CRISPR/Cas9 genome editing is a latest success in biotechnology that repurposes a natural biological system for a practical tool in genetic engineering. Site-specific DNA double strand breaks (DSB) induced by the CRISPR nuclease Cas9 allows endogenous cellular repair apparatus to generate desired repair products. Residence of Cas9 on cleaved DNA conceals the DNA ends from recognition by response and repair apparatus, delaying DNA damage response (DDR) and repair. Thus, tight-binding and long residence of Cas9 on DNA target are proposed as a new determinant of DSB repair pathway choice and may collaborate with other endogenous pathway choice regulators to control DSB repair. Accordingly, harnessing the binding and resident behavior of Cas9 not only broadens the application of CRISPR/dCas9 platform, which at least in part depends upon the tight binding and long residence of dCas9, but also minimizes the undesired outcomes of CRISPR/Cas9 genome editing.
In recent years, long non-coding RNAs (lncRNAs) have emerged as critical players in regulating biological processes at various levels. LncRNAs modulate the target genes through interacting with DNA, RNA, or protein, which have been found to regulate various physiological and pathological activities. Current studies have reported lncRNAs played an important role in lipid metabolism via diverse pathways. Dysregulation of lipid metabolism is closely related to various metabolic diseases, including obesity, fatty liver disease, and cancer. To further understand the mechanisms of lncRNAs in lipid metabolism, we focus on the role of lncRNAs in fatty acid and cholesterol biosynthesis, catabolism, and transferring, aiming to exploit relevant signaling pathways.
The ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR) are apical kinases that orchestrate the multifaceted DNA damage response (DDR) to a variety of genotoxic insults and regulate genomic stability. Whether RNA virus also manipulates the host’s DDR machine to facilitate replication is largely unknown. In this study, we revealed that single-stranded RNA virus replication specifically elicits host ATM- and ATR-mediated pathway activation and boosts their expression. The activated ATM and ATR are hijacked to the virus replication factory in the cytoplasm and facilitate viral gene expression and replication. Specific inhibitors targeting ATM and ATR strikingly block the viral proliferation and replication and inhibit expression of virus proteins. Our results reveal a novel, or otherwise noncanonical, conserved function of ATM/ATR outside DDR in promoting the replication of single-stranded RNA virus and provide an important mechanism of host–pathogen interactions.
Phosphatidylinositol-5-phosphate 4-kinases (PIP4K) are multiple cellular process regulatory lipid kinases. PIP4K2A and PIP4K2B are overexpressed in cancer. The recently reported kinase-independent role of PIP4K2A and PIP4K2B underscore the complexity of the underlying molecular changes and mechanisms involved. Here, we show proteome analysis of PIP4K2A, PIP4K2B and PIP4K2A/2B co-depleted osteosarcoma cells to reflect changes in protein expression and their post-translational modifications. PIP4K2A depletion mainly affects ribosome assembly, translation and cell proliferation. Proteins of the apoptotic process, DNA repair, and cell division are mainly affected in PIP4K2B knockdown cells. PIP4K2A/2B co-depletion affects proteins regulating vesicle transport, cell motility, RNA splicing, and cell division. PIP4K depletion also affects post-translational modifications (phosphorylation, acetylation) of proteins involved mainly in nucleosome organization, mRNA splicing, and DNA replication. In addition, we observed PIP4K2A and PIP4K2B overexpression in cells harbouring K-Ras G12V or G12D mutations. Collectively, our results show that single or co-depletion of the PIP4K isoforms regulate proteins participating in metabolism and maintenance of genome integrity. Given that PIP4K2A and PIP4K2B alter proteins related to genome instability and their role in cancer, these enzymes could be promising therapeutic targets for cells experiencing genotoxic stress conditions.
Hypoxia is one of the hallmarks of the solid cancer microenvironment that dominates cancer progression and exacerbation. Under the oxygen-deprived condition, cancer resists and circumvents all interventions for permanence. Genomic and genetic instability of hypoxic cancer remains to be insightfully investigated and interpreted, as in lung adenocarcinoma. Herein, non-small cell lung cancer (NSCLC) cell line A549 was exposed to hypoxic shots parallel with running non-hypoxic (normoxic) A549 cells. Based on the isolated total RNA, gene transcriptomic profiling was identified using microarray and analysed via Ingenuity Pathway Analysis (IPA). As well, wound healing and cytotoxicity of doxorubicin were performed to assess hypoxic lung cancer cell response. Gene expression analysis revealed that TP53 (p53) is the most activated signaling, along with suppression of chromosomal signalings (DNA replication and repair). Besides, TP53 and its dependent target CDKN1A/p21 (cell cycle down-regulator) were identified as the topmost significant upstream transcriptional regulators, commanding a 37-gene targetable panel for cancer survival. In addition, hypoxic A549 cells were more chemo-resistant and higher motile. Notably, other emerging alterations were detected regarding efflux transporters (ABC-As and ABC-G1) that have been selectively up-regulated over the rest of under-transcribed (ABC) transporters subfamilies. Together, these involved findings suggest that the p53 signaling could be a potential survival mediator of hypoxic NSCLC cells (A549), which triggered the prominent cell cycle down-regulation to frustrate apoptotic response in hypoxic NSCLC. Coupled with the impairing of DNA replication and repair signalings that promoted molecular alterations for survival. Represented by the remarkable epithelial-mesenchymal transition of hypo-proliferating hypoxic A549 cells by which was described at the transcriptional level alongside phenotypic level. Consequently, hypoxic A549 cells showed higher resistance to doxorubicin targeting DNA replication, regardless of any transcriptionally down-regulated (ABC) transporters.