ABSTRACT
Proper repair of DNA damage lesions is essential to maintaining genome integrity and preventing the development of human diseases, including cancer. Increasing evidence suggests the importance of the nuclear envelope in the spatial regulation of DNA repair, although the mechanisms of such regulatory processes remain poorly defined. Through a genome-wide synthetic viability screen for PARP-inhibitor resistance using an inducible CRISPR-Cas9 platform and BRCA1-deficient breast cancer cells, we identified a transmembrane nuclease (renamed NUMEN) that could facilitate compartmentalized and non-homologous end joining-dependent repair of double-stranded DNA breaks at the nuclear periphery. Collectively, our data demonstrate that NUMEN generates short 5' overhangs through its endonuclease and 3'â5' exonuclease activities, promotes the repair of DNA lesions-including heterochromatic lamina-associated domain breaks as well as deprotected telomeres-and functions as a downstream effector of DNA-dependent protein kinase catalytic subunit. These findings underline the role of NUMEN as a key player in DNA repair pathway choice and genome-stability maintenance, and have implications for ongoing research into the development and treatment of genome instability disorders.
Subject(s)
DNA Breaks, Double-Stranded , DNA Repair , Humans , DNA Repair/genetics , DNA-Binding Proteins/metabolism , DNA End-Joining Repair , Endonucleases/geneticsABSTRACT
Pachymic acid (PA), a bioactive ingredient isolated from Poria cocos Wolf, is reported with potential benefits of anti-inflammatory, anti-oxidative actions. It is reasoned that PA may play the potential benefits against cystitis glandularis (CG), an inflammation of the bladder tissue. In this study, we aimed to apply the network pharmacology and molecular docking analyses to reveal concrete anti-CG targets and mechanisms of PA, and then the bioinformatic findings were verified by using clinical and animal samples. The methodological data from network pharmacology approach showed that 303 and 243 reporting targets of CG and PA, and other 31 shared targets of CG and PA were identified. Subsequently, all top targets of PA against CG were screened out, including cyclooxygenase-2, epidermal growth factor receptor, tumor antigen p53 (TP53), tumor necrosis factor-alpha (TNF), interleukin-1 (IL-1) beta, proto-oncogene c-jun. Molecular docking data demonstrated that PA exerted potent bonding capacities with TNF, TP53 proteins in CG. In human study, the findings suggested that overactivated TNF-α expression and suppressed TP53 activation were detected in CG samples. In animal study, PA-treated mice showed reduced intravesical IL-1, IL-6 levels, and lactate dehydrogenase content, downregulated TNF-α and upregulated TP53 proteins in bladder samples. Taken together, our bioinformatics and experimental findings identify the key anti-CG biotargets and mechanisms of PA. More markedly, these pivotal pharmacological targets of PA against CG have been screened out and verified by using computational and experimental analyses.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Cystitis/drug therapy , Triterpenes/pharmacology , Tumor Necrosis Factor-alpha/chemistry , Tumor Suppressor Protein p53/chemistry , Aged , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/metabolism , Binding Sites , Computational Biology/methods , Cystitis/genetics , Cystitis/metabolism , Cystitis/pathology , Female , Gene Expression Regulation , Humans , Interleukin-1/genetics , Interleukin-1/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , L-Lactate Dehydrogenase/genetics , L-Lactate Dehydrogenase/metabolism , Male , Mice , Mice, Inbred ICR , Middle Aged , Molecular Docking Simulation , Protein Binding , Protein Conformation , Signal Transduction , Triterpenes/chemistry , Triterpenes/metabolism , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism , Tumor Suppressor Protein p53/agonists , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Urinary Bladder/drug effects , Urinary Bladder/metabolism , Urinary Bladder/pathologyABSTRACT
Myocardial ischemic injury is among the top 10 leading causes of death from cardiovascular diseases worldwide. Myocardial ischemia is caused mainly by coronary artery occlusion or obstruction. It usually occurs when the heart is insufficiently perfused, oxygen supply to the myocardium is reduced, and energy metabolism in the myocardium is abnormal. Pathologically, myocardial ischemic injury generates a large number of inflammatory cells, thus inducing a state of oxidative stress. This sharp reduction in the number of normal cells as a result of apoptosis leads to organ and tissue damage, which can be life-threatening. Therefore, effective methods for the treatment of myocardial ischemic injury and clarification of the underlying mechanisms are urgently required. Gaseous signaling molecules, such as NO, H2S, H2, and combined gas donors, have gradually become a focus of research. Gaseous signaling molecules have shown anti-apoptotic, anti-oxidative and anti-inflammatory effects as potential therapeutic agents for myocardial ischemic injury in a large number of studies. In this review, we summarize and discuss the mechanism underlying the protective effect of gaseous signaling molecules on myocardial ischemic injury.
ABSTRACT
In mammalian cells, long noncoding RNAs (lncRNAs) form complexes with proteins to execute various biological functions such as gene transcription, RNA processing and other signaling activities. However, methods to track endogenous lncRNA dynamics in live cells and screen for lncRNA interacting proteins are limited. Here, we report the development of CERTIS (CRISPR-mediated Endogenous lncRNA Tracking and Immunoprecipitation System) to visualize and isolate endogenous lncRNA, by precisely inserting a 24-repeat MS2 tag into the distal end of lncRNA locus through the CRISPR/Cas9 technology. In this study, we show that CERTIS effectively labeled the paraspeckle lncRNA NEAT1 without disturbing its physiological properties and could monitor the endogenous expression variation of NEAT1. In addition, CERTIS displayed superior performance on both short- and long-term tracking of NEAT1 dynamics in live cells. We found that NEAT1 and paraspeckles were sensitive to topoisomerase I specific inhibitors. Moreover, RNA Immunoprecipitation (RIP) of the MS2-tagged NEAT1 lncRNA successfully revealed several new protein components of paraspeckle. Our results support CERTIS as a tool suitable to track both spatial and temporal lncRNA regulation in live cells as well as study the lncRNA-protein interactomes.
Subject(s)
CRISPR-Cas Systems , Cell Tracking , Gene Expression Profiling , Gene Knock-In Techniques , Proteomics , RNA, Long Noncoding/metabolism , HEK293 Cells , Humans , RNA, Long Noncoding/geneticsABSTRACT
The scaffold protein Symplekin (Sympk) is involved in cytoplasmic RNA polyadenylation, transcriptional modulation, and the regulation of epithelial differentiation and proliferation via tight junctions. It is highly expressed in embryonic stem cells (ESCs), in which its role remains unknown. In this study, we found Sympk overexpression in mouse ESCs significantly increased colony formation, and Sympk deletion via CRISPR/Cas9 decreased colony formation. Sympk promoted ESC growth and its overexpression sustained ESC pluripotency, as assessed by teratoma and chimeric mouse formation. Genomic stability was preserved in these cells after long-term passage. The domain of unknown function 3453 (DUF3453) in Sympk was required for its interaction with the key pluripotent factor Oct4, and its depletion led to impaired colony formation. Sympk activated proliferation-related genes and suppressed differentiation-related genes. Our results indicate that Sympk interacts with Oct4 to promote self-renewal and pluripotency in ESCs and preserves genome integrity; accordingly, it has potential value for stem cell therapies. Stem Cells 2019;37:743-753.
