracting other microtubule regulators. This probably occurs through their action on microtubule ends, because catastrophe suppression does not require the EB domains needed for binding to known EB partners.     

Tumor-supportive and Osteoclastogenic Changes Induced by Breast Cancer-derived Factors Are Reversed by Inhibition of γ-Secretase

During breast cancer metastasis to bone, tumor cells home to bone marrow, likely targeting the stem cell niche, and stimulate osteoclasts, which mediate osteolysis required for tumor expansion. Although osteoblasts contribute to the regulation of the hematopoietic stem cell niche and control osteoclastogenesis through production of proresorptive cytokine RANKL (receptor activator of NF-κB ligand), their role in cancer metastases to bone is not fully understood. C57BL/6J mouse bone marrow cells were treated for 3–12 days with ascorbic acid (50 μg/ml) in the presence or absence of 10% medium conditioned by breast carcinoma cells MDA-MB-231, 4T1, or MCF7. Treatment with cancer-derived factors resulted in a sustained 40–60% decrease in osteoblast differentiation markers, compared with treatment with ascorbic acid alone, and induced an osteoclastogenic change in the RANKL/osteoprotegerin ratio. Importantly, exposure of bone cells to breast cancer-derived factors stimulated the subsequent attachment of cancer cells to immature osteoblasts. Inhibition of γ-secretase using pharmacological inhibitors DAPT and Compound E completely reversed cancer-induced osteoclastogenesis as well as cancer-induced enhancement of cancer cell attachment, identifying γ-secretase activity as a key mediator of these effects. Thus, we have uncovered osteoblasts as critical intermediary of premetastatic signaling by breast cancer cells and pinpointed γ-secretase as a robust target for developing therapeutics potentially capable of reducing both homing and progression of cancer metastases to bone.     

Effect of noise in processing of visual information

Background

Information transmission and processing in the nervous system has stochastic nature. Multiple factors contribute to neuronal trial-to-trial variability. Noise and variations are introduced by the processes at the molecular and cellular level (thermal noise, channel current noise, membrane potential variations, biochemical and diffusion noise at synapses etc). The stochastic processes are affected by different physical (temperature, electromagnetic field) and chemical (drugs) factors. The aim of this study was experimental investigation of hypotheses that increase in the noise level in the brain affects processing of visual information. Change in the noise level was introduced by an external factor producing excess noise in the brain.

Methods

An exposure to 450 MHz low-frequency modulated microwave radiation was applied to generate excess noise. Such exposure has been shown to increase diffusion, alter membrane resting potential, gating variables and intracellular Calcium efflux. Nine healthy volunteers passed the experimental protocol at the lower (without microwave) and the higher (with microwave) noise level. Two photos (visual stimuli) of unfamiliar, young male faces were presented to the subjects, one picture after another. The task was to identify later the photos from a group of six photos and to decide in which order they were presented. Each subject had a total of eight sessions at the lower and eight at the higher noise level. Each session consisted of 50 trials; altogether a subject made 800 trials, 400 at the lower and 400 at the higher noise level. Student t-test was applied for statistical evaluation of the results.

Results

Correct recognition of both stimuli in the right order was better at the lower noise level. All the subjects under investigation showed higher numbers of right answers in trials at the lower noise level. Average number of correct answers from n=400 trials with microwave exposure was 50.3, without exposure 54.4, difference 7.5%, p<0.002. No difference between results at the lower and the higher noise level was revealed in the case of only partly correct or incorrect answers.

Conclusions

Our experimental results showed that introduced excess noise reduced significantly ability of the nervous system in correct processing of visual information.

     

Semidominant effect of the l(1)ts403 (sbr10) mutation on nondisjunction of sex chromosomes in meiosis in Drosophila melanogaster females exposed to heat

The sbr gene of Drosophila melanogaster belongs to the NXF (nuclear export factor) family responsible for the mRNA transport from nucleus to cytoplasm. We have shown that in the heat-exposed (37 degrees C, 1 h) females, the l(1)ts403 (sbr10) mutation leads, in particular, to the high-frequency nondisjunction and loss of sex chromosomes in meiosis. For this trait, the incomplete dominance of the sbr10 mutation is observed. At the same time, the sbr10 mutation is recessive for many other traits of the heat-exposed flies: reduced viability, low fertility, impaired synthesis of the heat shock proteins, etc. The females heterozygous for the null allele (Df(1)vL4, a deletion eliminating gene srb) do not differ from females homozygous for the wild-type allele in frequency of the heat shock-induced nondisjunction and loss of sex chromosomes in meiosis. Because of this, the sbr10 mutation can be assigned to the gain-of-function alleles (those gaining the dominance function). Expression of the mutant sbr10 allele against the background of the wild-type allele suggests that in the heat shock-exposed females, the heat-modified product of this ts allele has a strong effect on sex chromosome disjunction in meiosis.     

