Short tRNA anticodon stem and mutant eRF1 allow stop codon reassignment.

Cognate tRNAs deliver specific amino acids to translating ribosomes according to the standard genetic code, and three codons with no cognate tRNAs serve as stop codons. Some protists have reassigned all stop codons as sense codons, neglecting this fundamental principle1-4. Here we analyse the in-frame stop codons in 7,259 predicted protein-coding genes of a previously undescribed trypanosomatid, Blastocrithidia nonstop. We reveal that in this species in-frame stop codons are underrepresented in genes expressed at high levels and that UAA serves as the only termination codon. Whereas new tRNAsGlu fully cognate to UAG and UAA evolved to reassign these stop codons, the UGA reassignment followed a different path through shortening the anticodon stem of tRNATrpCCA from five to four base pairs (bp). The canonical 5-bp tRNATrp recognizes UGG as dictated by the genetic code, whereas its shortened 4-bp variant incorporates tryptophan also into in-frame UGA. Mimicking this evolutionary twist by engineering both variants from B. nonstop, Trypanosoma brucei and Saccharomyces cerevisiae and expressing them in the last two species, we recorded a significantly higher readthrough for all 4-bp variants. Furthermore, a gene encoding B. nonstop release factor 1 acquired a mutation that specifically restricts UGA recognition, robustly potentiating the UGA reassignment. Virtually the same strategy has been adopted by the ciliate Condylostoma magnum. Hence, we describe a previously unknown, universal mechanism that has been exploited in unrelated eukaryotes with reassigned stop codons.

Effect of elevated temperature and hydrocortisone addition on the proliferation of fibroblasts.

Hyperthermia along with hydrocortisone (HC) are proven teratogens that can negatively influence embryo development during early pregnancy. Proliferation of cells is one of the main developmental processes during the early embryogenesis. This study was focused on testing the effect of elevated temperature and HC addition on proliferation of cells in in vitro cultures. The V79-4 cell line was treated with HC and cultured in vitro at 37 °C or 39 °C, respectively. To reveal the effect of both factors, the proliferation of cells cultured under different conditions was evaluated using various approaches (colony formation assay, generation of growth curves, computation of doubling times, and mitotic index estimation). Our results indicate that a short-term exposure to elevated temperature slightly stimulates and a long-term exposure suppresses cell proliferation. However, HC (0.1 mg/ml) acts as a stimulator of cell proliferation. Interestingly, the interaction of HC and long-term elevated temperature (39 °C) exposure results in at least partial compensation of the negative impact of elevated temperature by HC addition and in higher proliferation if compared with cells cultured at 39 °C without addition of HC.

Yeast applied readthrough inducing system (YARIS): an invivo assay for the comprehensive study of translational readthrough.

Stop codon readthrough-the decoding of a stop codon by a near-cognate tRNA-is employed by viruses to balance levels of enzymatic and structural proteins and by eukaryotic cells to enable isoform-specific protein synthesis in response to external stimuli. Owing to the prevalence of premature termination codons in human disease, readthrough has emerged as an attractive therapeutic target. A growing list of various features, for example the +4 nucleotide immediately following the stop codon, modulate readthrough levels, underscoring the need for systematic investigation of readthrough. Here, we identified and described a complete group of yeast tRNAs that induce readthrough in the stop-codon tetranucleotide manner when overexpressed, designated readthrough-inducing tRNAs (rti-tRNAs). These rti-tRNAs are the keystones of YARIS (yeast applied readthrough inducing system), a reporter-based assay enabling simultaneous detection of readthrough levels at all twelve stop-codon tetranucleotides and as a function of the complete set of rti-tRNAs. We demonstrate the utility of YARIS for systematic study of translation readthrough by employing it to interrogate the effects of natural rti-tRNA modifications, as well as various readthrough-inducing drugs (RTIDs). This analysis identified a variety of genetic interactions demonstrating the power of YARIS to characterize existing and identify novel RTIDs.

Embraced by eIF3: structural and functional insights into the roles of eIF3 across the translation cycle.

