mRNA Abundance In Cells Research Articles

The role of exosomal CDC6 in the hirudin-mediated suppression of the malignant phenotype of bladder cancer cells

BackgroundBladder cancer is a malignant tumor characterized by high recurrence and persistence due to the limited therapies that are currently available. Hirudin exerts a strong anticancer effect on several tumors. Thus, it is urgent to explore the biological function of hirudin in bladder cancer and the role of bladder cancer-derived exosomes in tumor inhibition. MethodsFirst, a network pharmacology analysis was performed to explore the relationships among hirudin, bladder cancer, and exosomes. Then, the effects of hirudin were examined by CCK-8 assay, flow cytometry, Transwell assay, and tumorigenic ability experiments in vitro. Exosomes derived from cells were identified with transmission electron microscopy, fluorescence labeling, and Western blotting and collected for further microarray analysis. Only CDC6 expression and mRNA abundance in hirudin-treated cells and exosomes was subjected to further validation using quantitative PCR and Western blotting. ResultsThrough network analysis, we found that hirudin affected bladder cancer, and this effect was related to exosomes. Our studies verified the effects of hirudin by revealing that hirudin inhibits malignant processes of bladder cancer cells in vitro, such as invasion, metastasis, and apoptosis. Similarly, the oncogenic effects of bladder cancer-derived exosomes were successfully isolated and identified. Via microarray assessment of the exosomes, we identified 600 differential mRNAs, of which the expression of the core target CDC6 was found to be significantly different in both The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. We further confirmed that hirudin suppresses CDC6 expression mRNA abundance in both cells and exosomes. ConclusionHirudin was able to decrease the expression of CDC6 in bladder cancer cells and exosomes, which effectively repressed the malignant processes of bladder cancer cells.

IRQC, a surveillance pathway for 40S ribosomal quality control during mRNA translation initiation.

Post-translational modification of ribosomal proteins enables rapid and dynamic regulation of protein biogenesis. Site-specific ubiquitylation of 40S ribosomal proteins uS10 and eS10 plays a key role during ribosome-associated quality control (RQC). Distinct, and previously functionally ambiguous, ubiquitylation events on the 40S proteins uS3 and uS5 are induced by diverse proteostasis stressors that impact translation activity. Here, we identify the ubiquitin ligase RNF10 and the deubiquitylating enzyme USP10 as the key enzymes that regulate uS3 and uS5 ubiquitylation. Prolonged uS3 and uS5 ubiquitylation results in 40S, but not 60S, ribosomal protein degradation in a manner independent of canonical autophagy. We show that blocking progression of either scanning or elongating ribosomes past the start codon triggers site-specific ubiquitylation events on ribosomal proteins uS5 and uS3. This study identifies and characterizes a distinct arm in the RQC pathway, initiation RQC (iRQC), that acts on 40S ribosomes during translation initiation to modulate translation activity and capacity.

Genes with 5' terminal oligopyrimidine tracts preferentially escape global suppression of translation by the SARS-CoV-2 Nsp1 protein.

Viruses rely on the host translation machinery to synthesize their own proteins. Consequently, they have evolved varied mechanisms to co-opt host translation for their survival. SARS-CoV-2 relies on a nonstructural protein, Nsp1, for shutting down host translation. However, it is currently unknown how viral proteins and host factors critical for viral replication can escape a global shutdown of host translation. Here, using a novel FACS-based assay called MeTAFlow, we report a dose-dependent reduction in both nascent protein synthesis and mRNA abundance in cells expressing Nsp1. We perform RNA-seq and matched ribosome profiling experiments to identify gene-specific changes both at the mRNA expression and translation levels. We discover that a functionally coherent subset of human genes is preferentially translated in the context of Nsp1 expression. These genes include the translation machinery components, RNA binding proteins, and others important for viral pathogenicity. Importantly, we uncovered a remarkable enrichment of 5′ terminal oligo-pyrimidine (TOP) tracts among preferentially translated genes. Using reporter assays, we validated that 5′ UTRs from TOP transcripts can drive preferential expression in the presence of Nsp1. Finally, we found that LARP1, a key effector protein in the mTOR pathway, may contribute to preferential translation of TOP transcripts in response to Nsp1 expression. Collectively, our study suggests fine-tuning of host gene expression and translation by Nsp1 despite its global repressive effect on host protein synthesis.

