CPEB2, CPEB3 and CPEB4 are coordinately regulated by miRNAs recognizing conserved binding sites in paralog positions of their 3′-UTRs

The cytoplasmic polyadenylation element binding-protein (CPEB) is an RNA-binding protein that participates in translational control. CPEB2, CPEB3 and CPEB4 are paralog proteins very similar among themselves referred as the CPEB2 subfamily. To gain insight into common mechanisms of regulation of the CPEB2 subfamily transcripts, we looked for putative cis-acting elements present in the 3′-UTRs of the three paralogs. We found different families of miRNAs predicted to target all subfamily members. Most predicted target sites for these families are located in paralog positions suggesting that these putative regulatory motifs were already present in the ancestral gene. We validated target sites for miR-92 and miR-26 in the three paralogs using mutagenesis of miRNA-binding sites in reporter constructs combined with over-expression and depletion of miRNAs. Both miR-92 and miR-26 induced a decrease in Luciferase activity associated to a reduction in mRNA levels of the reporter constructs. We also showed that the endogenous miRNAs co-regulate CPEB2, CPEB3 and CPEB4 transcripts, supporting our hypothesis that these genes have a common regulatory mechanism mediated by miRNAs. We also suggest that the ancestral pattern of miRNA-binding motifs was maintained throughout the generation of highly conserved elements in each of the 3′-UTRs

Histone deacetylase inhibitors suppress mechanical stress-induced expression of RUNX-2 and ADAMTS-5 through the inhibition of the MAPK signaling pathway in cultured human chondrocytes

Objective: To investigate the inhibitory effects and the regulatory mechanisms of histone deacetylase (HDAC) inhibitors on mechanical stress-induced gene expression of runt-related transcription factor (RUNX)-2 and a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS)-5 in human chondrocytes. Methods: Human chondrocytes were seeded in stretch chambers at a concentration of 5 x 10(4) cells/chamber. Cells were pre-incubated with or without HDAC inhibitors (MS-275 or trichostatin A; TSA) for 12 h, followed by uniaxial cyclic tensile strain (CTS) (0.5 Hz, 10% elongation), which was applied for 30 min using the ST-140-10 system (STREX, Osaka, Japan). Total RNA was extracted and the expression of RUNX-2, ADAMTS-5, matrix metalloproteinase (MMP)-3, and MMP-13 at the mRNA and protein levels were examined by real-time polymerase chain reaction (PCR) and immunocytochemistry, respectively. The activation of diverse mitogen-activated protein kinase (MAPK) pathways with or without HDAC inhibitors during CTS was examined by western blotting. Results: HDAC inhibitors (TSA: 10 nM, MS-275: 100 nM) suppressed CTS-induced expression of RUNX-2, ADAMTS-5, and MMP-3 at both the mRNA and protein levels within 1 h. CTS-induced activation of p38 MAPK (p38), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (INK) MAPKs was downregulated by both HDAC inhibitors. Conclusion: The CTS-induced expression of RUNX-2 and ADAMTS-5 was suppressed by HDAC inhibitors via the inhibition of the MAPK pathway activation in human chondrocytes. The results of the current study suggested a novel therapeutic role for HDAC inhibitors against degenerative joint disease such as osteoarthritis

Genomic and Transcriptional Co-Localization of Protein-Coding and Long Non-Coding RNA Pairs in the Developing Brain

Besides protein-coding mRNAs, eukaryotic transcriptomes include many long non-protein-coding RNAs (ncRNAs) of unknown function that are transcribed away from protein-coding loci. Here, we have identified 659 intergenic long ncRNAs whose genomic sequences individually exhibit evolutionary constraint, a hallmark of functionality. Of this set, those expressed in the brain are more frequently conserved and are significantly enriched with predicted RNA secondary structures. Furthermore, brain-expressed long ncRNAs are preferentially located adjacent to protein-coding genes that are (1) also expressed in the brain and (2) involved in transcriptional regulation or in nervous system development. This led us to the hypothesis that spatiotemporal co-expression of ncRNAs and nearby protein-coding genes represents a general phenomenon, a prediction that was confirmed subsequently by in situ hybridisation in developing and adult mouse brain. We provide the full set of constrained long ncRNAs as an important experimental resource and present, for the first time, substantive and predictive criteria for prioritising long ncRNA and mRNA transcript pairs when investigating their biological functions and contributions to development and disease

