A short splicing isoform of HBS1L links the cytoplasmic exosome and SKI complexes in humans.

The exosome complex is a major eukaryotic exoribonuclease that requires the SKI complex for its activity in the cytoplasm. In yeast, the Ski7 protein links both complexes, whereas a functional equivalent of the Ski7 has remained unknown in the human genome.Proteomic analysis revealed that a previously uncharacterized short splicing isoform of HBS1L (HBS1LV3) is the long-sought factor linking the exosome and SKI complexes in humans. In contrast, the canonical HBS1L variant, HBS1LV1, which acts as a ribosome dissociation factor, does not associate with the exosome and instead interacts with the mRNA surveillance factor PELOTA. Interestingly, both HBS1LV1 and HBS1LV3 interact with the SKI complex and HBS1LV1 seems to antagonize SKI/exosome supercomplex formation. HBS1LV3 contains a unique C-terminal region of unknown structure, with a conserved RxxxFxxxL motif responsible for exosome binding and may interact with the exosome core subunit RRP43 in a way that resembles the association between Rrp6 RNase and Rrp43 in yeast. HBS1LV3 or the SKI complex helicase (SKI2W) depletion similarly affected the transcriptome, deregulating multiple genes. Furthermore, half-lives of representative upregulated mRNAs were increased, supporting the involvement of HBS1LV3 and SKI2W in the same mRNA degradation pathway, essential for transcriptome homeostasis in the cytoplasm

Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system

Deciphering a function of a given protein requires investigating various biological aspects. Usually, the protein of interest is expressed with a fusion tag that aids or allows subsequent analyses. Additionally, downregulation or inactivation of the studied gene enables functional studies. Development of the CRISPR/Cas9 methodology opened many possibilities but in many cases it is restricted to non-essential genes. Recombinase-dependent gene integration methods, like the Flp-In system, are very good alternatives. The system is widely used in different research areas, which calls for the existence of compatible vectors and efficient protocols that ensure straightforward DNA cloning and generation of stable cell lines. We have created and validated a robust series of 52 vectors for streamlined generation of stable mammalian cell lines using the FLP recombinase-based methodology. Using the sequence-independent DNA cloning method all constructs for a given coding-sequence can be made with just three universal PCR primers. Our collection allows tetracycline-inducible expression of proteins with various tags suitable for protein localization, FRET, bimolecular fluorescence complementation (BiFC), protein dynamics studies (FRAP), co-immunoprecipitation, the RNA tethering assay and cell sorting. Some of the vectors contain a bidirectional promoter for concomitant expression of miRNA and mRNA, so that a gene can be silenced and its product replaced by a mutated miRNA-insensitive version. Our toolkit and protocols have allowed us to create more than 500 constructs with ease. We demonstrate the efficacy of our vectors by creating stable cell lines with various tagged proteins (numatrin, fibrillarin, coilin, centrin, THOC5, PCNA). We have analysed transgene expression over time to provide a guideline for future experiments and compared the effectiveness of commonly used inducers for tetracycline-responsive promoters. As proof of concept we examined the role of the exoribonuclease XRN2 in transcription termination by RNAseq

TEFM (c17orf42) is necessary for transcription of human mtDNA

Here we show that c17orf42, hereafter TEFM (transcription elongation factor of mitochondria), makes a critical contribution to mitochondrial transcription. Inactivation of TEFM in cells by RNA interference results in respiratory incompetence owing to decreased levels of H- and L-strand promoter-distal mitochondrial transcripts. Affinity purification of TEFM from human mitochondria yielded a complex comprising mitochondrial transcripts, mitochondrial RNA polymerase (POLRMT), pentatricopeptide repeat domain 3 protein (PTCD3), and a putative DEAD-box RNA helicase, DHX30. After RNase treatment only POLRMT remained associated with TEFM, and in human cultured cells TEFM formed foci coincident with newly synthesized mitochondrial RNA. Based on deletion mutants, TEFM interacts with the catalytic region of POLRMT, and in vitro TEFM enhanced POLRMT processivity on ss- and dsDNA templates. TEFM contains two HhH motifs and a Ribonuclease H fold, similar to the nuclear transcription elongation regulator Spt6. These findings lead us to propose that TEFM is a mitochondrial transcription elongation factor

Nuclear and mitochondrial genome responses in HeLa cells treated with inhibitors of mitochondrial DNA expression

The influence of mutations in the mitochondrial DNA (mtDNA) on the bioenergetic metabolism of the cell is still poorly understood. Many of the mutations in the mtDNA affect the expression of the mitochondrial genome. Investigations on cells from patients are not easy, especially as the mitochondrial DNA is heteroplasmic and this state is changed in culture. Moreover, the nuclear background and the mitochondrial haplotype may affect the behaviour of cells. Transfer of patient mitochondria to rho zero cell lines is also not optimal as these cells in general have many nuclear changes which may also affect cell behaviour. Thus, we decided to use inhibitors of mitochondrial genome expression, such as thiamphenicol, ethidium bromide and dideoxycytidine to investigate the bioenergetic metabolism of HeLa cells. We found that oxidative phosphorylation and glycolysis participate equally in ATP production in HeLa cells and that decreased activity of the respiratory chain leads to increased glycolysis and the reduction of cell growth. Insufficient ATP production in the oxidative phosphorylation process was not compensated by increased proliferation of the mitochondria. However, we were able to show that there are some mechanisms compensating limited expression of the mitochondrial genome within the mitochondria. Experiments with dideoxycytidine revealed that 10-fold decrease of the mtDNA copy number resulted in almost normal activity of cytochrome c oxidase. We found that mtDNA depletion is compensated mostly on the level of RNA metabolism in the mitochondria. Thus, our results are in agreement with the hypothesis that transcription initiation rather than mtDNA copy number is a rate limiting factor for expression of the mitochondrial genome

