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Hypoxia-induced alternative splicing in endothelial cells
(2012)
- BACKGROUND: Adaptation to low oxygen by changing gene expression is vitally important for cell survival and tissue development. The sprouting of new blood vessels, initiated from endothelial cells, restores the oxygen supply of ischemic tissues. In contrast to the transcriptional response induced by hypoxia, which is mainly mediated by members of the HIF family, there are only few studies investigating alternative splicing events. Therefore, we performed an exon array for the genome-wide analysis of hypoxia-related changes of alternative splicing in endothelial cells. METHODOLOGY/PRINCIPAL FINDINGS: Human umbilical vein endothelial cells (HUVECs) were incubated under hypoxic conditions (1% O(2)) for 48 h. Genome-wide transcript and exon expression levels were assessed using the Affymetrix GeneChip Human Exon 1.0 ST Array. We found altered expression of 294 genes after hypoxia treatment. Upregulated genes are highly enriched in glucose metabolism and angiogenesis related processes, whereas downregulated genes are mainly connected to cell cycle and DNA repair. Thus, gene expression patterns recapitulate known adaptations to low oxygen supply. Alternative splicing events, until now not related to hypoxia, are shown for nine genes: six which are implicated in angiogenesis-mediated cytoskeleton remodeling (cask, itsn1, larp6, sptan1, tpm1 and robo1); one, which is involved in the synthesis of membrane-anchors (pign) and two universal regulators of gene expression (cugbp1 and max). CONCLUSIONS/SIGNIFICANCE: For the first time, this study investigates changes in splicing in the physiological response to hypoxia on a genome-wide scale. Nine alternative splicing events, until now not related to hypoxia, are reported, considerably expanding the information on splicing changes due to low oxygen supply. Therefore, this study provides further knowledge on hypoxia induced gene expression changes and presents new starting points to study the hypoxia adaptation of endothelial cells.
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Conformational dynamics of the tetracycline-binding aptamer
(2011)
- The conformational dynamics induced by ligand binding to the tetracycline-binding aptamer is monitored via stopped-flow fluorescence spectroscopy and time-correlated single photon counting experiments. The fluorescence of the ligand is sensitive to changes within the tertiary structure of the aptamer during and after the binding process. In addition to the wild-type aptamer, the mutants A9G, A13U and A50U are examined, where bases important for regulation are changed to inhibit the aptamer’s function. Our results suggest a very fast two-step-mechanism for the binding of the ligand to the aptamer that can be interpreted as a binding step followed by a reorganization of the aptamer to accommodate the ligand. Binding to the two direct contact points A13 and A50 was found to occur in the first binding step. The exchange of the structurally important base A9 for guanine induces an enormous deceleration of the overall binding process, which is mainly rooted in an enhancement of the back reaction of the first binding step by several orders of magnitude. This indicates a significant loss of tertiary structure of the aptamer in the absence of the base A9, and underlines the importance of pre-organization on the overall binding process of the tetracycline-binding aptamer. Previous SectionNext Section
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Thermodynamic characterization of an engineered tetracycline-binding riboswitch
(2006)
- Riboswitches reflect a novel concept in gene regulation that is particularly suited for technological adaptation. Therefore, we characterized thermodynamically the ligand binding properties of a synthetic, tetracycline (tc)-binding RNA aptamer, which regulates gene expression in a dose-dependent manner when inserted into the untranslated region of an mRNA. In vitro, one molecule of tc is bound by one molecule of partially pre-structured and conformationally homogeneous apo-RNA. The dissociation constant of 770 pM, as determined by fluorimetry, is the lowest reported so far for a small molecule-binding RNA aptamer. Additional calorimetric analysis of RNA point mutants and tc derivatives identifies functional groups crucial for the interaction and including their respective enthalpic and entropic contributions we can propose detailed structural and functional roles for certain groups. The conclusions are consistent with mutational analyses in vivo and support the hypothesis that tc-binding reinforces the structure of the RNA aptamer, preventing the scanning ribosome from melting it efficiently.
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A fast and efficient translational control system for conditional expression of yeast genes
(2009)
- A new artificial regulatory system for essential genes in yeast is described. It prevents translation of target mRNAs upon tetracycline (tc) binding to aptamers introduced into their 5'UTRs. Exploiting direct RNA–ligand interaction renders auxiliary protein factors unnecessary. Therefore, our approach is strain independent and not susceptible to interferences by heterologous expressed regulatory proteins. We use a simple PCR-based strategy, which allows easy tagging of any target gene and the level of gene expression can be adjusted due to various tc aptamer-regulated promoters. As proof of concept, five differently expressed genes were targeted, two of which could not be regulated previously. In all cases, adding tc completely prevented growth and, as shown for Nop14p, rapidly abolished de novo protein synthesis providing a powerful tool for conditional regulation of yeast gene expression.
