Shutdown of achaete-scute homolog-1 expression by heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1 in hypoxia

The basic helix-loop-helix transcription factor hASH1, encoded by the ASCL1 gene, plays an important role in neurogenesis and tumor development. Recent findings indicate that the local oxygen tension is a critical determinant for the progression of neuroblastomas. Here we investigated the molecular mechanisms underlying the oxygen-dependent expression of hASH1 in neuroblastoma cells. Exposure of human neuroblastoma-derived Kelly cells to 1% O2 significantly decreased ASCL1 mRNA and hASH1 protein levels. Using reporter gene assays, we show that the response of hASH1 to hypoxia is mediated mainly by post-transcriptional inhibition via the ASCL1 mRNA 5'- and 3'-UTRs, while additional inhibition of the ASCL1 promoter was observed under prolonged hypoxia. By RNA pull-down experiments followed by MALDI/TOF-MS analysis, we identified heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1 and hnRNP-R as interactors binding directly to the ASCL1 mRNA 5'- and 3'-UTRs and influencing its expression. We further demonstrate that hnRNP-A2/B1 is a key positive regulator of ASCL1, findings that were also confirmed by analysis of a large compilation of gene expression data. Our data suggest that a prominent down-regulation of hnRNP-A2/B1 during hypoxia is associated with the post-transcriptional suppression of hASH1 synthesis. This novel post-transcriptional mechanism for regulating hASH1 levels will have important implications in neural cell fate development and disease

The Association of Fatigue With Decreasing Regularity of Locomotion During an Incremental Test in Trained and Untrained Healthy Adults

Fatigue is a key factor that affects human motion and modulates physiology, biochemistry, and performance. Prolonged cyclic human movements (locomotion primarily) are characterized by a regular pattern, and this extended activity can induce fatigue. However, the relationship between fatigue and regularity has not yet been extensively studied. Wearable sensor methodologies can be used to monitor regularity during standardized treadmill tests (e.g., the widely used Bruce test) and to verify the effects of fatigue on locomotion regularity. Our study on 50 healthy adults [27 males and 23 females; <40 years; five dropouts; and 22 trained (T) and 23 untrained (U) subjects] showed how locomotion regularity follows a parabolic profile during the incremental test, without exception. At the beginning of the trial, increased walking speed in the absence of fatigue is associated with increased regularity (regularity index, RI, a. u., null/unity value for aperiodic/periodic patterns) up until a peak value (RI = 0.909 after 13.8 min for T and RI = 0.915 after 13.4 min for U subjects; median values, n. s.) and which is then generally followed (after 2.8 and 2.5 min, respectively, for T/U, n. s.) by the walk-to-run transition (at 12.1 min for both T and U, n. s.). Regularity then decreases with increased speed/slope/fatigue. The effect of being trained was associated with significantly higher initial regularity [0.845 (T) vs 0.810 (U), p < 0.05 corrected], longer test endurance [23.0 min (T) vs 18.6 min (U)], and prolonged decay of locomotor regularity [8.6 min (T) vs 6.5 min (U)]. In conclusion, the monitoring of locomotion regularity can be applied to the Bruce test, resulting in a consistent time profile. There is evidence of a progressive decrease in regularity following the walk-to-run transition, and these features unveil significant differences among healthy trained and untrained adult subjects

Targetfinder.org: a resource for systematic discovery of transcription factor target genes

Targetfinder.org (http://targetfinder.org/) provides a web-based resource for finding genes that show a similar expression pattern to a group of user-selected genes. It is based on a large-scale gene expression compendium (>1200 experiments, >13 000 genes). The primary application of Targetfinder.org is to expand a list of known transcription factor targets by new candidate target genes. The user submits a group of genes (the ‘seed’), and as a result the web site provides a list of other genes ranked by similarity of their expression to the expression of the seed genes. Additionally, the web site provides information on a recovery/cross-validation test to check for consistency of the provided seed and the quality of the ranking. Furthermore, the web site allows to analyse affinities of a selected transcription factor to the promoter regions of the top-ranked genes in order to select the best new candidate target genes for further experimental analysis

