Pre-mRNA splicing is critical for cells, as defects in this process can lead to altered open reading frames and defective proteins, potentially causing neurodegenerative diseases and cancer. Introns ...are removed in the nucleus and splicing is documented by the addition of exon-junction-complexes (EJCs) at exon-exon boundaries. This "memory" of splicing events is important for the ribosome, which translates the RNAs in the cytoplasm. In case a stop codon was detected before an EJC, translation is blocked and the RNA is eliminated by the nonsense-mediated decay (NMD). In the model organism
, two guard proteins, Gbp2 and Hrb1, have been identified as nuclear quality control factors for splicing. In their absence, intron-containing mRNAs leak into the cytoplasm. Their presence retains transcripts until the process is completed and they release the mRNAs by recruitment of the export factor Mex67. On transcripts that experience splicing problems, these guard proteins recruit the nuclear RNA degradation machinery. Interestingly, they continue their quality control function on exported transcripts. They support NMD by inhibiting translation and recruiting the cytoplasmic degradation factors. In this way, they link the nuclear and cytoplasmic quality control systems. These discoveries are also intriguing for humans, as homologues of these guard proteins are present also in multicellular organisms. Here, we provide an overview of the quality control mechanisms of pre-mRNA splicing, and present Gbp2 and Hrb1, as well as their human counterparts, as important players in these pathways.
Splicing expands, reshapes, and regulates the transcriptome of eukaryotic organisms. Despite its importance, key questions remain unanswered, including the following: Can splicing evolve when ...organisms adapt to new challenges? How does evolution optimize inefficiency of introns' splicing and of the splicing machinery? To explore these questions, we evolved yeast cells that were engineered to contain an inefficiently spliced intron inside a gene whose protein product was under selection for an increased expression level. We identified a combination of mutations in Cis (within the gene of interest) and in Trans (in mRNA-maturation machinery). Surprisingly, the mutations in Cis resided outside of known intronic functional sites and improved the intron's splicing efficiency potentially by easing tight mRNA structures. One of these mutations hampered a protein's domain that was not under selection, demonstrating the evolutionary flexibility of multi-domain proteins as one domain functionality was improved at the expense of the other domain. The Trans adaptations resided in two proteins, Npl3 and Gbp2, that bind pre-mRNAs and are central to their maturation. Interestingly, these mutations either increased or decreased the affinity of these proteins to mRNA, presumably allowing faster spliceosome recruitment or increased time before degradation of the pre-mRNAs, respectively. Altogether, our work reveals various mechanistic pathways toward optimizations of intron splicing to ultimately adapt gene expression patterns to novel demands.
One important task of eukaryotic cells is to translate only mRNAs that were correctly processed to prevent the production of truncated proteins, found in neurodegenerative diseases and cancer. ...Nuclear quality control of splicing requires the SR-like proteins Gbp2 and Hrb1 in S. cerevisiae, where they promote the degradation of faulty pre-mRNAs. Here we show that Gbp2 and Hrb1 also function in nonsense mediated decay (NMD) of spliced premature termination codon (PTC)-containing mRNAs. Our data support a model in which they are in a complex with the Upf-proteins and help to transmit the Upf1-mediated PTC recognition to the transcripts ends. Most importantly they appear to promote translation repression of spliced transcripts that contain a PTC and to finally facilitate degradation of the RNA, presumably by supporting the recruitment of the degradation factors. Therefore, they seem to control mRNA quality beyond the nuclear border and may thus be global surveillance factors. Identification of SR-proteins as general cellular surveillance factors in yeast will help to understand the complex human system in which many diseases with defects in SR-proteins or NMD are known, but the proteins were not yet recognized as general RNA surveillance factors.
