Search

Abstract Background We analysed 48 non-redundant antibiotic target proteins from all bacteria, 22 antibiotic target proteins from E. coli only and 4243 non-drug targets from E. coli to identify differences in their properties and to predict new potential drug targets. Results When compared to non-targets, bacterial antibiotic targets tend to be long, have high β-sheet and low α-helix contents, are polar, are found in the cytoplasm rather than in membranes, and are usually enzymes, with ligases particularly favoured. Sequence features were used to build a support vector machine model for E. coli proteins, allowing the assignment of any sequence to the drug target or non-target classes, with an accuracy in the training set of 94%. We identified 319 proteins (7%) in the non-target set that have target-like properties, many of which have unknown function. 63 of these proteins have significant and undesirable similarity to a human protein, leaving 256 target like proteins that are not present in humans. Conclusions We suggest that antibiotic discovery programs would be more likely to succeed if new targets are chosen from this set of target like proteins or their homologues. In particular, 64 are essential genes where the cell is not able to recover from a random insertion disruption.

Glucocorticoids (GC) like dexamethasone (Dex) are potent anti-inflammatory agents with diverse cellular functions including the potentiation of the activity of AU-rich elements (AREs). AREs are cis-acting instability sequence elements located in the 3'UTRs of many inflammatory mediator mRNAs. Here, available RNA-seq data were used to investigate the effect of GCs on the ARE-mRNA-transcriptome. At a global scale, ARE-mRNAs had a tendency to be downregulated after GC-treatment of the A549 lung cancer cell-line, but with notable cases of upregulation. mRNA stability experiments indicated that not only the downregulated, but also the upregulated ARE-mRNAs are destabilized by Dex-treatment. Several of the most upregulated ARE-mRNAs code for anti-inflammatory mediators including the established GC targets DUSP1 and ZFP36; both code for proteins that target ARE-containing mRNAs for destruction. GCs are widely used in the treatment of COVID-19 patients; we show that ARE-mRNAs are more likely to regulate in opposite directions between Dex-treatment and SARS-CoV-2 infections compared to non-ARE mRNAs. The effect of GC treatment on ARE-mRNA abundance was also investigated in blood monocytes of COVID-19 patients. The results were heterogeneous; however, in agreement with in vitro observations, ZFP36 and DUSP1 were often amongst the most differentially expressed mRNAs. The results of this study propose a universal destabilization of ARE-mRNAs by GCs, but a diverse overall outcome in vitro likely due to induced transcription or due to the heterogeneity of COVID-19 patient's responses in vivo., (© 2024. The Author(s).)

Background: AU-rich elements (AREs) are located in the 3'UTRs of 22% of human mRNAs, including most transiently expressed inflammatory mediators. By default, AREs mark mRNAs for decay and translational inhibition, but this activity can be temporarily inhibited in case of infection to allow the onset of inflammation. Morbidity and mortality in COVID-19 patients have been associated with dysregulated inflammation, a process that may include aberrant ARE activity., Results: RNA-seq data from available transcriptomic studies were analyzed to investigate a possible differential expression of mRNAs that contain AREs in the context of SARS-CoV-2 infections. ARE-mRNAs turned out to be significantly overrepresented among the upregulated mRNAs after SARS-CoV-2 infection (up to 42%). In contrast, ARE-mRNAs were underrepresented (16%) in the downregulated group. Consequently, at a global scale, ARE-mRNAs are significantly more upregulated after SARS-CoV-2 infection compared to non-ARE mRNAs. This observation was apparent in lung cell line models such as A549 and Calu-3 and with infections with other respiratory viruses and cell lines. Most importantly, at the clinical level, the elevated ARE-mRNA response appeared strongest in blood cells of COVID-19 patients with mild disease. It diminished with disease severity and was least apparent in patients in need of intubation and respiratory-related death. Gene function and clustering analysis suggest that the ARE-response is rather global and the upregulated ARE-mRNAs in patients with mild disease do not particularly cluster in specific functional groups., Conclusions: Compared to the rest of the transcriptome, ARE-containing mRNAs are preferentially upregulated in response to viral infections at a global level. In the context of COVID-19, they are most upregulated in mild disease. Due to their large number, their levels measured by RNA-seq may provide a reliable indication of COVID-19 severity., (© 2022. The Author(s).)

