Conserved DNA sequence features underlie pervasive RNA polymerase pausing

Pausing of transcribing RNA polymerase is regulated and creates opportunities to control gene expression. Research in metazoans has so far mainly focused on RNA polymerase II (Pol II) promoter-proximal pausing leaving the pervasive nature of pausing and its regulatory potential in mammalian cells unclear. Here, we developed a pause detecting algorithm (PDA) for nucleotide-resolution occupancy data and a new native elongating transcript sequencing approach, termed nested NET-seq, that strongly reduces artifactual peaks commonly misinterpreted as pausing sites. Leveraging PDA and nested NET-seq reveal widespread genome-wide Pol II pausing at single-nucleotide resolution in human cells. Notably, the majority of Pol II pauses occur outside of promoter-proximal gene regions primarily along the gene-body of transcribed genes. Sequence analysis combined with machine learning modeling reveals DNA sequence properties underlying widespread transcriptional pausing including a new pause motif. Interestingly, key sequence determinants of RNA polymerase pausing are conserved between human cells and bacteria. These studies indicate pervasive sequence-induced transcriptional pausing in human cells and the knowledge of exact pause locations implies potential functional roles in gene expression.