Additional file 4: of RIP-Chip analysis supports different roles for AGO2 and GW182 proteins in recruiting and processing microRNA targets

Overview of gene expression levels in IP and FT samples. Focus on the enriched genes in AGO2-IP and GW182-IP vs FT samples. The reported expression levels are computed as the average values of the three performed experimental replicates. (PDF 1423 kb)

Cellular stress induces cap-independent alpha-enolase/MBP-1 translation.

AbstractMyc promoter-binding protein-1 (MBP-1) is a shorter protein variant of the glycolytic enzyme alpha-enolase. Although several lines of evidence indicate that MBP-1 acts as a tumor suppressor, the cellular mechanisms and signaling pathways underlying MBP-1 expression still remain largely elusive. To dissect these pathways, we used the SkBr3 breast cancer cell line and non-tumorigenic HEK293T cells ectopically overexpressing alpha-enolase/MBP-1. Here, we demonstrate that induced cell stresses promote MBP-1 expression through the AKT/PERK/eIF2α signaling axis. Our results contribute to shedding light on the molecular mechanisms underlying MBP-1 expression in non-tumorigenic and cancer c…

Assignment of enolase processed pseudogene (ENO1P) to human chromosome 1 bands 1q41→q42

Myc Promoter-Binding Protein-1 (MBP-1) Is a Novel Potential Prognostic Marker in Invasive Ductal Breast Carcinoma

BackgroundAlpha-enolase is a glycolytic enzyme that catalyses the formation of phosphoenolpyruvate in the cell cytoplasm. α-Enolase and the predominantly nuclear Myc promoter-binding protein-1 (MBP-1) originate from a single gene through the alternative use of translational starting sites. MBP-1 binds to the P2 c-myc promoter and competes with TATA-box binding protein (TBP) to suppress gene transcription. Although several studies have shown an antiproliferative effect of MBP-1 overexpression on several human cancer cells, to date detailed observations of α-enolase and MBP-1 relative expression in primary tumors versus normal tissues and their correlation with clinicopathological features ha…

Negative transcriptional control of ERBB2 gene by MBP-1 and HDAC1: diagnostic implications in breast cancer

Abstract Background The human ERBB2 gene is frequently amplified in breast tumors, and its high expression is associated with poor prognosis. We previously reported a significant inverse correlation between Myc promoter-binding protein-1 (MBP-1) and ERBB2 expression in primary breast invasive ductal carcinoma (IDC). MBP-1 is a transcriptional repressor of the c-MYC gene that acts by binding to the P2 promoter; only one other direct target of MBP-1, the COX2 gene, has been identified so far. Methods To gain new insights into the functional relationship linking MBP-1 and ERBB2 in breast cancer, we have investigated the effects of MBP-1 expression on endogenous ERBB2 transcript and protein lev…

DNA amplification in sea urchin oocytes.

In situ hybridization of ribosomal RNA withParacentrotus lividus ovaries suggests that ribosomal DNA undergoes amplification in the mononucleolate oocytes of this sea urchin.

The gene encoding the transcriptional repressor BERF-1 maps to a region of conserved synteny on mouse chromosome 16 and human chromosome 3 and a related pseudogene maps to mouse chromosome 8.

We have recently identified and characterized a Kruppel-like zinc finger protein (BERF-1), that functions as a repressor of β enolase gene transcription. By interspecific backcross analysis the gene encoding BERF-1 was localized 4.7 cM proximal to the <i>Mtv6</i> locus on mouse chromosome 16, and an isolated pseudogene was localized to mouse chromosome 8, about 5.3 cM distal to the D8Mit4 marker. Nucleotide sequence identity and chomosome location indicate that the gene encoding BERF-1 is the mouse homologue (<i>Zfp148</i>) of ZNF148 localized to human chromosome 3q21, a common translocation site in acute myeloid leukemia patients.

