Antisense Transcription

Excerpts from Nascent RNA Sequencing Reveals Widespread Pausing and Divergent Initiation at Human Promoters

"Transcription of coding and noncoding RNA molecules by eukaryotic RNA polymerases requires their collaboration with hundreds of transcription factors to direct and control polymerase recruitment, initiation, elongation, and termination."

"transcriptionally engaged Pol II that has accumulated between ∼20 and 50 bases downstream of transcription start sites (TSSs) (5, 6), indicating that transcription can be regulated at the stage of elongation as well as the recruitment and initiation stages."

"This promoter-proximal pausing or stalling (8) is proposed to be an important post-initiation, rate-limiting target for gene regulation."

"A global run-on-sequencing (GRO-seq) assay: to map and quantify transcriptionally engaged polymerase density genome-wide. These measurements provide a snapshot of genome-wide transcription and directly evaluate promoter-proximal pausing on all genes. We used nuclear run-on assays (NRO) to extend nascent RNAs that are associated with transcriptionally engaged polymerases under conditions where new initiation is prohibited."

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Excerpts from "Nascent RNA Sequencing Reveals Widespread Pausing and Divergent Initiation at Human Promoters" on GRO-seq (detailed in Figure S1: http://www.sciencemag.org/content/suppl/2008/12/04/1162228.DC1/Core.SOM.pdf)

5’-7meG is explained in Five-prime cap as "in eukaryotes, the 5′ cap (cap-0), found on the 5′ end of an mRNA molecule, consists of a guanine nucleotide connected to mRNA via an unusual 5′ to 5′ triphosphate linkage. This guanosine is methylated on the 7 position directly after capping in vivo by a methyltransferase. It is referred to as a 7-methylguanylate cap, abbreviated m⁷G or 5'-7meG".

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Nuclear run-on experiments explained in Nuclear Run‐on Assays

"The nuclear run‐on assay is used to measure the transcriptional activity of selected endogenous genes. Nuclei are isolated from appropriate cells using techniques that keep engaged RNA polymerase complexes bound to genomic DNA. Subsequent incubation with the four ribonucleotide triphosphates, one of which is radiolabelled, allows polymerase complexes to progress several hundred base pairs along the genome, producing short, radioactive RNA molecules. After purification, the RNA species of interest are detected by hybridization to appropriate DNA sequences immobilized on a membrane. The amount of specifically hybridized RNA is proportional to the number of engaged polymerase complexes and therefore reflects the transcriptional activity of the gene in the intact cell."

"Divergent transcription is a mark for most active promoters."

"Active promoters are typically marked by histone modifications such as di- and trimethylation of H3-Lys⁴ (H3K4me2 and H3K4me3) as well as acetylation of histone H3 and H4 (H3ac and H4ac). These modifications show a bimodal distribution around TSSs, with the trough representing a nucleosome-free region encompassing the TSS."

"The majority of promoters experience initiation in the upstream direction but that these divergent polymerases do not productively elongate transcripts."

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Global nuclear run-on experiments are explained in Wiki.

"GRO-seq involves the labeling of newly synthesized transcripts with bromouridine (BrU). Cells or nuclei are incubated with BrUTP in the presence of Sarkoysl which prevents the attachment of RNA polymerase to the DNA. Therefore only RNA polymerase that are already on the DNA before the addition of Sarkosyl will produce new transcripts that will be labeled with BrU. The labeled transcripts are captured with anti-BrdU antibody labeled magnetic beads, converted to cDNAs and then sequenced by Next Generation DNA sequencing. The sequencing reads are then aligned to the genome and number of reads per transcript provide an accurate estimate of the number of transcripts synthesized."

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Cryptic promoters are promoters situated within genes, according to Gene regulation by antisense transcription.

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Excerpts from Wiki

"Bidirectional promoters are short (<1 kbp), intergenic regions of DNA between the 5' ends of the genes in a bidirectional gene pair."

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As in Nascent RNA Sequencing Reveals Widespread Pausing and Divergent Initiation at Human Promoters, promoters that produce mRNAs in only one direction are referred as a class of divergent promoters. So a subclass of bidirectional promoters are divergent promoters.