Next-generation sequencing-based genome diagnostics across clinical genetics centers: Implementation choices and their effects

Implementation of next-generation DNA sequencing (NGS) technology into routine diagnostic genome care requires strategic choices. Instead of theoretical discussions on the consequences of such choices, we compared NGS-based diagnostic practices in eight clinical genetic centers in the Netherlands, based on genetic testing of nine pre-selected patients with cardiomyopathy. We highlight critical implementation choices, including the specific contributions of laboratory and medical specialists, bioinformaticians and researchers to diagnostic genome care, and how these affect interpretation and reporting of variants. Reported pathogenic mutations were consistent for all but one patient. Of the two centers that were inconsistent in their diagnosis, one reported to have found 'no causal variant', thereby underdiagnosing this patient. The other provided an alternative diagnosis, identifying another variant as causal than the other centers. Ethical and legal analysis showed that informed consent procedures in all centers were generally adequate for diagnostic NGS applications that target a limited set of genes, but not for exome- and genome-based diagnosis. We propose changes to further improve and align these procedures, taking into account the blurring boundary between diagnostics and research, and specific counseling options for exome- and genome-based diagnostics. We conclude that alternative diagnoses may infer a certain level of 'greediness' to come to a positive diagnosis in interpreting sequencing results. Moreover, there is an increasing interdependence of clinic, diagnostics and research departments for comprehensive diagnostic genome care. Therefore, we invite clinical geneticists, physicians, researchers, bioinformatics experts and patients to reconsider their role and position in future diagnostic genome care

Mutations in KEOPS-Complex Genes Cause Nephrotic Syndrome with Primary Microcephaly

Galloway-Mowat syndrome (GAMOS) is an autosomal-recessive disease characterized by the combination of early-onset nephrotic syndrome (SRNS) and microcephaly with brain anomalies. Here we identified recessive mutations in OSGEP, TP53RK, TPRKB, and LAGE3, genes encoding the four subunits of the KEOPS complex, in 37 individuals from 32 families with GAMOS. CRISPR-Cas9 knockout in zebrafish and mice recapitulated the human phenotype of primary microcephaly and resulted in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibited cell proliferation, which human mutations did not rescue. Furthermore, knockdown of these genes impaired protein translation, caused endoplasmic reticulum stress, activated DNA-damage-response signaling, and ultimately induced apoptosis. Knockdown of OSGEP or TP53RK induced defects in the actin cytoskeleton and decreased the migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identified four new monogenic causes of GAMOS, describe a link between KEOPS function and human disease, and delineate potential pathogenic mechanisms

Analysis of the \u3b2-adducin gene: new insights into gene structure and function

Adducins are a family of membrane skeleton proteins encoded by three related genes (ADD1, ADD2 and ADD3 or a-, b- and g-adducin genes). Both ADD1 and ADD3 are ubiquitously expressed, while the b-adducin gene shows a pattern of expression restricted to brain and haematopoietic tissues. Adducin is found as either a heterodimer or heterotetramer of a/b or a/g subunit composition in most tissues. Human erythrocytes mainly contain a/b heterodimers, whereas the g-subunit is also found at low levels in mouse red blood cells (RBCs). [...

The human oncoprotein PRAME unveiled : interaction networks and genomic targets

Contains fulltext : 100889.pdf (publisher's version ) (Open Access)Radboud Universiteit Nijmegen, 21 januari 2013Promotor : Stunnenberg, H.G.192 p