Search results for "Nucleases"
showing 10 items of 147 documents
The application of the CRISPR-Cas9 genome editing machinery in food and agricultural science: Current status, future perspectives, and associated cha…
2019
The recent progress in genetic engineering has brought multiple benefits to the food and agricultural industry by enhancing the essential characteristics of agronomic traits. Powerful tools in the field of genome editing, such as siRNA-mediated RNA interference for targeted suppression of gene expression and transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs) for DNA repair have been widely used for commercial purposes. However, in the last few years, the discovery of the CRISPR-Cas9 system has revolutionized genome editing and has attracted attention as a powerful tool for several industrial applications. Herein, we review current progresses in the uti…
A Comparison of Techniques to Evaluate the Effectiveness of Genome Editing
2018
Genome editing using engineered nucleases (meganucleases, zinc finger nucleases, transcription activator-like effector nucleases) has created many recent breakthroughs. Prescreening for efficiency and specificity is a critical step prior to using any newly designed genome editing tool for experimental purposes. The current standard screening methods of evaluation are based on DNA sequencing or use mismatch-sensitive endonucleases. They can be time-consuming and costly or lack reproducibility. Here, we review and critically compare standard techniques with those more recently developed in terms of reliability, time, cost, and ease of use.
The exonuclease Xrn1 activates transcription and translation of mRNAs encoding membrane proteins
2019
The highly conserved 5’–3’ exonuclease Xrn1 regulates gene expression in eukaryotes by coupling nuclear DNA transcription to cytosolic mRNA decay. By integrating transcriptome-wide analyses of translation with biochemical and functional studies, we demonstrate an unanticipated regulatory role of Xrn1 in protein synthesis. Xrn1 promotes translation of a specific group of transcripts encoding membrane proteins. Xrn1-dependence for translation is linked to poor structural RNA contexts for translation initiation, is mediated by interactions with components of the translation initiation machinery and correlates with an Xrn1-dependence for mRNA localization at the endoplasmic reticulum, the trans…
PARP inhibition enhances tumor cell-intrinsic immunity in ERCC1-deficient non-small cell lung cancer.
2018
The cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) pathway detects cytosolic DNA to activate innate immune responses. Poly(ADP-ribose) polymerase inhibitors (PARPi) selectively target cancer cells with DNA repair deficiencies such as those caused by BRCA1 mutations or ERCC1 defects. Using isogenic cell lines and patient-derived samples, we showed that ERCC1-defective non-small cell lung cancer (NSCLC) cells exhibit an enhanced type I IFN transcriptomic signature and that low ERCC1 expression correlates with increased lymphocytic infiltration. We demonstrated that clinical PARPi, including olaparib and rucaparib, have cell-autonomous immunomodulatory properties in ERCC1-defecti…
Exosomal miRNA analysis in Non-Small Cell Lung Cancer (NSCLC) patients' plasma through qPCR : a feasible liquid biopsy tool
2016
Abstract: The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective tyrosine kinase inhibitors (TKIs). To optimize the use of these TKIs, the discovery of new biomarkers for early detection and disease progression is mandatory. These plasma-isolated exosomes can be used as a non-invasive and repeatable way for the detection and followup of these biomarkers. One ml of plasma from 12 NSCLC patients, with different mutations and treatments (and 6 healthy donors as controls), were used as exosome sources. After RNAse treatment, in order to degrade circulating miRNAs, the exosomes were isolated with a comm…
Prediction of Chromatin Accessibility in Gene-Regulatory Regions from Transcriptomics Data
2017
AbstractThe epigenetics landscape of cells plays a key role in the establishment of cell-type specific gene expression programs characteristic of different cellular phenotypes. Different experimental procedures have been developed to obtain insights into the accessible chromatin landscape including DNase-seq, FAIRE-seq and ATAC-seq. However, current downstream computational tools fail to reliably determine regulatory region accessibility from the analysis of these experimental data. In particular, currently available peak calling algorithms are very sensitive to their parameter settings and show highly heterogeneous results, which hampers a trustworthy identification of accessible chromatin…
Telomere Length Determines TERRA and R-Loop Regulation through the Cell Cycle
2017
Maintenance of a minimal telomere length is essential to prevent cellular senescence. When critically short telomeres arise in the absence of telomerase, they can be repaired by homology-directed repair (HDR) to prevent premature senescence onset. It is unclear why specifically the shortest telomeres are targeted for HDR. We demonstrate that the non-coding RNA TERRA accumulates as HDR-promoting RNA-DNA hybrids (R-loops) preferentially at very short telomeres. The increased level of TERRA and R-loops, exclusively at short telomeres, is due to a local defect in RNA degradation by the Rat1 and RNase H2 nucleases, respectively. Consequently, the coordination of TERRA degradation with telomere r…
A detailed experimental study of a DNA computer with two endonucleases
2017
Abstract Great advances in biotechnology have allowed the construction of a computer from DNA. One of the proposed solutions is a biomolecular finite automaton, a simple two-state DNA computer without memory, which was presented by Ehud Shapiro’s group at the Weizmann Institute of Science. The main problem with this computer, in which biomolecules carry out logical operations, is its complexity – increasing the number of states of biomolecular automata. In this study, we constructed (in laboratory conditions) a six-state DNA computer that uses two endonucleases (e.g. AcuI and BbvI) and a ligase. We have presented a detailed experimental verification of its feasibility. We described the effe…
PIWIL3 Forms a Complex with TDRKH in Mammalian Oocytes.
2019
P-element induced wimpy testis (PIWIs) are crucial guardians of genome integrity, particularly in germ cells. While mammalian PIWIs have been primarily studied in mouse and rat, a homologue for the human PIWIL3 gene is absent in the Muridae family, and hence the unique function of PIWIL3 in germ cells cannot be effectively modeled by mouse knockouts. Herein, we investigated the expression, distribution, and interaction of PIWIL3 in bovine oocytes. We localized PIWIL3 to mitochondria, and demonstrated that PIWIL3 expression is stringently controlled both spatially and temporally before and after fertilization. Moreover, we identified PIWIL3 in a mitochondrial-recruited three-membered complex…
Dom34 Links Translation to Protein O-mannosylation.
2016
In eukaryotes, Dom34 upregulates translation by securing levels of activatable ribosomal subunits. We found that in the yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans, Dom34 interacts genetically with Pmt1, a major isoform of protein O-mannosyltransferase. In C. albicans, lack of Dom34 exacerbated defective phenotypes of pmt1 mutants, while they were ameliorated by Dom34 overproduction that enhanced Pmt1 protein but not PMT1 transcript levels. Translational effects of Dom34 required the 5′-UTR of the PMT1 transcript, which bound recombinant Dom34 directly at a CA/AC-rich sequence and regulated in vitro translation. Polysomal profiling revealed that Dom34 stimu…