Gamma enolase expression as early marker of neuronal differentiation of murine neuroblastoma cells N-115

In this study we determined the levels of gamma enolase mRNA in mouse neuroblastoma cell line N-115 at early period of induction of differentiation by serum withdrawal. The expression of gamma enolase was examined by Northern blot analysis of total RNA extracted from cells induced for different lengths of time. We found a 3-fold increase in the level of gamma enolase mRNA after 24 hours of induction of differentiation and higher levels were detected in cells induced for longer time, reaching a 10-fold increase after four days.

Palmitoylation is a post-translational modification of Alix regulating the membrane organization of exosome-like small extracellular vesicles.

Abstract Background Virtually all cell types have the capacity to secrete nanometer-sized extracellular vesicles, which have emerged in recent years as potent signal transducers and cell-cell communicators. The multifunctional protein Alix is a bona fide exosomal regulator and skeletal muscle cells can release Alix-positive nano-sized extracellular vesicles, offering a new paradigm for understanding how myofibers communicate within skeletal muscle and with other organs. S-palmitoylation is a reversible lipid post-translational modification, involved in different biological processes, such as the trafficking of membrane proteins, achievement of stable protein conformations, and stabilization…

Conserved alternative splicing in the 5'-untranslated region of the muscle-specific enolase gene. Primary structure of mRNAs, expression and influence of secondary structure on the translation efficiency.

We report here the isolation and characterization of cDNAs covering the 5'-end region of mouse and rat mRNAs that encode the beta or muscle-specific isoform of the glycolytic enzyme enolase. As previously determined for humans, two classes of beta-enolase transcripts with distinct sequences in their 5'-untranslated regions are present in both mouse and rat muscles. A mechanism of alternative splicing, conserved from mouse to man, generates the two forms of mRNA. Secondary-structure predictions indicated that, in all cases, a more stable secondary structure could exist in the 5' end of the message with the longer leader. In vitro transcripts containing defined human or mouse 5'-untranslated …

Additional file 6: of RIP-Chip analysis supports different roles for AGO2 and GW182 proteins in recruiting and processing microRNA targets

Wilcoxon test p-values summary. Wilcoxon test p-values (log10) obtained by comparing the variable values associated with the enriched/underrepresented genes sets. Three different miRNA target prediction tools (Targetscan, PITA and miRanda) were used to compute the necessary binding sites (BS) matrices. The BS matrices used to compute the p-values in the last panel were obtained by considering BS predicted by at least two of the three prediction tools. In each panel, the variables computed with the three AGO2 IN profiles were used to distinguish enriched and underrepresented genes in AGO2-IP vs FT and the variables computed with the three GW182 IN profiles were used to distinguish enriched a…

The Kelch protein NS1-BP interacts with alpha-enolase/MBP-1 and is involved in c-Myc gene transcriptional control

Alpha-enolase is a key glycolytic enzyme that plays a functional role in several physiological processes depending on the cellular localization. The enzyme is mainly localized in the cytoplasm whereas an alternative translated form, named MBP-1, is predominantly nuclear. The MBP-1 protein has been characterized as a c-Myc promoter binding protein that negatively controls transcription. In the present study, we identified the kelch protein NS1-BP as one of the alpha-enolase/MBP-1 partners by using a yeast two-hybrid screening. Although NS1-BP has been originally described as a protein mainly localized in the nucleus, we provide evidence that NS1-BP also interacts with actin in human cells, a…

Phenols content and 2-D electrophoresis protein pattern: a promising tool to monitor Posidonia meadows health state

Abstract Background The endemic seagrass Posidonia oceanica (L.) Delile colonizes soft bottoms producing highly productive meadows that play a crucial role in coastal ecosystems dynamics. Human activities and natural events are responsible for a widespread meadows regression; to date the identification of "diagnostic" tools to monitor conservation status is a critical issue. In this study the feasibility of a novel tool to evaluate ecological impacts on Posidonia meadows has been tested. Quantification of a putative stress indicator, i.e. phenols content, has been coupled to 2-D electrophoretic protein analysis of rhizome samples. Results The overall expression pattern from Posidonia rhizom…