Recent years have witnessed major progress in development of novel therapeutic agents such as chemotherapy, targeted therapy and immune checkpoint inhibitors for breast cancer. However, cancer-related death remains high especially in triple-negative breast cancer (TNBC) due limited therapeutic options. Development of targeted therapies for TNBC requires better understanding of biology and signaling networks that promote disease progression. Fascin, an actin bundling protein, was identified as a key regulator of many signaling pathways that contribute to breast cancer progression. Herein, fascin ShRNA was used to generate stable fascin knockdown (FSCN1 KD ) in the MDA-MB-231 TNBC cell line and then were subjected to comprehensive mRNA and miRNA transcriptome analysis. We identified 129 upregulated and 114 downregulated mRNA transcripts, while 14 miRNAs were differentially expressed in FSCN1 KD . Ingenuity pathway analysis (IPA) was used to predict the impact of differentially expressed transcripts on signaling pathways and functional categories and to construct miRNA-mRNA regulatory networks in the context of FSCN1 knockdown. Compared to FSCN1 KD , fascin-positive (FSCN1 CON ) breast cancer cells showed enrichment in genes promoting cellular proliferation, migration, survival, DNA replication and repair. Expression of FSCN1 high (identified in BRCA dataset from TCGA) in conjunction with elevated expression of the top 10 upregulated or decreased expression of the top 10 downregulated genes (identified in our FSCN1 CON vs. FSCN1 KD ) correlates with worst survival outcome. Taken together, these data confirmed fascin's role in promoting TNBC progression, and identified a novel opportunity for therapeutic interventions via targeting those FSCN1-related transcripts.

Active cancer-associated fibroblasts (CAFs) are major components of the tumor microenvironment, which promote carcinogenesis and modulate response to therapy. Therefore, targeting these cells or reducing their paracrine pro-carcinogenic effects could be of great therapeutic value. To this end, we sought to investigate the effect of eugenol, a natural phenolic molecule, on active breast CAFs. We have shown that decitabine (5-Aza-2'-deoxycytidine, DAC) and eugenol inhibit the expression of the DNA methyltransferase genes DNMT1 and DNMT3A at both the protein and mRNA levels in breast CAF cells. While the effect of eugenol was persistent, DAC had only a transient inhibitory effect on the mRNA level of both DNMT genes. Furthermore, eugenol and DAC suppressed the invasive/migratory and proliferative potential of CAF cells as well as their paracrine pro-carcinogenic effects both in vitro and in humanized orthotopic tumor xenografts. Interestingly, these inhibitory effects of decitabine and eugenol were mediated through E2F1 downregulation. Indeed, ectopic expression of E2F1 upregulated both genes and attenuated the effects of eugenol. Additionally, we provide clear evidence that eugenol, like DAC, strongly modulates the methylation pattern in active CAF cells, through methylating several oncogenes and demethylating various important tumor suppressor genes, which affected their mRNA expression levels. Importantly, the E2F1 promoter was also hypermethylated and the gene downregulated in response to eugenol. Together, these findings show that the active features of breast CAF cells can be normalized through eugenol-dependent targeting of DNMT1/DNMT3A and the consequent modulation in gene methylation., (© 2021 Wiley Periodicals LLC.)

