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

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.</jats:p

Prediction of Tuber Damage from Harvesting and Processing Machine Working Units Based on the Recording of Impact Parameters

The paper presents the development of an empirical mathematical model of the potato tuber damage index that considers the relationship between impact parameters, such as peak acceleration, velocity change, and the experimental coefficient. This coefficient was developed using statistical analysis methods for four isolated surfaces and two potato varieties, Hermes and Saturna. The IRD 400 device was used to measure impacts, which recorded peak accelerations and changes in impact velocity at initial velocities in the 2.43–4.43 m·s−1 range. The study’s results indicate that the variety, type of surface, and initial impact velocity had a statistically significant effect on the tuber damage index; the type of surface and initial impact velocity had a statistically significant effect on the peak acceleration values (p < 0.05). The increase in peak acceleration with increases in the impact velocity confirms the hypothesis that the maximum force and the resulting internal stresses of the tuber are key elements causing damage due to the impact. The highest values of the tuber damage index occurred during impacts with steel surfaces and conveyor bars. The developed model allows for the faster prediction of potential damage in harvesting and post-harvest processing conditions than traditional measurement methods. To fully use the proposed model in Agriculture 4.0, further research should be conducted to improve recording devices for the measurement of impact parameters in real time

The Usefulness of Soil Penetration Resistance Measurements for Improving the Efficiency of Cultivation Technologies

The research results of soil penetration resistance (SPR) tests carried out on sandy clay using four cone probes with different dimensions of the measuring tip are presented in this study. It was indicated that the values of SPR can be used to diagnose the cultivation layer and, on this basis, determine whether it is necessary to cultivate it and select tools for the required treatment. Tests were carried out on three levels of soil density, 1.37, 1.43 and 1.51 g&#8729;cm&minus;3, and two moisture contents, 7.64% and 10.4%. The results show that the probe with the smallest cone with apex angles of 30&deg; and 60&deg; on the least dense soil indicated higher SPR by over 50% more than other probes with the highest cone and the same opening angles. The change in cone opening angle from 30&deg; to 60&deg; led to an increase in probe indications in the range of 10&ndash;25%, depending on the diameter of the cone tip. The statistical analysis shows that values of probe indications were statistically significant and were influenced by soil density, probe cone tip dimensions, the surface of the base and the apex angle. The values of SPR are fundamental in diagnosing the quality of the soil&rsquo;s top layer, determining the necessity of breaking it up, and selecting the optimal tools for this procedure. To improve the efficiency of agricultural crop cultivation technologies. This is particularly important when carrying out cultivation procedures in an environmentally friendly manner. The measurements will help support the introduction of sustainable farming practices, including direct seeding, no-till cultivation, or precision agriculture, reducing soil degradation and increasing environmental benefits