In vitro methionine supplementation during lipopolysaccharide stimulation modulates immunometabolic gene network expression in isolated polymorphonuclear cells from lactating Holstein cows

Methionine (Met) is one of the 2 most limiting amino acids for milk production in dairy cow diets. The accepted "ideal" ratio of lysine (Lys) to Met (L:M) when formulating diets is 3:1. However, blood from cows fed corn silage-based diets without supplemental rumen-protected Met averages approximately 3.6:1 L:M. Recent in vivo research on cattle immunonutrition has revealed that the immune system could benefit from greater Met supply. To study more closely the effects of different L:M ratios, blood polymorphonuclear cells (PMN) were isolated from 5 Holstein cows in mid-lactation (238 ± 20 d postpartum, 33.8 ± 3.8 kg of milk/d; mean ± SD). The PMN were incubated at 3 different levels of L:M (3.6:1, 2.9:1, or 2.4:1) and stimulated with lipopolysaccharide (LPS) at either 0 or 50 μg/mL for 2 h at 37°C. Target genes were associated with cytokines, pathogen recognition, nuclear receptors, killing mechanisms, and Met and glutathione metabolism. Data were subjected to ANOVA using PROC MIXED in SAS, with L:M, LPS, and their interaction as fixed effects. Stimulation with LPS upregulated genes related to cytokines (IL1B, TNF, IL10 and IL6) and nuclear receptors, including nuclear factor kappa B (NFKB1) and glucocorticoid receptor (NR3C1), and downregulated the mRNA abundance of chemokine receptor 1 (CXCR1), lysozyme (LYZ) and glutathione reductase (GSR). A linear decrease was observed in the mRNA abundance of TNF when L:M was decreased. A similar response was observed for interleukin-1 receptor-associated kinase 1 (IRAK1) and NFKB1 abundance in cells stimulated with LPS (linear effect). A linear increase of LYZ mRNA expression as L:M decreased was detected in unstimulated cells. Furthermore, a decrease in L:M led to a linear decrease of superoxide dismutase 1 (SOD1) mRNA abundance in cells challenged with LPS. Overall, LPS challenge triggered the activation of isolated PMN from mid-lactation cows. However, data suggest the use of a shorter incubation time to capture the peak response and not the resolution of the inflammatory response as in the present study. Our results indicate a possible involvement of Met in modulating PMN inflammatory and oxidative stress status and in helping the resolution of inflammation after initial stimulation.

