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

TLDR

RNA polymerases are highly regulated molecular machines. The authors introduce GRO‑seq, a method that maps the position, amount, and orientation of transcriptionally engaged RNA polymerases across the genome. GRO‑seq involves isolating nuclear run‑on RNA, sequencing it on a large scale, and mapping the reads back to the genome to locate active polymerases. The study finds that about 30 % of human genes exhibit promoter‑proximal polymerase peaks, transcription often extends beyond pre‑mRNA 3′ cleavage, antisense transcription is common, and most promoters harbor an upstream, opposite‑orientation polymerase that is linked to active genes but fails to elongate beyond the promoter, suggesting that polymerase interactions across broad promoter regions govern transcriptional orientation and efficiency.

Abstract

RNA polymerases are highly regulated molecular machines. We present a method (global run-on sequencing, GRO-seq) that maps the position, amount, and orientation of transcriptionally engaged RNA polymerases genome-wide. In this method, nuclear run-on RNA molecules are subjected to large-scale parallel sequencing and mapped to the genome. We show that peaks of promoter-proximal polymerase reside on approximately 30% of human genes, transcription extends beyond pre-messenger RNA 3' cleavage, and antisense transcription is prevalent. Additionally, most promoters have an engaged polymerase upstream and in an orientation opposite to the annotated gene. This divergent polymerase is associated with active genes but does not elongate effectively beyond the promoter. These results imply that the interplay between polymerases and regulators over broad promoter regions dictates the orientation and efficiency of productive transcription.