Cancer stem cells (CSCs), a rare population of tumor cells with high self-renewability potential, have gained increasing attention due to their contribution to chemoresistance and metastasis. We have previously demonstrated a critical role for the actin-bundling protein (fascin) in mediating breast cancer chemoresistance through activation of focal adhesion kinase (FAK). The latter is known to trigger the β-catenin signaling pathway. Whether fascin activation of FAK would ultimately trigger β-catenin signaling pathway has not been elucidated. Here, we assessed the effect of fascin manipulation in breast cancer cells on triggering β-catenin downstream targets and its dependence on FAK. Gain and loss of fascin expression showed its direct effect on the constitutive expression of β-catenin downstream targets and enhancement of self-renewability. In addition, fascin was essential for glycogen synthase kinase 3β inhibitor-mediated inducible expression and function of the β-catenin downstream targets. Importantly, fascin-mediated constitutive and inducible expression of β-catenin downstream targets, as well as its subsequent effect on CSC function, was at least partially FAK dependent. To assess the clinical relevance of the in vitro findings, we evaluated the consequence of fascin, FAK, and β-catenin downstream target coexpression on the outcome of breast cancer patient survival. Patients with coexpression of fascin high and FAK high or high β-catenin downstream targets showed the worst survival outcome, whereas in fascin low , patient coexpression of FAK high or high β-catenin targets had less significant effect on the survival. Altogether, our data demonstrated the critical role of fascin-mediated β-catenin activation and its dependence on intact FAK signaling to promote breast CSC function. These findings suggest that targeting of fascin-FAK-β-catenin axis may provide a novel therapeutic approach for eradication of breast cancer from the root., (Copyright © 2020 Barnawi, Al-Khaldi, Bakheet, Fallatah, Alaiya, Ghebeh and Al-Alwan.)

Adenylate-uridylate (AU)-rich elements (AREs) are sequence instability elements that are known to be located in the 3' untranslated regions (UTR) in thousands of human transcripts. AREs regulate the expression of many genes at the post-transcriptional level, and they are essential for many normal cellular functions. We conducted a transcriptome-wide screen for AREs and found that they are most abundant in introns, with up to 25% of introns containing AREs corresponding to 58% of human genes. Clustering studies of ARE size, complexity, and distribution revealed that, in introns, longer AREs with two or more overlapping repeats are more abundant than in the 3'UTR, and only introns can contain very long AREs with 6-14 overlapping AUUUA pentamers. We found that intronic sites of the ARE binding proteins HuR/ELAVL1, ZFP36/TTP, AUF1, and BRF1/ZFP36L1 overlap with the intronic AREs with HuR being most abundant. Accordingly, RNA-IP experiments demonstrated a specific association of HuR with reporter and endogenous pre-mRNAs that contain intronic AREs. Moreover, HuR knockdown led to a significant general reduction in the mRNA levels of genes that contain intronic AREs and to a specific reduction in the expression of ARE-intronic reporters. The data represent bioinformatics analysis for key RNA-binding proteins interactions with intronic AREs and provide experimental evidence for HuR binding to AREs. The widespread distribution of intronic AREs and their particular association with HuR and HuR binding sites indicates that more than half of human genes can be regulated post-transcriptionally by AREs., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Here we present an updated version of the AU-Rich Element Database (ARED-Plus) that is freely available at http://brp.kfshrc.edu.sa/ared. AREs are conserved sequence elements that were first discovered in the 3'UTR of mammalian transcripts. Over the past years, we compiled a series of ARE databases that revealed the extent and wide distribution of ARE-containing genes. For this update, we adopted an optimized search algorithm with improved specificity and sensitivity in ARE selection. The designation of the different ARE clusters was simplified by directly correlating the number of the ARE cluster to the number of overlapping AUUUA pentamers. Additionally, the new database was expanded to include genes with intronic AREs (pre-mRNAs) and their characteristics since recent observations reported their abundance and biological significance. Several enhancements were incorporated such as customized column view, additional search options and live search functionalities. The new version includes links to AREsite and AREScore, two related ARE assessment algorithms for further evaluation of the ARE characteristics. ARED-Plus now contains an updated repertoire of AREs in the human transcriptome that may be useful in several research fields., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Defects in AU-rich elements (ARE)-mediated posttranscriptional control can lead to several abnormal processes that underlie carcinogenesis. Here, we performed a systematic analysis of ARE-mRNA expression across multiple cancer types. First, the ARE database (ARED) was intersected with The Cancer Genome Atlas databases and others. A large set of ARE-mRNAs was over-represented in cancer and, unlike non-ARE-mRNAs, correlated with the reversed balance in the expression of the RNA-binding proteins tristetraprolin (TTP, ZFP36) and HuR (ELAVL1). Serial statistical and functional enrichment clustering identified a cluster of 11 overexpressed ARE-mRNAs (CDC6, KIF11, PRC1, NEK2, NCAPG, CENPA, NUF2, KIF18A, CENPE, PBK, TOP2A) that negatively correlated with TTP/HuR mRNA ratios and was involved in the mitotic cell cycle. This cluster was upregulated in a number of solid cancers. Experimentally, we demonstrated that the ARE-mRNA cluster is upregulated in a number of tumor breast cell lines when compared with noninvasive and normal-like breast cancer cells. RNA-IP demonstrated the association of the ARE-mRNAs with TTP and HuR. Experimental modulation of TTP or HuR expression led to changes in the mitosis ARE-mRNAs. Posttranscriptional reporter assays confirmed the functionality of AREs. Moreover, TTP augmented mitotic cell-cycle arrest as demonstrated by flow cytometry and histone H3 phosphorylation. We found that poor breast cancer patient survival was significantly associated with low TTP/HuR mRNA ratios and correlated with high levels of the mitotic ARE-mRNA signature. These results significantly broaden the role of AREs and their binding proteins in cancer, and demonstrate that TTP induces an antimitotic pathway that is diminished in cancer. Cancer Res; 76(14); 4068-80. ©2016 AACR., (©2016 American Association for Cancer Research.)

