A high-throughput 3' UTR reporter screening identifies microRNA interactomes of cancer genes.

Introduction: Despite the established contribution of deregulated microRNA (miRNA) function to carcinogenesis, relatively few miRNA-cancer gene interactions have been validated, making it difficult to appreciate the true complexity of miRNA-cancer gene regulatory networks.

Results: In this effort, we identify miRNA interactomes of 17 well-established cancer genes, involved in various cancer types, through a miRNome-wide 3' UTR reporter screening. Using a novel and performant strategy for high-throughput screening data analysis, we identify 390 interactions, quadrupling the size of the known miRNA interactome for the cancer genes under investigation.

Regulation of G Protein-Coupled Receptors by Ubiquitination.

G protein-coupled receptors (GPCRs) comprise the largest family of membrane receptors that control many cellular processes and consequently often serve as drug targets. These receptors undergo a strict regulation by mechanisms such as internalization and desensitization, which are strongly influenced by posttranslational modifications. Ubiquitination is a posttranslational modification with a broad range of functions that is currently gaining increased appreciation as a regulator of GPCR activity.

Characterization of the interaction between the dopamine D4 receptor, KLHL12 and β-arrestins.

Dopamine receptors are G protein-coupled receptors involved in regulation of cognition, learning, movement and endocrine signaling. The action of G protein-coupled receptors is highly regulated by multifunctional proteins, such as β-arrestins which can control receptor desensitization, ubiquitination and signaling. Previously, we have reported that β-arrestin 2 interacts with KLHL12, a BTB-Kelch protein which functions as an adaptor in a Cullin3-based E3 ligase complex and promotes ubiquitination of the dopamine D4 receptor.

Dopamine D4 receptor ubiquitination.

Ubiquitination is a post-translational modification that targets proteins for degradation but can also regulate other cellular processes such as endocytosis, trafficking and DNA repair. We investigate ubiquitination of the dopamine D4receptor (D4R) which belongs to the superfamily of G protein-coupled receptors (GPCR). Several polymorphic variants of the D4R exist, which differ in the number of 16-amino acid repeats in the third intracellular loop (IC3) of the receptor.

KLHL12 Promotes Non-Lysine Ubiquitination of the Dopamine Receptors D4.2 and D4.4, but Not of the ADHD-Associated D4.7 Variant.

Dopamine D4 Receptor Polymorphism: The dopamine D4 receptor has an important polymorphism in its third intracellular loop that is intensively studied and has been associated with several abnormal conditions, among others, attention deficit hyperactivity disorder.

Klhl12 Promotes Ubiquitination Of The Dopamine D4 Receptor On Non-lysine Residues: In previous studies we have shown that KLHL12, a BTB-Kelch protein, specifically interacts with the polymorphic repeats of the dopamine D4 receptor and enhances its ubiquitination, which, however, has no influence on receptor degradation. In this study we provide evidence that KLHL12 promotes ubiquitination of the dopamine D4 receptor on non-lysine residues.

Novel biological insights in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive type of blood cancer that accounts for about 15% of pediatric and 25% of adult acute lymphoblastic leukemia (ALL) cases. It is considered as a paradigm for the multistep nature of cancer initiation and progression. Genetic and epigenetic reprogramming events, which transform T-cell precursors into malignant T-ALL lymphoblasts, have been extensively characterized over the past decade.

ZEB2 drives immature T-cell lymphoblastic leukaemia development via enhanced tumour-initiating potential and IL-7 receptor signalling.

Early T-cell precursor leukaemia (ETP-ALL) is a high-risk subtype of human leukaemia that is poorly understood at the molecular level. Here we report translocations targeting the zinc finger E-box-binding transcription factor ZEB2 as a recurrent genetic lesion in immature/ETP-ALL. Using a conditional gain-of-function mouse model, we demonstrate that sustained Zeb2 expression initiates T-cell leukaemia.

Characterization of a set of tumor suppressor microRNAs in T cell acute lymphoblastic leukemia.

