2021/07/14 by Umut Gerlevik, Philipp Lorenz, Anna R Lamstaes +10 · 1 voice · 1 citation
Biochemistry, Genetics and Molecular Biology · #RNA Research and Splicing #RNA and protein synthesis mechanisms #RNA modifications and cancer
paper · pdf · doi:10.1101/2021.07.14.452379
openalex publication_date 2021/07/14 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/14
Genomes are pervasively transcribed, leading to stable and unstable transcripts that influence 3-dimensional genome organisation and gene regulation. High sensitivity and nucleotide resolution are required to resolve mammalian nascent transcriptomes. Here, we exploit the sensitivity of 4-thio-uridine (4sU) metabolic pulse-labelling to develop two nucleotide-resolution methods: S ingle- N ucleotide resolution 4s U sequencing (SNU-Seq) and s ize-fractionated 4sU-Seq (sf4sU-Seq). sf4sU-Seq involves gel isolation of abundant 4sU-labelled promoter proximal nascent transcripts, enabling nucleotide resolution mapping of transcription start sites and promoter proximal pauses (PPPs) on the same transcript. SNU-Seq exploits 3’ end RNA-Seq, using bacterial poly(A) polymerase (bPAP) to polyadenylate the 3’ ends of nascent transcripts and create oligo(dT)-primed libraries. The artificial poly(A) tail marks the precise position of polymerase on a transcription unit. SNU-Seq read levels are similar at pre-mRNAs and enhancers genome-wide and read spikes in pre-mRNA outputs map pauses, PPPs and polyadenylation sites. SNU-Seq enables discovery of thousands of unannotated regions of divergent transcription and helps define hundreds of the more than 10,000 regions of primed non-transcribed acetylated open chromatin that induce divergent nascent transcripts within 0.5h of IFN-γ treatment in Hep3B cells. Thus, combining chromatin analysis with SNU-Seq reveals the transcriptional responsiveness of an epigenetically primed human genome. HIGHLIGHTS SNU-Seq maps nascent transcripts with bp resolution, high sensitivity and low cost sf4sU-Seq resolves TSS and PPP at the same gene, complementing SNU-Seq 1000’s of divergently transcribed enhancers resolved by SNU-Seq Rapid IFNγ dependent transcriptional induction from primed Hep3B epigenome GRAPHICAL ABSTRACT