Kinetics and Dynamics of the Stiff and Flexible Tines with the Duckfoot and the Coulter after Impact with Stones Embedded in Compacted Soil. Part II

The kinetics and dynamics of the stiff and flexible tines with the duckfoot and the coulter after impact with stones embedded in compacted soil were examined. The beak of the duckfoot was positioned in the axis of the row of stones embedded in the soil at the depth of stones thickness. The coulter covered the stone or impact the edge of the stone halfway along its length. The tools worked at a speed of 0.83–2.22 m·s−1 and a working depth of 0.05–0.10 m. The results of specific parameters were compared to the response of the tools to loads in soil without stones. For both soil conditions, the kinetics of the flexible tine was 24 times more reactive, and the dynamic loads were two times lower than for the stiff tine. The responses of both tines were suppressed along with the working depth because of the more favorable place of impact of the duckfoot beak with the stone. Along with the working speed, for a stiff tine, the specific accelerations decreased significantly, by ten times, and the specific forces increased slightly, by 1.6 times. Among the two systems of setting the coulter, the impact of the cutting edge of the coulter with the stone in the middle of its length was more unfavorable than the work of the coulter covering the stone

Qatar-Sudan Archaeological Project: Excavations at the Ghazali monastery from 2014 to 2016

The excavation report covers eight months of fieldwork at the site of Ghazali, which resulted in the clearing of the entire monastery and the discovery of three annexes located on the north and west of the complex. The spiritual part of the monastery included two churches located in the southeastern corner of the complex, a household compound on the west side and a refectory and dormitory in between. Conservation work focused on the reconstruction and restoration of water storage installations in Room Y, as well as north of the North Church. Excavation outside the monastic walls brought the discovery of an iron smelting area with several well-preserved furnaces. Exploration of the monks’ cemetery uncovered regular box superstructures and an intriguing variety of substructures from simple vertical pit tombs to elaborate vaulted chambers

Effect of Stone Impacts on Various Ground Engaging Tools (Flexible/Stiff Tines and Coulter): Part I

Analysis of the state of knowledge showed a gap in the description of tool–stone feedback. Therefore, the aim of this study was to investigate tool–stone interactions. Spherical-like silicate stones were hit by stiff and flexible tines with a duckfoot or a coulter. The tools worked with various parameters in the depth range of 0.05–0.10 m and a speed of 0.83–2.22 m·s–1. The characteristics of stone movement were specific to the type of tool and were described by the Numerical Stone Movement Scale developed for the purpose of the research. After the impact with the stiff tine, the stones were thrown the greatest distance of 0.26–1.08 m, and these distances were strongly dependent on the working speed and slightly dependent on the working depth. Large vibrations of the flexible tine and the location of the contact point of the tine in relation to the centre of the stone thickness contributed to the random behaviour of stones that were slightly moved, rotated or displaced. The specific work required to remove the stone reflected the distance travelled by the stone as well as the specific force which largely contributed to increasing the differences in this work between both tines

Nucleus- and plastid-targeted annexin 5 promotes reproductive development in Arabidopsis and is essential for pollen and embryo formation

Abstract Background Pollen development is a strictly controlled post-meiotic process during which microspores differentiate into microgametophytes and profound structural and functional changes occur in organelles. Annexin 5 is a calcium- and lipid-binding protein that is highly expressed in pollen grains and regulates pollen development and physiology. To gain further insights into the role of ANN5 in Arabidopsis development, we performed detailed phenotypic characterization of Arabidopsis plants with modified ANN5 levels. In addition, interaction partners and subcellular localization of ANN5 were analyzed to investigate potential functions of ANN5 at cellular level. Results Here, we report that RNAi-mediated suppression of ANN5 results in formation of smaller pollen grains, enhanced pollen lethality, and delayed pollen tube growth. ANN5 RNAi knockdown plants also displayed aberrant development during the transition from the vegetative to generative phase and during embryogenesis, reflected by delayed bolting time and reduced embryo size, respectively. At the subcellular level, ANN5 was delivered to the nucleus, nucleolus, and cytoplasm, and was frequently localized in plastid nucleoids, suggesting a likely role in interorganellar communication. Furthermore, ANN5-YFP co-immunoprecipitated with RABE1b, a putative GTPase, and interaction in planta was confirmed in plastidial nucleoids using FLIM-FRET analysis. Conclusions Our findings let us to propose that ANN5 influences basal cell homeostasis via modulation of plastid activity during pollen maturation. We hypothesize that the role of ANN5 is to orchestrate the plastidial and nuclear genome activities via protein-protein interactions however not only in maturing pollen but also during the transition from the vegetative to the generative growth and seed development