Protein synthesis is mediated via numerous molecules including the ribosome, mRNA, tRNAs, as well as translation initiation, elongation and release factors. Some of these factors play several roles throughout the entire process to ensure proper assembly of the preinitiation complex on the right mRNA, accurate selection of the initiation codon, errorless production of the encoded polypeptide and its proper termination. Perhaps, the most intriguing of these multitasking factors is the eukaryotic initiation factor eIF3. Recent evidence strongly suggests that this factor, which coordinates the progress of most of the initiation steps, does not come off the initiation complex upon subunit joining, but instead it remains bound to 80S ribosomes and gradually falls off during the first few elongation cycles to: (1) promote resumption of scanning on the same mRNA molecule for reinitiation downstream-in case of translation of upstream ORFs short enough to preserve eIF3 bound; or (2) come back during termination on long ORFs to fine tune its fidelity or, if signaled, promote programmed stop codon readthrough. Here, we unite recent structural views of the eIF3-40S complex and discus all known eIF3 roles to provide a broad picture of the eIF3's impact on translational control in eukaryotic cells.

Centromeres Off the Hook: Massive Changes in Centromere Size and Structure Following Duplication of CenH3 Gene in Fabeae Species.

In most eukaryotes, centromere is determined by the presence of the centromere-specific histone variant CenH3. Two types of chromosome morphology are generally recognized with respect to centromere organization. Monocentric chromosomes possess a single CenH3-containing domain in primary constriction, whereas holocentric chromosomes lack the primary constriction and display dispersed distribution of CenH3. Recently, metapolycentric chromosomes have been reported in Pisum sativum, representing an intermediate type of centromere organization characterized by multiple CenH3-containing domains distributed across large parts of chromosomes that still form a single constriction. In this work, we show that this type of centromere is also found in other Pisum and closely related Lathyrus species, whereas Vicia and Lens genera, which belong to the same legume tribe Fabeae, possess only monocentric chromosomes. We observed extensive variability in the size of primary constriction and the arrangement of CenH3 domains both between and within individual Pisum and Lathyrus species, with no obvious correlation to genome or chromosome size. Search for CenH3 gene sequences revealed two paralogous variants, CenH3-1 and CenH3-2, which originated from a duplication event in the common ancestor of Fabeae species. The CenH3-1 gene was subsequently lost or silenced in the lineage leading to Vicia and Lens, whereas both genes are retained in Pisum and Lathyrus. Both of these genes appear to have evolved under purifying selection and produce functional CenH3 proteins which are fully colocalized. The findings described here provide the first evidence for a highly dynamic centromere structure within a group of closely related species, challenging previous concepts of centromere evolution.

Sprouty gene dosage influences temporal-spatial dynamics of primary enamel knot formation.

BACKGROUND: The mouse embryonic mandible comprises two types of tooth primordia in the cheek region: progressive tooth primordia of prospective functional teeth and rudimentary tooth primordia in premolar region - MS and R2. Mice lacking Sprouty genes develop supernumerary tooth in front of the lower M1 (first molar) primordium during embryogenesis. We focused on temporal-spatial dynamics of Sonic Hedgehog expression as a marker of early odontogenesis during supernumerary tooth development. RESULTS: Using mouse embryos with different dosages of Spry2 and Spry4 genes, we showed that during the normal development of M1 in the mandible the sooner appearing Shh signaling domain of the R2 bud transiently coexisted with the later appearing Shh expression domain in the early M1 primordium. Both domains subsequently fused together to form the typical signaling center representing primary enamel knot (pEK) of M1 germ at embryonic day (E) 14.5. However, in embryos with lower Spry2;Spry4 gene dosages, we observed a non-fusion of original R2 and M1 Shh signaling domains with consequent formation of a supernumerary tooth primordium from the isolated R2 bud. CONCLUSIONS: Our results bring new insight to the development of the first lower molar of mouse embryos and define simple tooth unit capable of individual development, as well as determine its influence on normal and abnormal development of the tooth row which reflect evolutionarily conserved tooth pattern. Our findings contribute significantly to existing knowledge about supernumerary tooth formation.

Pharmacological effects of mTORC1/C2 inhibitor in a preclinical model of NASH progression.