Nucleolin binds to a subset of selenoprotein mRNAs and regulates their expression

Selenium, an essential trace element, is incorporated into selenoproteins as selenocysteine (Sec), the 21st amino acid. In order to synthesize selenoproteins, a translational reprogramming event must occur since Sec is encoded by the UGA stop codon. In mammals, the recoding of UGA as Sec depends on the selenocysteine insertion sequence (SECIS) element, a stem-loop structure in the 3′ untranslated region of the transcript. The SECIS acts as a platform for RNA-binding proteins, which mediate or regulate the recoding mechanism. Using UV crosslinking, we identified a 110 kDa protein, which binds with high affinity to SECIS elements from a subset of selenoprotein mRNAs. The crosslinking activity was purified by RNA affinity chromatography and identified as nucleolin by mass spectrometry analysis. In vitro binding assays showed that purified nucleolin discriminates among SECIS elements in the absence of other factors. Based on siRNA experiments, nucleolin is required for the optimal expression of certain selenoproteins. There was a good correlation between the affinity of nucleolin for a SECIS and its effect on selenoprotein expression. As selenoprotein transcript levels and localization did not change in siRNA-treated cells, our results suggest that nucleolin selectively enhances the expression of a subset of selenoproteins at the translational level

Aldosterone and vasopressin affect α- and γ-ENaC mRNA translation

Vasopressin and aldosterone play key roles in the fine adjustment of sodium and water re-absorption in the nephron. The molecular target of this regulation is the epithelial sodium channel (ENaC) consisting of α-, β- and γ-subunits. We investigated mRNA-specific post-transcriptional mechanisms in hormone-dependent expression of ENaC subunits in mouse kidney cortical collecting duct cells. Transcription experiments and polysome gradient analysis demonstrate that both hormones act on transcription and translation. RNA-binding proteins (RBPs) and mRNA sequence motifs involved in translational control of γ-ENaC synthesis were studied. γ-ENaC–mRNA 3′-UTR contains an AU-rich element (ARE), which was shown by RNA affinity chromatography to interact with AU-rich element binding proteins (ARE-BP) like HuR, AUF1 and TTP. Some RBPs co-localized with γ-ENaC mRNA in polysomes in a hormone-dependent manner. Reporter gene co-expression experiments with luciferase γ-ENaC 3′-UTR constructs and ARE-BP expression plasmids demonstrate the importance of RNA–protein interaction for the up-regulation of γ-ENaC synthesis. We document that aldosterone and the V2 receptor agonist dDAVP act on synthesis of α- and γ-ENaC subunits mediated by RBPs as effectors of translation but not by mRNA stabilization. Immunoprecipitation and UV-crosslinking analysis of γ-ENaC–mRNA/HuR complexes document the significance of γ-ENaC–mRNA–3′-UTR/HuR interaction for hormonal control of ENaC synthesis

Enhanced translation by Nucleolin via G-rich elements in coding and non-coding regions of target mRNAs

RNA-binding proteins (RBPs) regulate gene expression at many post-transcriptional levels, including mRNA stability and translation. The RBP nucleolin, with four RNA-recognition motifs, has been implicated in cell proliferation, carcinogenesis and viral infection. However, the subset of nucleolin target mRNAs and the influence of nucleolin on their expression had not been studied at a transcriptome-wide level. Here, we globally identified nucleolin target transcripts, many of which encoded cell growth- and cancer-related proteins, and used them to find a signature motif on nucleolin target mRNAs. Surprisingly, this motif was very rich in G residues and was not only found in the 3′-untranslated region (UTR), but also in the coding region (CR) and 5′-UTR. Nucleolin enhanced the translation of mRNAs bearing the G-rich motif, since silencing nucleolin did not change target mRNA stability, but decreased the size of polysomes forming on target transcripts and lowered the abundance of the encoded proteins. In summary, nucleolin binds G-rich sequences in the CR and UTRs of target mRNAs, many of which encode cancer proteins, and enhances their translation