Evidence for Necroptosis as a Mechanism of Islet Amyloid–Induced Beta-Cell Death

Islet amyloid deposition occurs in the majority of individuals with type 2 diabetes (T2D) and contributes to beta-cell death by mechanisms which are not fully understood. Necroptosis is an immunogenic form of cell death dependent on tumor necrosis factor receptor 1 (TNFR1) signaling, receptor interacting protein kinase 3 (RIPK3) oligomerization, and mixed lineage kinase domain-like (MLKL) phosphorylation. We previously showed mRNA and protein levels of TNFa, a TNFR1 ligand, are increased in islets by amyloid deposition in vitro and in vivo, raising the possibility that amyloid formation induces necroptosis. Thus, we sought to determine whether the components of necroptosis are present in beta cells, and if islet amyloid-induced TNFa expression elicits beta-cell necroptosis. We observed protein expression of TNFR1, RIPK3, and MLKL in the INS-1 beta-cell line, indicating necroptosis components are present in beta cells. In human islet amyloid polypeptide (hIAPP) transgenic mouse islets cultured in 16.7 mM glucose, an in vitro model of islet amyloidosis, we found necroptosis signaling to be increased compared to amyloid-free wild type (WT) islets. This manifested as increased oligomerization of RIPK3 protein (trimers: 2.7 ± 0.2 [hIAPP] vs. 1.0 [WT], n=3, p<0.01) and phosphorylation of the RIPK3 target MLKL (1.8 ± 0.3 vs. 1.0, n=3, p=0.06), without a change in TNFR1 protein expression (1.1 ± 0.1 vs. 1.0, n=2, p=0.74). As further support that amyloid induces immunogenic cell death, immune cell mRNA abundance was increased in vivo in amyloid containing hIAPP transgenic islets vs. WT islets (macrophages: Emr1, 1.6 ± 0.2 [hIAPP] vs. 1.1 ± 0.1 [WT], n=4, p=0.04, T cells: Cd3, 2.7 ± 0.5 vs. 1.0 ± 0.2, n=4, p=0.03, and B cells: Cd20, 2.3 ± 0.2 vs. 1.2 ± 0.5, n=2, p=0.17). These data suggest that necroptosis may be a previously unrecognized mechanism of beta-cell death in response to islet amyloid formation. Further studies are needed to determine whether inhibition of necroptosis signaling may reduce beta-cell death in T2D. Disclosure A.T. Templin: None. M.F. Hogan: None. N. Esser: None. S. Zraika: None. R.L. Hull: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company. S.E. Kahn: Advisory Panel; Self; Boehringer Ingelheim GmbH, Elcelyx Therapeutics, Inc., Eli Lilly and Company, Intarcia Therapeutics, Inc., Janssen Research & Development, Merck & Co., Inc., Novo Nordisk A/S.

Small interfering RNA-mediated regulation of gene expression and its role as a plant reverse genetic tool

In the era of genome sequencing, various techniques have been conceptualized for identifying the functions of annotated genes in model as well as crop systems. One of them is the use of small RNAs in the characterization of gene function. Small RNAs regulate gene expression in eukaryotic organisms in a post-transcriptional manner. Small interfering RNAs (siRNAs) are a group of short, double-stranded RNA (dsRNA) molecules that bind to and inhibit the translation of specific and complimentary mRNAs, thereby knocking-down target gene expression. The siRNA-mediated gene regulation that has initially evolved as a part of innate immunity in plants against invading pathogens also has assumed significance in plant development and has become one of the important tools in regulating mRNA abundance in cells in a post-transcriptional manner. The process of silencing of genes begins with the folding of complimentary RNAs and the production of small dsRNAs by the activity of type III ribonucleases. These small RNAs carry sequence-specific effectors of RNA silencing pathways that negatively regulate expression of genes, viruses, repetitive DNA sequences and transposons. In the current review, we briefly describe the siRNA-mediated regulation of gene expression and its merits and limitations as a reverse genetic tool for functional genomic studies in plants.

Regulation of a muralytic enzyme-encoding gene by two non-coding RNAs

ABSTRACTNon-coding regulatory RNAs fine-tune gene expression post-transcriptionally. In the streptomycetes, rpfA – encoding a muralytic enzyme required for establishing and exiting dormancy – is flanked by non-coding regulatory RNA elements both upstream (riboswitch) and downstream [antisense small RNA (sRNA)]. In Streptomyces coelicolor, the upstream riboswitch decreases rpfA transcript abundance in response to the second messenger cyclic di-AMP, itself involved in cell wall metabolism and dormancy. There is, however, no obvious expression platform associated with this riboswitch and consequently, its mechanism of action is entirely unknown. Using in vitro transcription assays, we discovered that the rpfA riboswitch promoted premature transcription termination in response to cyclic di-AMP. Through an extensive mutational analysis, we determined that attenuation required ligand binding and involved an unusual extended stem-loop region unique to a subset of rpfA riboswitches in the actinobacteria. At the other end of the rpfA gene, an antisense sRNA, termed Scr3097, is expressed opposite the predicted rpfA terminator. Using northern blotting, we found that Scr3097 accumulation mirrored that of the rpfA mRNA. In liquid culture, we detected Scr3097 exclusively in exponential-phase cells, and in plate-grown culture, we observed the sRNA primarily in differentiating cultures. Using mutational analyses, we found that the sRNA increased rpfA mRNA abundance in cells. Taken together, our work revealed multiple regulatory RNAs controlling rpfA expression in the streptomycetes.