The posttranscriptional control of gene expression by microRNAs (miRNAs) is highly redundant, and compensatory effects limit the consequences of the inactivation of individual miRNAs. This implies that only a few miRNAs can function as effective tumor suppressors. It is also the basis of our strategy to define functionally relevant miRNA target genes that are not under redundant control by other miRNAs.

The Notch driven long non-coding RNA repertoire in T-cell acute lymphoblastic leukemia.

Genetic studies in T-cell acute lymphoblastic leukemia have uncovered a remarkable complexity of oncogenic and loss-of-function mutations. Amongst this plethora of genetic changes, NOTCH1 activating mutations stand out as the most frequently occurring genetic defect, identified in more than 50% of T-cell acute lymphoblastic leukemias, supporting a role as an essential driver for this gene in T-cell acute lymphoblastic leukemia oncogenesis. In this study, we aimed to establish a comprehensive compendium of the long non-coding RNA transcriptome under control of Notch signaling.

The H3K27me3 demethylase UTX is a gender-specific tumor suppressor in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of leukemia that is mainly diagnosed in children and shows a skewed gender distribution toward males. In this study, we report somatic loss-of-function mutations in the X-linked histone H3K27me3 demethylase ubiquitously transcribed X (UTX) chromosome, in human T-ALL. Interestingly, UTX mutations were exclusively present in male T-ALL patients and allelic expression analysis revealed that UTX escapes X-inactivation in female T-ALL lymphoblasts and normal T cells.

Expressed repeat elements improve RT-qPCR normalization across a wide range of zebrafish gene expression studies.

The selection and validation of stably expressed reference genes is a critical issue for proper RT-qPCR data normalization. In zebrafish expression studies, many commonly used reference genes are not generally applicable given their variability in expression levels under a variety of experimental conditions. Inappropriate use of these reference genes may lead to false interpretation of expression data and unreliable conclusions.

RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer.

The translational control of oncoprotein expression is implicated in many cancers. Here we report an eIF4A RNA helicase-dependent mechanism of translational control that contributes to oncogenesis and underlies the anticancer effects of silvestrol and related compounds. For example, eIF4A promotes T-cell acute lymphoblastic leukaemia development in vivo and is required for leukaemia maintenance.

MicroRNA-128-3p is a novel oncomiR targeting PHF6 in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia arises from the leukemic transformation of developing thymocytes and results from cooperative genetic lesions. Inactivation of the PHF6 gene is frequently observed in T-cell acute lymphoblastic leukemia, suggesting an important tumor suppressive role for PHF6 in the pathobiology of this leukemia. Although the precise function of PHF6 is still unknown, this gene is most likely involved in chromatin regulation, a strongly emerging theme in T-cell acute lymphoblastic leukemia.

The need for transparency and good practices in the qPCR literature.

Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.

Effective Alu repeat based RT-Qpcr normalization in cancer cell perturbation experiments.

Background: Measuring messenger RNA (mRNA) levels using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) is common practice in many laboratories. A specific set of mRNAs as internal control reference genes is considered as the preferred strategy to normalize RT-qPCR data. Proper selection of reference genes is a critical issue, especially in cancer cells that are subjected to different in vitro manipulations.

EVI1 inhibits apoptosis induced by antileukemic drugs via upregulation of CDKN1A/p21/WAF in human myeloid cells.

Overexpression of ecotropic viral integration site 1 (EVI1) is associated with aggressive disease in acute myeloid leukemia (AML). Despite of its clinical importance, little is known about the mechanism through which EVI1 confers resistance to antileukemic drugs. Here, we show that a human myeloid cell line constitutively overexpressing EVI1 after infection with a retroviral vector (U937_EVI1) was partially resistant to etoposide and daunorubicin as compared to empty vector infected control cells (U937_vec).

A cooperative microRNA-tumor suppressor gene network in acute T-cell lymphoblastic leukemia (T-ALL).