Transient response genes regulate critical biological responses that include cell proliferation, signal transduction events, and responses to exogenous agents such as inflammatory stimuli, microbes, and radiation. An important feature that ensures a timely response is the short half-life of the messenger RNA (mRNA), which is thought to be predominantly mediated by adenylate uridylate-rich sequence elements (AREs) in the 3' untranslated region (3' UTR). The repertoire and extent of transient response genes in the human genome are not known. We used a computational approach to delineate those genes that code for transient ARE mRNAs. We utilized a 3' UTR-specific ARE motif to retrieve and cluster 3'-end ESTs using a refined extraction protocol. With the availability of the entire human genome, we were able to utilize ARE EST clusters for further mining and computational prediction of ARE genes. The described approaches led to the finding of more than 1500 ARE genes in the human genome. In particular, "hidden" ARE mRNAs and alternative forms due to 3'UTR completeness, variant polyadenylation, and splicing were uncovered.

We evaluated the expression of over 900 AU-rich element (ARE)-containing transcripts in primary human T lymphocytes following stimulation with anti-CD3 and anti-CD28 antibodies and found that approximately 48% of these transcripts were regulated following T cell activation. We identified approximately 145 ARE-containing transcripts that were rapidly induced and then rapidly disappeared within 1 h after activation. Another 250 ARE-containing transcripts expressed in resting T cells were rapidly turned off within 30 min after activation. The rates of transcript disappearance correlated well with rapid mRNA decay measured following transcriptional arrest with actinomycin D. We identified a subset of ARE-containing transcripts that were rapidly induced following T cell activation that were also induced following lipopolysaccharide stimulation of THP-1 monocytes, and these transcripts exhibited rapid decay in both cell types. Our results suggest that ARE-mediated mRNA decay plays an important role in the precisely coordinated down-regulation of gene expression following immune cell activation.

