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Where does transcription start? 5'-RACE adapted to next-generation sequencing
Fleur A D Leenen1, Sara Vernocchi1, Oliver E Hunewald2
1Department 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.
Nucleic Acids Research
|November 29, 2015
Summary
Transcription initiation is variable, not fixed at a single start site. This study used Next-Generation Sequencing to reveal multiple transcription start site (TSS) loci, impacting gene expression and protein levels.
Area of Science:
- Molecular Biology
- Genetics
- Epigenetics
Background:
- Transcription initiation is a complex process crucial for gene regulation.
- Previous studies often assumed fixed transcription start sites (TSSs).
- The micro-variability of TSSs and its functional impact remain underexplored.
Purpose of the Study:
- To investigate the variability and complexity of transcription initiation.
- To adapt RNA ligase-mediated rapid amplification of 5' cDNA ends (5'-RACE) for Next-Generation Sequencing (NGS) to map TSSs.
- To analyze TSS distribution in simple (ADRB2R) and complex (GR) genes.
Main Methods:
- Oligo-labeling of 5'-m(7)G-capped mRNA.
- Application of 5'-RACE coupled with NGS.
- Analysis of transcription initiation sites in ADRB2R and GR genes.
- Investigating the effects of epigenetic modification (5-azacytidine) and inducers (dexamethasone, Interferon-γ) on GR transcription.
Main Results:
- Identified transcription initiation from loci of 4-10 adjacent nucleotides, not fixed TSSs.
- ADRB2R utilized a single locus with 4 adjacent TSSs.
- Unstimulated GR exhibited 358 TSSs across 38 loci, primarily in 5' UTRs.
- Epigenetic demethylation and induced transcription expanded the number and distribution of GR TSSs.
- In vitro, TSS microvariability regulated mRNA translation efficiency and protein isoform abundance.
Conclusions:
- Transcription initiation is characterized by microvariability at specific loci, challenging the fixed TSS model.
- The number and location of TSSs are dynamically regulated by epigenetic states and external stimuli.
- TSS microvariability plays a functional role in modulating mRNA translation and protein isoform diversity.
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