Identification of Suitable Normalizing Genes for Quantitative Real-Time RT-PCR Analysis of Gene Expression in Fetal Mouse Gonads

In biological research, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assays are commonly employed to study mRNA abundance in cells and tissues. This type of assay usually relies on assessing transcript abundance relative to constitutively expressed endogenous reference genes. Therefore, it is important that the reference genes themselves are stably expressed in the cells or tissues analyzed, independent of factors such as age, sex, disease or experimental manipulations. Since no gene is expressed at the same level in all cells at all times, suitable reference genes must be identified for the specific cellular system or tissue being investigated. Here, we sought to identify stably expressed endogenous reference genes during embryonic gonad development in the mouse. We measured the transcript abundance of 10 frequently employed normalizing genes, of which 4 were stably expressed in fetal gonads from 11.5 to 14.5 dpc irrespective of sex. Based on our analysis, we suggest that Rn18s, Rps29, Tbp and Sdha are suitable reference genes for qRT-PCR expression studies during early gonad differentiation in the mouse.

Alternative polyadenylation signals in the 3′ non-coding region of a voltage-gated potassium channel gene are major determinants of mRNA isoform expression

We investigated the role of the 3′ non-coding region of a mouse voltage-gated potassium channel mRNA (mKv1.4 mRNA) in post-transcriptional regulation of gene expression. In contrast to an earlier report from studies carried out in Xenopus oocytes, we found that 3′ non-coding region sequences of mKv1.4 mRNAs did not significantly affect expression of a heterologous reporter RNA in vitro or in mammalian cells/cell lines. Instead, our data revealed a possible role for alternative polyadenylation mediated by distinct determinants ∼ 0.2 kb and ∼ 1.2 kb downstream of the Kv1.4 coding region. The use of the downstream polyadenylation signal correlated with the synthesis of a larger Kv1.4 mRNA isoform that was more abundantly expressed than the smaller mRNA species, whose expression was regulated by the upstream polyadenylation signal. Our results suggest that the relative strengths of the polyadenylation signals are major determinants of overall Kv1.4 mRNA abundance in cells.

Oestrogen receptor α is required for biochanin A-induced apolipoprotein A-1 mRNA expression in HepG2 cells

Epidemiological studies have indicated that soya consumption may produce a better plasma lipid profile. The effect may be attributed to the phyto-oestrogens in soya. The red clover (Trifolium pratense) isoflavone biochanin A has a chemical structure similar to those phyto-oestrogens found in soya beans, and is marketed as a nutraceutical for alleviating postmenopausal symptoms. In the present study we investigated the effect of biochanin A on the mRNA expression of ApoA-1 in the hepatic cell line HepG2. Real-time PCR revealed that biochanin A increased ApoA-1 mRNA abundance in cells expressing oestrogen receptor (ER) alpha. Without ERalpha transfection, biochanin A had no effect on mRNA abundance. In order to study the transcriptional control, a fragment of the 5'-flanking region of the ApoA-1 gene was amplified and inserted in a firefly luciferase reporter plasmid. The reporter assay indicated that the transactivation of the ApoA-1 promoter was induced by biochanin A in HepG2 cells transfected with the ERalpha expression plasmid. This induction was reduced by the anti-oestrogen ICI 182,780, whereas the inhibitors of protein kinase (PK) C, PKA, or mitogen-activated kinase (ERK) had no suppressive effect. The present study illustrated that biochanin A might up regulate hepatic apoA-1 mRNA expression through an ER-dependent pathway.