Study of early and transient response gene expression is important for understanding the mechanisms of response to growth stimuli and exogenous agents such as microbes, stress, and radiation. Many of the cytokines, proto-oncogenes, and other transiently expressed gene products are encoded by mRNAs that contain AU-rich elements (AREs) in their 3' untranslated regions (UTRs). In this article, we describe an approach to selectively synthesize ARE-containing cDNA (ARE-cDNA) using an innovative combination of culture treatment, thermostabilization of reverse transcriptase (RT) by the disaccharide trehalose, and use of optimized ARE-specific oligomers. The monocytic cell line, THP-1, was treated with cycloheximide and endotoxin to enrich for ARE-mediated gene expression followed by the RT procedure. Selection of ARE-cDNA with simultaneous suppression of abundant cDNA was made possible using the procedure as monitored by the preferential expression of IL-8, an ARE-cDNA molecule, over the abundant housekeeping cDNA, beta-actin. The use of trehalose dramatically reversed cDNA abundance, resulting in almost complete suppression of housekeeping cDNA. Finally, construction of specialized ARE-cDNA libraries confirmed the selectivity of ARE-cDNAs and the presence of rare genes. The ability to reverse the abundance of housekeeping and other highly expressed genes toward ARE genes facilitates the discovery and study of rare early response and transiently expressed genes.

Adenylate/uridylate-rich element (ARE)-mediated mRNA turnover is an important regulatory component of gene expression for innate and specific immunity, in the hematopoietic system, in cellular growth regulation, and for many other cellular processes. This diversity is reflected in the distribution of AREs in the human genome, which we have established as a database of more than 900 ARE-containing genes that may utilize AREs as a means of controlling cellular mRNA levels. The p38 mitogen-activated protein kinase (MAP kinase) pathway has been implicated in regulating the stability of nine ARE-containing transcripts. Here we explored the entire spectrum of ARE-containing genes for p38-dependent regulation of ARE-mediated mRNA turnover with a custom cDNA array containing probes for 950 ARE mRNAs. The human monocytic cell line THP-1 treated with lipopolysaccharide (LPS) was used as a reproducible cellular model system that allowed us to precisely control the conditions of mRNA induction and decay in the absence and presence of the p38 inhibitor SB203580. This approach allowed us to establish an LPS-induced ARE mRNA expression profile in human monocytes and determine the half-lives of 470 AU-rich mRNAs. Most importantly, we identified 42 AU-rich genes, previously unrecognized, that show p38-dependent mRNA stabilization. In addition to a number of cytokines, several interesting novel AU-rich transcripts likely to play a role in macrophage activation by LPS exhibited p38-dependent transcript stabilization, including macrophage-specific colony-stimulating factor 1, carbonic anhydrase 2, Bcl2, Bcl2-like 2, and nuclear factor erythroid 2-like 2. Finally, the identification of the p38-dependent upstream activator MAP kinase kinase 6 as a member of this group identifies a positive feedback loop regulating macrophage signaling via p38 MAP kinase-dependent transcript stabilization.

Messenger RNAs that have the stability determinants, adenylate uridylate-rich elements (AREs), in their 3' untranslated region (UTR) code for key products that regulate early and transient biological responses. We used a computational laboratory approach for amplification of large, including full-length, protein-coding regions for ARE genes. Statistical analysis of the initiation regions in the 5' UTR of ARE-mRNAs was performed. Accordingly, several 5' primers and a single universal 3' primer that targeted the initiation consensuses and ARE regions, respectively, were designed. Using optimized conditions, the primers were able to enrich and amplify large protein-coding regions for the ARE gene family. The selective amplification of ARE cDNAs was verified using specific polymerase chain reactions (PCRs) to known ARE mRNA molecules and monitoring the abundance of the non-ARE beta-actin signal. A mini-library from the amplified ARE products was constructed for further confirmation of ARE selection. Distinct ARE amplified cDNA pools were selectively generated by distinct 5' primers. The biological utility of the method was shown with differential display. The up-regulation of several ARE-mRNAs, including the full-length coding region of the small inducible cytokine A4 (SCYA4) gene, was shown in endotoxin-stimulated monocytic cells. The integrated computational and laboratory approach should lead to enhanced capability for discovery and expression analysis of early and transient response genes.