Human airway epithelial extracellular vesicle miRNA signature is altered upon asthma development

Background: miRNAs are master regulators of signaling pathways critically involved in asthma and are transferred between cells in extracellular vesicles (EV). We aimed to investigate whether the miRNA content of EV secreted by primary normal human bronchial epithelial cells (NHBE) is altered upon asthma development. Methods: NHBE cells were cultured at air-liquid interface and treated with interleukin (IL)-13 to induce an asthma-like phenotype. EV isolations by precipitation from basal culture medium or apical surface wash were characterized by nanoparticle tracking analysis, transmission electron microscopy, and Western blot, and EV-associated miRNAs were identified by a RT-qPCR-based prof…

Detecting significant features in modeling microRNA-target interactions

MicroRNAs (miRNAs) are small non-coding RNA molecules mediating the translational repression and degradation of target mRNAs in the cell. Mature miRNAs are used as a template by the RNA-induced silencing complex (RISC) to recognize the complementary mRNAs to be regulated. Up to 60% of human genes are putative targets of one or more miRNAs. Several prediction tools are available to suggest putative miRNA targets, however, only a small part of the interaction pairs has been validated by experimental approaches. The analysis of the expression profile of the RNA fraction immunoprecipitated (IP) with the RISC proteins is an established method to detect which genes are actually regulated by the R…

Additional file 7: of RIP-Chip analysis supports different roles for AGO2 and GW182 proteins in recruiting and processing microRNA targets

Summary of miRNA expression profiles shuffling effects. ROC analysis was performed to evaluate the performance of F6 and F4d variables, computed with simulated miRNA profiles, in distinguishing enriched/underrepresented genes in AGO2 or GW182-IP samples. Each panel reports the AUC values obtained with simulated variables. Each boxplot refers to AUC values obtained with a specific set of simulations, where the expression profile of a set of miRNAs was shuffled. The boxplot in the center was obtained by shuffling all miRNAs. The boxplots from the center to the right refer to simulations where all the miRNAs were shuffled with the exception of n top expressed miRNAs, n increasing in the right …

Conserved Structure and Promoter Sequence Similarity in the Mouse and Human Genes Encoding the Zinc Finger Factor BERF-1/BFCOL1/ZBP-89

Abstract We have characterized the genomic structure of the mouse Zfp148 gene encoding Beta-Enolase Repressor Factor-1 (BERF-1), a Kruppel-like zinc finger protein involved in the transcriptional regulation of several genes, which is also termed ZBP-89, BFCOL1. The cloned Zfp148 gene spans 110 kb of genomic DNA encompassing the 5′-end region, 9 exons, 8 introns, and the 3′-untranslated region. The promoter region displays the typical features of a housekeeping gene: a high G+C content and the absence of canonical TATA and CAAT boxes consistent with the multiple transcription initiation sites determined by primary extension analysis. Computer-assisted search in the human genome database allo…

RIP-Chip analysis supports different roles for AGO2 and GW182 proteins in recruiting and processing microRNA targets.

Background MicroRNAs (miRNAs) are small non-coding RNA molecules mediating the translational repression and degradation of target mRNAs in the cell. Mature miRNAs are used as a template by the RNA-induced silencing complex (RISC) to recognize the complementary mRNAs to be regulated. To discern further RISC functions, we analyzed the activities of two RISC proteins, AGO2 and GW182, in the MCF-7 human breast cancer cell line. Methods We performed three RIP-Chip experiments using either anti-AGO2 or anti-GW182 antibodies and compiled a data set made up of the miRNA and mRNA expression profiles of three samples for each experiment. Specifically, we analyzed the input sample, the immunoprecipita…

Additional file 5: of RIP-Chip analysis supports different roles for AGO2 and GW182 proteins in recruiting and processing microRNA targets