The primary cause of breast cancer mortality is the metastatic invasion of cancerous stem cells (CSC). Cluster of differentiation 44 (CD44) is a well-known CSC marker in various cancers, as well as a ...key role player in metastasis and relapse of breast cancer. CD44 is a cell-membrane embedded protein, and it interacts with different proteins to regulate cancer cell behavior. Transcription factor forkhead box protein A2 (FOXA2) acts as an important regulator in multiple cancers, including breast cancer. However, the biological significance of CD44-FOXA2 association in breast cancer metastasis remains unclear. Herein, we observed that CD44 expression was higher in metastatic lymph nodes compared to primary tumors using a flow cytometric analysis. CD44 overexpression in breast cancer cell lines significantly promoted cell migration and invasion abilities, whereas the opposite effects occurred upon the knockdown of CD44. The stem cell array analysis revealed that FOXA2 expression was upregulated in CD44 knockdown cells. However, the knockdown of FOXA2 in CD44 knockdown cells reversed the effects on cell migration and invasion. Furthermore, we found that CD44 mediated FOXA2 localization in breast cancer cells through the AKT pathway. Moreover, the immunofluorescence assay demonstrated that AKT inhibitor wortmannin and AKT activator SC79 treatment in breast cancer cells impacted FOXA2 localization. Collectively, this study highlights that CD44 promotes breast cancer metastasis by downregulating nuclear FOXA2.
The neuroactive alkaloids in betel quid (BQ) can induce BQ addiction. We conducted a case–control study to investigate the effects of BQ-associated DSM-5 symptoms, pathological behaviors, and BQ use ...disorder (BUD) on oral squamous cell carcinoma (OSCC) risk. A total of 233 patients with newly diagnosed and histopathologically confirmed OSCC and 301 sex- and age-matched controls were included. BQ-related DSM-5 symptoms in the 12 months prior to disease onset were used to measure psychiatric characteristics and BUD. Compared with nonchewers, chewers with the symptoms of unsuccessful cutdown of BQ consumption, neglecting major roles, social or interpersonal problems, abandoning or limiting activities, hazardous use, and continued use despite the awareness of the dangers had a 54.8-, 49.3-, 49.9-, 40.4-, 86.2-, and 42.9-fold higher risk of developing OSCC, respectively. Mild-to-moderate and severe BUD were, respectively, associated with a 8.2–8.5- and 42.3-fold higher OSCC risk, compared with BQ nonuse. Risky BQ use of pathological behavior was associated with a 12.5-fold higher OSCC risk in chewers with no BUD or mild BUD and a 65.0-fold higher risk in chewers with moderate-to-severe BUD (p for risk heterogeneity between the two BUD groups, 0.041). In conclusion, BQ-associated DSM-5 symptoms, pathological behaviors, and BUD severity are associated with the impact of BQ chewing on OSCC development. The pathological behavior of risky BQ use enhances OSCC risk in chewers with moderate-to-severe BUD. Preventing BUD in new BQ users and treating BUD in chewers who already have the disorder are two priorities in areas where BQ chewing is prevalent.
A microfluidic chip is proposed to separate microparticles using cross-flow filtration
enhanced with hydrodynamic focusing. By exploiting a buffer flow from the side, the
microparticles in the sample ...flow are pushed on one side of the microchannels,
lining up to pass through the filters. Meanwhile a larger pressure gradient in the filters
is obtained to enhance separation efficiency. Compared with the traditional cross-flow
filtration, our proposed mechanism has the buffer flow to create a moving
virtual boundary for the sample flow to actively push all the particles to reach the
filters for separation. It further allows higher flow rates. The device
only requires soft lithograph fabrication to create microchannels and a novel pressurized
bonding technique to make high-aspect-ratio filtration structures. A mixture of
polystyrene microparticles with 2.7 μm and 10.6 μm
diameters are successfully separated. 96.2 ± 2.8% of the large particle are recovered with
a purity of 97.9 ± 0.5%, while 97.5 ± 0.4% of the small particle are depleted with a
purity of 99.2 ± 0.4% at a sample throughput of 10 μl/min. The experiment
is also conducted to show the feasibility of this mechanism to separate biological
cells with the
sample solutions of spiked PC3 cells in whole blood. By virtue of its high separation efficiency, our
device offers a label-free separation technique and potential integration with other components,
thereby serving as a promising tool for continuous cell filtration and analysis
applications.
In addition to its essential role in ribonucleotide reduction, ribonucleotide reductase (RNR) small subunit, RRM2, has been known to play a critical role in determining tumor malignancy. ...Overexpression of RRM2 significantly enhances the invasive and metastatic potential of tumor. Angiogenesis is critical to tumor malignancy; it plays an essential role in tumor growth and metastasis. It is important to investigate whether the angiogenic potential of tumor is affected by RRM2.