Knowledge of the benefits of mTOR inhibition concerning adipogenesis and inflammation has recently encouraged the investigation of a new generation of mTOR inhibitors for non-alcoholic steatohepatitis (NASH). We investigated whether treatment with a specific mTORC1/C2 inhibitor (Ku-0063794; KU) exerted any beneficial impacts on experimentally-induced NASH in vitro and in vivo. The results indicated that KU decreases palmitic acid-induced lipotoxicity in cultivated primary hepatocytes, thus emerging as a successful candidate for testing in an in vivo NASH dietary model, which adopted the intraperitoneal KU dosing route rather than oral application due to its significantly greater bioavailability in mice. The pharmacodynamics experiments commenced with the feeding of male C57BL/6 mice with a high-fat atherogenic western-type diet (WD) for differing intervals over several weeks aimed at inducing various phases of NASH. In addition to the WD, the mice were treated with KU for 3 weeks or 4 months. Acute and chronic KU treatments were observed to be safe at the given concentrations with no toxicity indications in the mice. KU was found to alleviate NASH-related hepatotoxicity, mitochondrial and oxidative stress, and decrease the liver triglyceride content and TNF-α mRNA in at least one set of in vivo experiments. The KU modulated liver expression of selected metabolic and oxidative stress-related genes depended upon the length and severity of the disease. Although KU failed to completely reverse the histological progression of NASH in the mice, we demonstrated the complexity of mTORC1/C2 signaling regulation and suggest a stratified therapeutic management approach throughout the disease course.

Pharmacokinetics of Intramuscularly Administered Thermoresponsive Polymers.

Aqueous solutions of some polymers exhibit a lower critical solution temperature (LCST); that is, they form phase-separated aggregates when heated above a threshold temperature. Such polymers found many promising (bio)medical applications, including in situ thermogelling with controlled drug release, polymer-supported radiotherapy (brachytherapy), immunotherapy, and wound dressing, among others. Yet, despite the extensive research on medicinal applications of thermoresponsive polymers, their biodistribution and fate after administration remained unknown. Thus, herein, they studied the pharmacokinetics of four different thermoresponsive polyacrylamides after intramuscular administration in mice. In vivo, these thermoresponsive polymers formed depots that subsequently dissolved with a two-phase kinetics (depot maturation, slow redissolution) with half-lives 2 weeks to 5 months, as depot vitrification prolonged their half-lives. Additionally, the decrease of TCP of a polymer solution increased the density of the intramuscular depot. Moreover, they detected secondary polymer depots in the kidneys and liver; these secondary depots also followed two-phase kinetics (depot maturation and slow dissolution), with half-lives 8 to 38 days (kidneys) and 15 to 22 days (liver). Overall, these findings may be used to tailor the properties of thermoresponsive polymers to meet the demands of their medicinal applications. Their methods may become a benchmark for future studies of polymer biodistribution.

Physiological and fitness differences between cytotypes vary with stress in a grassland perennial herb.

BACKGROUND AND AIMS: Understanding the consequences of polyploidization is a major step towards assessing the importance of this mode of speciation. Most previous studies comparing different cytotypes, however, did so only within a single environment and considered only one group of traits. To take a step further, we need to explore multiple environments and a wide range of traits. The aim of this study was to assess response of diploid and autotetraploid individuals of Knautia arvensis (Dipsacaceae) to two stress conditions, shade or drought. METHODS: We studied eleven photosynthetic, morphological and fitness parameters of the plants over three years in a common garden under ambient conditions and two types of stress. KEY RESULTS: The results indicate strong differences in performance and physiology between cytotypes in ambient conditions. Interestingly, higher fitness in diploids contrasted with more efficient photosynthesis in tetraploids in ambient conditions. However, stress, especially drought, strongly reduced fitness and disrupted function of the photosystems in both cytotypes reducing the between cytotype differences. The results indicate that drought stress reduced function of the photosynthetic processes in both cytotypes but particularly in tetraploids, while fitness reduction was stronger in diploids. CONCLUSIONS: The photosynthesis related traits show higher plasticity in polyploids as theoretically expected, while the fitness related traits show higher plasticity in diploids especially in response to drought. This suggests that between cytotype comparisons need to consider multiple traits and multiple environments to understand the breath of possible responses of different cytotypes to stress. They also show that integrating results based on different traits is not straightforward and call for better mechanistic understanding of the relationships between species photosynthetic activity and fitness. Still, considering multiple environments and multiple species traits is crucial for understanding the drivers of niche differentiation between cytotypes in future studies.