Persistent backtracking by RNA polymerase II

RNA polymerase II (pol II) can backtrack during transcription elongation, exposing the 3’ end of nascent RNA. Nascent RNA sequencing can approximate the location of backtracking events that are quickly resolved; however, the extent and genome wide distribution of more persistent backtracking is unknown. Consequently, we developed a novel method to directly sequence backtracked RNA. Our data shows that pol II slides backwards more than 20 nucleotides in human cells and can persist in this backtracked state. Persistent backtracking mainly occurs where pol II pauses near promoters and intron-exon junctions, and is enriched in genes involved in translation, replication, and development, where gene expression is inhibited if these events are unresolved. Histone genes are highly prone to persistent backtracking, where the accumulation and resolution of such events is required for timely expression during cell division. These results demonstrate that persistent backtracking has the potential to affect diverse gene expression programs. LORAXseq, crosslinked footprinting, and mNETseq was performed in cells lacking TFIIS to measure the genomic distribution of persistent backtracking and nascent transcription.