We examined the expression of antiangiogenic thrombospondin-1 (TSP-1) and proangiogenic vascular endothelial growth factor (VEGF) in two RRM2-overexpressing KB cells: KB-M2-D and KB-HURs. We found that TSP-1 was significantly decreased in both KB-M2-D and KB-HURs cells compared to the parental KB and mock transfected KB-V. Simultaneously, RRM2-overexpressing KB cells showed increased production of VEGF mRNA and protein. In contrast, attenuating RRM2 expression via siRNA resulted in a significant increased TSP-1 expression in both KB and LNCaP cells; while the expression of VEGF by the two cells was significantly decreased under both normoxia and hypoxia. In comparison with KB-V, overexpression of RRM2 had no significant effect on proliferation in vitro, but it dramatically accelerated in vivo subcutaneous growth of KB-M2-D. KB-M2-D possessed more angiogenic potential than KB-V, as shown in vitro by its increased chemotaxis for endothelial cells and in vivo by the generation of more vascularized tumor xenografts.
These findings suggest a positive role of RRM2 in tumor angiogenesis and growth through regulation of the expression of TSP-1 and VEGF.
Lung cancer is one of the leading causes of cancer mortality worldwide. As it is often first diagnosed only when cancer metastasis has already occurred, the development of effective biomarkers for ...the risk prediction of cancer metastasis, followed by stringent monitoring and the early treatment of high-risk patients, is essential for improving patient survival. Cancer cells exhibit alterations in metabolic pathways that enable them to maintain rapid growth and proliferation, which are quite different from the metabolic pathways of normal cells. Fumarate hydratase (FH, fumarase) is a well-known tricarboxylic acid cycle enzyme that catalyzes the reversible hydration/dehydration of fumarate to malate. The current study sought to investigate the relationship between FH expression levels and the outcome of patients with lung cancer. FH was knocked down in lung cancer cells using shRNA or overexpressed using a vector, and the effect on migration ability was assessed. Furthermore, the role of AMP-activated protein kinase (AMPK) phosphorylation and disabled homolog 2 in the underlying mechanism was investigated using an AMPK inhibitor approach. The results showed that in lung cancer tissues, low FH expression was associated with lymph node metastasis, tumor histology and recurrence. In addition, patients with low FH expression exhibited a poor overall survival in comparison with patients having high FH expression. When FH was overexpressed in lung cancer cells, cell migration was reduced with no effect on cell proliferation. Furthermore, the level of phosphorylated (p-)AMPK, an energy sensor molecule, was upregulated when FH was knocked down in lung cancer cells, and the inhibition of p-AMPK led to an increase in the expression of disabled homolog 2, a tumor suppressor protein. These findings suggest that FH may serve as an effective biomarker for predicting the prognosis of lung cancer and as a therapeutic mediator. Key words: lung cancer, fumarate hydratase, AMPK, disabled homolog 2, migration
Members of the β-nitrostyrene family are known to suppress tumor growth, with the underlying mechanisms of β-nitrostyrene remain mostly unclear. Herein, we synthesized a β-nitrostyrene derivative, ...3′-hydroxy-4′-methoxy-β-methyl-β-nitrostyrene (CYT-Rx20), and explored its anticancer activities in human lung cancer cells in vitro and in vivo.
Cell viability was measured by XTT assay. Apoptosis was detected by Annexin V/PI staining. Caspase activation was determined by western blotting. ROS (reactive oxygen species), MMP (mitochondrial membrane potential) and mitochondrial mass were determined by flow cytometry. GSH level was detected by ELISA assay.
In this study, we found that CYT-Rx20 significantly reduced cell viability, accompanied by G2/M arrest in lung cancer cells. Increased protein levels of cleaved-caspase families indicated apoptotic cell death upon CYT-Rx20 treatment. Furthermore, increased level of intracellular reactive oxygen species (ROS), loss of mitochondrial membrane potential (ΔΨm), glutathione (GSH) depletion and inhibition of GSH reductase were observed after CYT-Rx20 treatment. The effects of CYT-Rx20 on cell viability and the loss of ΔΨm were significantly reversed when cells were pretreated with thiol antioxidants NAC, GSH, or 2-ME. Finally, xenograft animal study demonstrated that CYT-Rx20 significantly suppressed lung tumor growth in vivo.
Our data demonstrated that CYT-Rx20 triggered apoptotic cell death in lung cancer cells and suppressed lung tumor growth through GSH depletion, suggesting that CYT-Rx20 may have the potential to be further developed as an anticancer compound for treating lung cancer.
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