For a long time RNA polymerases did not get the credit they actually deserve. They have been viewed as muscle-cars at a drag race: Waiting for a start signal to race along the gene only stopping for the finish line. This view, however, has markedly changed with the development of high-throughput sequencing techniques such as RNA Polymerase II (Pol II) ChIP, global run-on sequencing (GRO-seq) and precision nuclear run-on and sequencing (PRO-seq).
With these methods researchers painted a much more complex picture of transcription in which RNA Polymerases pause at transcriptional start sites (TSS), initiate divergent transcription at promoters, and link splicing outcomes with elongation rates. However, the consequences of these remarkable observations remained unclear.
A team of talented researchers led by Stirling Churchman wanted to venture deeper into the mysteries of transcription in human cells but faced a problem: the established methods lacked the required resolution. They had previously established a high-throughput sequencing technique termed native elongation transcript sequencing (NET-seq) in yeast that allows mapping of the strand-specific localization of Pol II with single-nucleotide resolution. Transferring this technology to human cells they were able to map transcriptional activity over the entire genome with unmet precision and made some astonishing discoveries.
Here is what they found:
- The majority of expressed genes displayed proximal-promoter pausing as well as antisense transcription upstream of promoters and downstream of polyadenylation sites.
- 77% of gene exhibited divergent transcription at promoter-proximal regions.
- NET-seq identified a new class of antisense transcription originating downstream of sense transcription termed convergent transcription.
- DNAse-seq revealed that converged transcription reflects sense and antisense transcription, which initiates locally and undergoes promoter-proximal pausing flanking the +1 nucleosome.
- Converged transcription was associated with low-level expressed genes and might contribute to transcriptional regulation.
- Transcription factor binding can directly regulate transcriptional elongation in a strand- and direction-specific manner.
- Pol II pauses at retained exon boundaries suggesting that alternative splicing is coupled to transcriptional elongation.
Conclusively, the researchers state that NET-seq should be easily adaptable to any human cell type or tissue and will allow the high resolution analysis of transcriptional regulation across different cell types, responses to signaling pathways, and cellular differentiation.
Head over to Cell, April 2015 to learn how to catch RNA Polymerases.