Venn diagram of lists of enriched genes. The considered lists are: AGO2-IP (UP_AGO2 set), the list of enriched genes detected by Fan et al. [13] (UP_AGO2_Fan) and our list of enriched genes in GW182-IP sample (UP_GW182). The reported p-values refer to the closest intersection set of genes and are computed with one tail Fisher-test. (PDF 34 kb)

The kelch protein NS1-BP interacts with alpha-enolase/MBP-1 and is involved in c-Myc gene transcriptional control

Additional file 1: of RIP-Chip analysis supports different roles for AGO2 and GW182 proteins in recruiting and processing microRNA targets

Analysis of miRNA expression in AGO2 and GW182-IP samples. a) miRNA expression level in AGO2-IP samples (average value from the three performed experiments) vs the expression level in IN samples (average value from the three performed experiments). The Pearson correlation values reported on the top of the picture were computed by using all the expressed miRNA, and the top 100 or 50 expressed miRNAs. The colored points refer to miRNA that have been validated by RT-PCR data. Green points refer to hsa-miR-141-3p, hsa-miR-21-5p, hsa-let-7f-5p, hsa-miR-16-5p, hsa-miR-24-3p, hsa-miR-27a-3p, hsa-miR-23a-3p. The red point refers to hsa-miR-1260a. b) Comparison of IP/IN ratios obtained by RT-PCR dat…

ENO1 gene product binds to the c-myc promoter and acts as a transcriptional repressor: relationship with Myc promoter-binding protein 1 (MBP-1).

The Myc promoter-binding protein-1 (MBP-1) is a 37-38 kDa protein that binds to the c-myc P2 promoter and negatively regulates transcription of the protooncogene. MBP-1 cDNA shares 97% similarity with the cDNA encoding the glycolytic enzyme alpha-enolase and both genes have been mapped to the same region of human chromosome 1, suggesting the hypothesis that the two proteins might be encoded by the same gene. We show here data indicating that a 37 kDa protein is alternatively translated from the full-length alpha-enolase mRNA. This shorter form of alpha-enolase is able to bind the MBP-1 consensus sequence and to downregulate expression of a luciferase reporter gene under the control of the c…

Additional file 8: of RIP-Chip analysis supports different roles for AGO2 and GW182 proteins in recruiting and processing microRNA targets

Empirical Cumulative Distribution Function of 3â UTR and coding region length of IP-Enriched genes. Enriched genes in AGO (1â 4) and in GW182 protein family IP selected by considering log2 IP-Enrichment of transcript greater than 1. Data are downloaded from Landthaler et al. [14]. The Empirical Cumulative Distribution Function of the 3â UTR length (top) and coding region length (bottom) of genes enriched exclusively by AGO-IP (red line), GW182-IP (blue line) and both IPs (black line) are reported. The reported p-value is computed by performing a Wilcoxon test to compare the length distributions of genes enriched exclusively in AGO-IP and in GW182-IP. (PDF 145 kb)

Pro-invasive stimuli and the interacting protein Hsp70 favour the route of alpha-enolase to the cell surface

AbstractCell surface expression of alpha-enolase, a glycolytic enzyme displaying moonlighting activities, has been shown to contribute to the motility and invasiveness of cancer cells through the protein non-enzymatic function of binding plasminogen and enhancing plasmin formation. Although a few recent records indicate the involvement of protein partners in the localization of alpha-enolase to the plasma membrane, the cellular mechanisms underlying surface exposure remain largely elusive. Searching for novel interactors and signalling pathways, we used low-metastatic breast cancer cells, a doxorubicin-resistant counterpart and a non-tumourigenic mammary epithelial cell line. Here, we demon…

Negative Regulation of β Enolase Gene Transcription in Embryonic Muscle Is Dependent upon a Zinc Finger Factor That Binds to the G-rich Box within the Muscle-specific Enhancer