Mutation-selection networks of cancer initiation: tumor suppressor genes and chromosomal instability

In this paper, we derive analytic solutions of stochastic mutation-selection networks that describe early events of cancer formation. A main assumption is that cancer is initiated in tissue compartments, where only a relatively small number of cells are at risk of mutating into cells that escape from homeostatic regulation. In this case, the evolutionary dynamics can be approximated by a low-dimensional stochastic process with a linear Kolmogorov forward equation that can be solved analytically. Most of the time, the cell population is homogeneous with respect to relevant mutations. Occasionally, such homogeneous states are connected by 'stochastic tunnels'. We give a precise analysis of the existence of tunnels and calculate the rate of tunneling. Finally, we calculate the conditions for chromosomal instability (CIN) to precede inactivation of the first tumor suppressor gene. In this case, CIN is an early event and a driving force of cancer progression. The techniques developed in this paper can be used to study arbitrarily complex mutation-selection networks of the somatic evolution of cancer.     

Molecular evolution of rDNA external transcribed spacer and phylogeny of sect. Petota (genus Solanum)

The 5(') external transcribed spacer (ETS) region of ribosomal DNA of 30 species of Solanum sect. Petota and the European Solanum dulcamara were compared. Two structural elements can be distinguished in the ETS: (i). a variable region (VR), demonstrating significant structural rearrangements and (ii). a conservative region (CR), evolving mainly by base substitutions. In VR, a conservative element (CE) with similarity to the ETS of distantly related Nicotiana is present. The ancestral organization of ETS (variant A) was found for non-tuber-bearing species of ser. Etuberosa, tuber-bearing wild potatoes of Central American ser. Bulbocastana, Pinnatisecta, and Polyadenia and S. dulcamara. Duplication of CE took place in the ETS of species from ser. Commersoniana and Circaeifolia (variant B). South American diploids and Mexican polyploids from superser. Rotata also possess two CE, and additionally two duplications around CE1 are present in VR (variant C). Three major lineages could be distinguished: non-tuber-bearing species of ser. Etuberosa, tuber-bearing Central American diploids and all South American species radiated from a common ancestor at early stages of evolution, indicating a South American origin of the tuber-bearing species. Later, Central and South American diploids evolved further as independent lineages. South American species form a monophyletic group composed of series with both stellata and rotata flower morphology. Solanum commersonii represents a sister taxon for all rotata species, whereas ser. Circaeifolia diverged earlier. Two main groups, C1 and C2, may be distinguished for species possessing ETS variant C. C1 contains ser. Megistacroloba, Conicibaccata, Maglia, and Acaulia, whereas all diploids of ser. Tuberosa are combined into C2. A closer relationship of Solanum chacoense (ser. Yungasensa) to the C2 group was found. The origin of polyploid species Solanum maglia, Solanum acaule, Solanum tuberosum, Solanum iopetalum, and Solanum demissum is discussed.     

Contextual Features of Higher Plant mRNA 5"-Untranslated Regions

Computer analysis of nucleotide sequences of 5"-untranslated regions (5"-UTR) of higher plant mRNA adopted from the EMBL nucleotide sequence database was carried out. It was demonstrated that the average nucleotide frequencies of the leader sequences and adjacent regions of basal promoters are similar, whereas introns and 3"-UTR have a higher content of T and a lower content of C. A particular 5"-UTR contextual feature is a misbalance in the content of complementary nucleotides, probably caused by negative influence of the stable secondary structure on the translation properties of the leader sequence. Approximately 20% of 5"-UTR possess AUG triplets, i.e., twice as much as it has been estimated earlier. The properties of the open reading frames of the leader sequence (uORF) and presumable causes of their high content in 5"-UTR of eukaryotic mRNAs are discussed. The nature of correlation between some features of uORFs and protein-coding gene sequences is analyzed. It is demonstrated that in effectively translated mRNAs the leader AUG triplets are more frequently located in a nonoptimal context, whereas the terminating codons of uORFs more frequently exist in the optimal one. A hypothesis is put forward that the efficiency of termination at the uORF stop codon might substantially interfere with the mRNA translation activity.     

Dark recovery of diploid yeast cells after simultaneous exposure to UV-irradiation and hyperthermia

Quantitative regularities of dark recovery of wild-type diploid yeast cells of Saccharomyces cerevisiae simultaneously treated with UV-light (254 nm) and high temperatures (53-56 degrees C) were studied. Under this combined action, the constant of recovery, which defines the probability of elimination of the UV-radiation induced damage per unit of time, did not depend on the temperature of irradiation. It was shown that both the irreversible component of cell damage and the number of cells that died without division gradually increased as the temperature of exposure increased. It is concluded, on this basis, that the mechanism of synergistic interaction of UV-radiation and hyperthermia is related not to the inhibition of dark recovery itself, but to the increase in the shape of irreversibly damaged cells incapable of recovering from the induced damage.