title: Where does transcription start? 5'-RACE adapted to next-generation sequencing.
abstract: The variability and complexity of the transcription initiation process was examined by adapting RNA ligase-mediated rapid amplification of 5' cDNA ends (5'-RACE) to Next-Generation Sequencing (NGS). We oligo-labelled 5'-m(7)G-capped mRNA from two genes, the simple mono-exonic Beta-2-Adrenoceptor (ADRB2R)and the complex multi-exonic Glucocorticoid Receptor (GR, NR3C1), and detected a variability in TSS location that has received little attention up to now. Transcription was not initiated at a fixed TSS, but from loci of 4 to 10 adjacent nucleotides. Individual TSSs had frequencies from <0.001% to 38.5% of the total gene-specific 5' m(7)G-capped transcripts. ADRB2R used a single locus consisting of 4 adjacent TSSs. Unstimulated, the GR used a total of 358 TSSs distributed throughout 38 loci, that were principally in the 5' UTRs and were spliced using established donor and acceptor sites. Complete demethylation of the epigenetically sensitive GR promoter with 5-azacytidine induced one new locus and 127 TSSs, 12 of which were unique. We induced GR transcription with dexamethasone and Interferon-γ, adding one new locus and 185 additional TSSs distributed throughout the promoter region. In-vitro the TSS microvariability regulated mRNA translation efficiency and the relative abundance of the different GRN-terminal protein isoform levels.
authors.affiliation: Department of Infection and Immunity, Luxembourg Institute of Health, Esch-Sur-Alzette L-4354, Grand-Duchy of Luxembourg Department of Immunology, Research Institute of Psychobiology, University of Trier, Trier D-54290, Germany.
authors.forename: Fleur A D
authors.initials: FA
authors.lastname: Leenen
authors.affiliation: Department of Infection and Immunity, Luxembourg Institute of Health, Esch-Sur-Alzette L-4354, Grand-Duchy of Luxembourg Department of Immunology, Research Institute of Psychobiology, University of Trier, Trier D-54290, Germany.
authors.forename: Sara
authors.initials: S
authors.lastname: Vernocchi
authors.affiliation: Department of Infection and Immunity, Luxembourg Institute of Health, Esch-Sur-Alzette L-4354, Grand-Duchy of Luxembourg.
authors.forename: Oliver E
authors.initials: OE
authors.lastname: Hunewald
authors.affiliation: Department of Infection and Immunity, Luxembourg Institute of Health, Esch-Sur-Alzette L-4354, Grand-Duchy of Luxembourg.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Schmitz
authors.affiliation: Department of Infection and Immunity, Luxembourg Institute of Health, Esch-Sur-Alzette L-4354, Grand-Duchy of Luxembourg.
authors.forename: Anne M
authors.initials: AM
authors.lastname: Molitor
authors.affiliation: Department of Infection and Immunity, Luxembourg Institute of Health, Esch-Sur-Alzette L-4354, Grand-Duchy of Luxembourg Department of Immunology, Research Institute of Psychobiology, University of Trier, Trier D-54290, Germany Claude.Muller@lih.lu.
authors.forename: Claude P
authors.initials: CP
authors.lastname: Muller
authors.affiliation: Department of Infection and Immunity, Luxembourg Institute of Health, Esch-Sur-Alzette L-4354, Grand-Duchy of Luxembourg.
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Turner
mesh_terms: D020121:5' Untranslated Regions; D001374:Azacitidine / Q000494:pharmacology; D045744:Cell Line, Tumor; D003907:Dexamethasone / Q000494:pharmacology; D005091:Exons; D056426:Genetic Loci; D014644:Genetic Variation; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007438:Introns; D021141:Nucleic Acid Amplification Techniques; D009841:Oligonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012315:RNA Caps / Q000235:genetics / Q000378:metabolism; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics* / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000235:genetics* / Q000378:metabolism; D013194:Staining and Labeling; D024363:Transcription Initiation Site; D061785:Transcription Initiation, Genetic / Q000187:drug effects
chemical_list: D020121:5' Untranslated Regions; C553443:ADRB2 protein, human; C497713:NR3C1 protein, human; D009841:Oligonucleotides; D020033:Protein Isoforms; D012315:RNA Caps; D018343:Receptors, Adrenergic, beta-2; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone; D007371:Interferon-gamma; D001374:Azacitidine