The Cytosolic Protein G0S2 Maintains Quiescence in Hematopoietic Stem Cells

Bone marrow hematopoietic stem cells (HSCs) balance proliferation and differentiation by integrating complex transcriptional and post-translational mechanisms regulated by cell intrinsic and extrinsic factors. We found that transcripts of G0/G1 switch gene 2 (G0S2) are enriched in lineage− Sca-1+ c-kit+ (LSK) CD150+ CD48− CD41− cells, a population highly enriched for quiescent HSCs, whereas G0S2 expression is suppressed in dividing LSK CD150+ CD48− cells. Gain-of-function analyses using retroviral expression vectors in bone marrow cells showed that G0S2 localizes to the mitochondria, endoplasmic reticulum, and early endosomes in hematopoietic cells. Co-transplantation of bone marrow cells transduced with the control or G0S2 retrovirus led to increased chimerism of G0S2-overexpressing cells in femurs, although their contribution to the blood was reduced. This finding was correlated with increased quiescence in G0S2-overexpressing HSCs (LSK CD150+ CD48−) and progenitor cells (LS−K). Conversely, silencing of endogenous G0S2 expression in bone marrow cells increased blood chimerism upon transplantation and promoted HSC cell division, supporting an inhibitory role for G0S2 in HSC proliferation. A proteomic study revealed that the hydrophobic domain of G0S2 interacts with a domain of nucleolin that is rich in arginine-glycine-glycine repeats, which results in the retention of nucleolin in the cytosol. We showed that this cytosolic retention of nucleolin occurs in resting, but not proliferating, wild-type LSK CD150+ CD48− cells. Collectively, we propose a novel model of HSC quiescence in which elevated G0S2 expression can sequester nucleolin in the cytosol, precluding its pro-proliferation functions in the nucleolus

Expression proteomics of UPF1 knockdown in HeLa cells reveals autoregulation of hnRNP A2/B1 mediated by alternative splicing resulting in nonsense-mediated mRNA decay

BACKGROUND: In addition to acting as an RNA quality control pathway, nonsense-mediated mRNA decay (NMD) plays roles in regulating normal gene expression. In particular, the extent to which alternative splicing is coupled to NMD and the roles of NMD in regulating uORF containing transcripts have been a matter of debate. RESULTS: In order to achieve a greater understanding of NMD regulated gene expression we used 2D-DiGE proteomics technology to examine the changes in protein expression induced in HeLa cells by UPF1 knockdown. QPCR based validation of the corresponding mRNAs, in response to both UPF1 knockdown and cycloheximide treatment, identified 17 bona fide NMD targets. Most of these were associated with bioinformatically predicted NMD activating features, predominantly upstream open reading frames (uORFs). Strikingly, however, the majority of transcripts up-regulated by UPF1 knockdown were either insensitive to, or even down-regulated by, cycloheximide treatment. Furthermore, the mRNA abundance of several down-regulated proteins failed to change upon UPF1 knockdown, indicating that UPF1`s role in regulating mRNA and protein abundance is more complex than previously appreciated. Among the bona fide NMD targets, we identified a highly conserved AS-NMD event within the 3` UTR of the HNRNPA2B1 gene. Overexpression of GFP tagged hnRNP A2 resulted in a decrease in endogenous hnRNP A2 and B1 mRNA with a concurrent increase in the NMD sensitive isoforms. CONCLUSIONS: Despite the large number of changes in protein expression upon UPF1 knockdown, a relatively small fraction of them can be directly attributed to the action of NMD on the corresponding mRNA. From amongst these we have identified a conserved AS-NMD event within HNRNPA2B1 that appears to mediate autoregulation of HNRNPA2B1 expression levels

CMS physics technical design report : Addendum on high density QCD with heavy ions

Peer reviewe

L'Auto-vélo : automobilisme, cyclisme, athlétisme, yachting, aérostation, escrime, hippisme / dir. Henri Desgranges

23 juin 19181918/06/23 (A19,N6362)