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′-m7G-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′ m7G-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 GR N-terminal protein isoform levels.
Highlights
The genome does encode mRNA and protein sequences but it contains the temporal, spatial and quantitative instructions for their expression
Transcription is initiated from a transcription start site (TSS) after completing the assembly of the competent transcription initiation complex on the associated promoter
Whilst some alternative 5 UTR first exons may be similar in length and nucleotide sequence, e.g. the Pcdh and UGT1 gene clusters [6], most alternative 5 UTR first exons differ in length and sequence
Summary
The genome does encode mRNA and protein sequences but it contains the temporal, spatial and quantitative instructions for their expression This elaborate regulation occurs principally at the transcriptional level, determining both gene expression and transcript diversity. Many genes possess a 5 UTR containing multiple alternative first exons, each with its own alternative promoter as a second level of transcriptional complexity. Whilst some alternative 5 UTR first exons may be similar in length and nucleotide (nt) sequence, e.g. the Pcdh and UGT1 gene clusters [6], most alternative 5 UTR first exons differ in length and sequence These complex 5 UTRs evolved through processes such as gene duplication by recombination, retroposition, intronic deletions, etc.
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