Green fluorescent protein is a quantitative reporter of gene expression in individual eukaryotic cells

Green fluorescent protein (GFP) has gained widespread use as a tool to visualize spatial and temporal patterns of gene expression in vivo. However, it is not generally accepted that GFP can also be used as a quantitative reporter of gene expression. We report that GFP is a reliable reporter of gene expression in individual eukaryotic cells when fluorescence is measured by flow cytometry. Two pieces of evidence support this conclusion: GFP fluorescence increases in direct proportion to the GFP gene copy number delivered to cells by a replication-defective adenovirus vector, Ad.CMV-GFP, and the intensity of GFP fluorescence is directly proportional to GFP mRNA abundance in cells. This conclusion is further supported by the fact that the induction of GFP gene expression from two inducible promoters (i.e., the TRE and ICP0 promoters) is readily detected by flow cytometric measurement of GFP fluorescent intensity. Collectively, the results presented herein indicate that GFP fluorescence is a reliable and quantitative reporter of underlying differences in gene expression.

Differential expression of Na:K:2Cl cotransporter, glucose transporter 1, and aquaporin 1 in freshly isolated and cultured bovine corneal tissues.

Little is known about whether culturing corneal limiting layers causes changes in the expression of their membrane transporter proteins from those present in fresh tissues. Accordingly, we compared mRNA abundance of three well-described types of transporters: water channel aquaporin 1 (AQP1), glucose transporter (GLUT1), and Na:K:2Cl cotransporter (NKCC), as well as NKCC protein levels in fresh bovine corneal epithelium and endothelium with those in their cultured counterparts. Abundance of mRNA encoding AQP1, GLUT1, and NKCC was quantified by a lysate nuclease protection assay. NKCC transcription was further characterized by Northern blotting. All data were normalized to cell DNA and protein contents. In the fresh epithelium, in all three cases mRNA levels were two to four times higher than in the endothelium. Expression of AQP1 and GLUT1 was 10 to 12 times higher than that of NKCC. After the third passage, the endothelial cell mRNA abundance in each case decreased 2- to 3-fold. Passage-dependent decreases were also observed in NKCC protein expression in the epithelial cells. In both corneal layers, there was a qualitative correlation between NKCC mRNA and protein levels. Both in fresh and cultured epithelial and endothelial cells, a shark NKCC1 DNA probe hybridized with mRNAs of two different lengths (about 5.0-5.5 and 7.0-7.5 kb). An anti-NKCC T4 monoclonal antibody recognized two major proteins with apparent molecular masses of 190 to 200 and 150 to 160 kDa. In summary, membrane transporter function in culture may not be always indicative of their role in fresh tissue since in cultured cells AQP1, GLUT1, and NKCC mRNA levels declined. Furthermore, in both epithelial and endothelial cells, there is expression of two different proteins and mRNAs that possibly encode for secretory (NKCC1) and absorptive (NKCC2) isoforms.

Medium tonicity regulates expression of the Na(+)- and Cl(-)-dependent betaine transporter in Madin-Darby canine kidney cells by increasing transcription of the transporter gene.

Betaine is one of the major compatible osmolytes accumulated by kidney derived Madin-Darby canine kidney cells cultured in hypertonic medium. Betaine is accumulated by Na(+)- and Cl(-)-dependent uptake from the medium. To gain insight into the mechanism by which hypertonicity evokes an increase in the Vmax of the betaine transporter in Madin-Darby canine kidney cells, we measured the relative abundance of mRNA for the transporter in cells shifted to a hypertonic medium and found parallel increases in mRNA abundance and cotransporter activity. The increase in mRNA levels preceded the increase in transporter activity slightly. Transcription of the gene for the transporter rose rapidly and to the same relative extent as mRNA abundance in cells shifted to hypertonic medium, indicating that transcription of the gene for the cotransporter plays a major role in regulating the accumulation of betaine in response to hypertonicity.