We have previously identified a muscle-specific enhancer within the first intron of the human beta enolase gene. Present in this enhancer are an A/T-rich box that binds MEF-2 protein(s) and a G-rich box (AGTGGGGGAGGGGGCTGCG) that interacts with ubiquitously expressed factors. Both elements are required for tissue-specific expression of the gene in skeletal muscle cells. Here, we report the identification and characterization of a Kruppel-like zinc finger protein, termed beta enolase repressor factor 1, that binds in a sequence-specific manner to the G-rich box and functions as a repressor of the beta enolase gene transcription in transient transfection assays. Using fusion polypeptides of b…

An integrated humoral and cellular response is elicited in pancreatic cancer by alpha-enolase, a novel pancreatic ductal adenocarcinoma-associated antigen

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with a very poor 5-year survival rate. alpha-Enolase is a glycolytic enzyme that also acts as a surface plasminogen receptor. We find that it is overexpressed in PDAC and present on the cell surface of PDAC cell lines. The clinical correlation of its expression with tumor status has been reported for lung and hepatocellular carcinoma. We have previously demonstrated that sera from PDAC patients contain IgG autoantibodies to alpha-enolase. The present work was intended to assess the ability of alpha-enolase to induce antigen-specific T cell responses. We show that alpha-enolase-pulsed dendritic cells (DC) specifically stimulate healt…

Additional file 9: of RIP-Chip analysis supports different roles for AGO2 and GW182 proteins in recruiting and processing microRNA targets

Summary of miRNA expression profiles switch between experiment replicas. ROC analysis of F6&F4d SVM model trained with variables calculated with miRNA expression profiles from each of the three anti-AGO2 RIP experiments. SVM models were used to classify the top 1000 and the bottom 1000 genes with respect to the IP/FT mRNA expression ratio, computed for each of the three AGO2 RIP experiments. (PDF 653 kb)

The PVT-1 oncogene is a Myc protein target that is overexpressed in transformed cells

The human PVT-1 gene is located on chromosome 8 telomeric to the c-Myc gene and it is frequently involved in the translocations occurring in variant Burkitt's lymphomas and murine plasmacytomas. It has been proposed that PVT-1 regulates c-Myc gene transcription over a long distance. To get new insights into the functional relationships between the two genes, we have investigated PVT-1 and c-Myc expression in normal human tissues and in transformed cells. Our findings indicate that PVT-1 expression is restricted to a relative low number of normal tissues compared to the wide distribution of c-Myc mRNA, whereas the gene is highly expressed in many transformed cell types including neuroblastom…

Additional file 3: of RIP-Chip analysis supports different roles for AGO2 and GW182 proteins in recruiting and processing microRNA targets

Summary of enriched and underrepresented genes. Summary of enriched and underrepresented genes in AGO2 and GW182-IP vs FT comparisons performed by SAMR (column 2–3). The enrichment results obtained with the REA algorithm are reported in columns 4–5. Columns 6 and 7 report the 3’UTR and Coding region (CR) lengths respectively. In columns 8–21 we report the number of binding sites predicted by Targetscan in the 3’UTR and the Coding region of seven highly expressed miRNAs. (XLS 294 kb)

Additional file 2: of RIP-Chip analysis supports different roles for AGO2 and GW182 proteins in recruiting and processing microRNA targets

Gene set enrichment analysis results with seven top expressed miRNA predicted targets sets. Predicted targets of miRNAs (column 1) were predicted with three different target prediction tools (column 2). The total number of predicted targets is indicated in column 3. Five lists of genes were analyzed. For each list of genes the number of genes in common with the predicted targets and the associated hypergeometric test pvalue are provided. The total number of genes considered in the analysis is 16,392. The five considered lists are: a list of genes enriched in AGO2 IP sample from [13]; lists of genes enriched in AGO2 IP vs IN and IP vs FT samples; lists of genes enriched in GW182 IP vs IN and…