Investigation of The Nascent Human Transcriptome

In order to better understand the transcription cycle in higher eukaryotes as well as the role of the of various non-coding, transient RNAs, we developed SNU-Seq (single nucleotide resolution 4sU-seq) in human cells. SNU-Seq is based on metabolic labelling using 4-thiouridine and generates nascent transcription profiles at single nucleotide resolution. This technique exhibits high sensitivity for non-coding transcripts and enabled us to characterise transcriptional pausing behaviour, elongation dynamics, poly(A)-sites, and exon-intron boundaries. When combined with size-selection, SNU-Seq precisely maps the promoter proximal pause and, through k-means clustering, reveals different classes of genes with distinct pausing behaviour. Ontology analysis identifies developmental TFs, MYC, and TBP as the key transcription factors that are enriched at genes that exhibit high degrees of promoter-proximal pausing. Finally, a pipeline is presented for using SNU-Seq to map transcription start sites and identify more than 1000 novel, transcriptionally active human TSSs.