The importance of individual microRNAs (miRNAs) has been established in specific cancers. However, a comprehensive analysis of the contribution of miRNAs to the pathogenesis of any specific cancer is lacking. Here we show that in T-cell acute lymphoblastic leukemia (T-ALL), a small set of miRNAs is responsible for the cooperative suppression of several tumor suppressor genes.

EVI1-mediated down regulation of MIR449A is essential for the survival of EVI1 positive leukaemic cells.

Chromosomal rearrangements involving the MECOM (MDS1 and EVI1 complex) locus are recurrent genetic events in myeloid leukaemia and are associated with poor prognosis. In this study, we assessed the role of MECOM locus protein EVI1 in the transcriptional regulation of microRNAs (miRNAs) involved in the leukaemic phenotype. For this, we profiled expression of 366 miRNAs in 38 MECOM-rearranged patient samples, normal bone marrow controls and MECOM (EVI1) knock down/re-expression models.

Dopamine D4 receptor oligomerization--contribution to receptor biogenesis.

Dopamine D(4) receptors (D(4) Rs) are G protein-coupled receptors that play a role in attention and cognition. In the present study, we investigated the dimerization properties of this receptor. Western blot analysis of the human D(4.

IL-1β potently stabilizes IL-6 mRNA in human astrocytes.

Uncontrolled expression of IL-6 in the central nervous system is associated with neurodegenerative pathology and glioma development. Astrocytes are the predominant source of IL-6 in the central nervous system, and they are characteristically susceptible to synergistic IL-6 expression. Combined β-adrenergic and TNF-receptor triggering induces synergistic IL-6 expression in 1321N1 cells via a transcriptional enhancer mechanism.

The dopamine D4 receptor: biochemical and signalling properties.

Dopamine is an important neurotransmitter that regulates several key functions in the brain, such as motor output, motivation and reward, learning and memory, and endocrine regulation. Dopamine does not mediate fast synaptic transmission, but rather modulates it by triggering slow-acting effects through the activation of dopamine receptors, which belong to the G-protein-coupled receptor superfamily. Besides activating different effectors through G-protein coupling, dopamine receptors also signal through interaction with a variety of proteins, collectively termed dopamine receptor-interacting proteins.

KLHL12-mediated ubiquitination of the dopamine D4 receptor does not target the receptor for degradation.

In previous studies, we identified KLHL12 as a novel interaction partner of the dopamine D4 receptor that functions as an adaptor in a Cullin3-based E3 ubiquitin ligase complex to target the receptor for ubiquitination. In this study, we show that KLHL12 promotes poly-ubiquitination of the receptor by performing ubiquitination assays in eukaryotic cells. Furthermore, we demonstrate that KLHL12 not only interacts with both immature, ER-associated and mature, plasma membrane-associated D4 receptors, but also promotes ubiquitination of both receptor subpools.

Resistance of the dopamine D4 receptor to agonist-induced internalization and degradation.

Dopamine receptors are G-protein-coupled receptors involved in the control of motivation, learning, and fine-tuning of motor movement, as well as modulation of neuroendocrine signalling. Stimulation of G-protein-coupled receptors normally results in attenuation of signalling through desensitization, followed by internalization and down-regulation of the receptor. These processes allow the cell to regain homeostasis after exposure to extracellular stimuli and offer protection against excessive signalling.

BTB Protein KLHL12 targets the dopamine D4 receptor for ubiquitination by a Cul3-based E3 ligase.

Dopamine receptors belong to the superfamily of G-protein-coupled receptors and are subdivided into D1-type (D1 and D5) and D2-type (D2, D3, and D4) receptors. The D4 receptor has a remarkable polymorphism in its third intracellular loop, which is under intensive investigation and which has been associated with, among other conditions, attention deficit hyperactivity disorder. Here, we demonstrate that KLHL12, a BTB-Kelch protein, specifically binds to this polymorphic region of the D4 receptor through its Kelch domain.