Mammalian NET-seq reveals genome-wide nascent transcription coupled to RNA processing

Takayuki Nojima, Tomás Gomes, Ana Rita Fialho Grosso, Hiroshi Kimura, Michael J. Dye, Somdutta Dhir, Maria Carmo-Fonseca, Nicholas J. Proudfoot

Research output: Contribution to journalArticlepeer-review

240 Citations (Scopus)

Abstract

Transcription is a highly dynamic process. Consequently, we have developed native elongating transcript sequencing technology for mammalian chromatin (mNET-seq), which generates single-nucleotide resolution, nascent transcription profiles. Nascent RNA was detected in the active site of RNA polymerase II (Pol II) along with associated RNA processing intermediates. In particular, we detected 5′splice site cleavage by the spliceosome, showing that cleaved upstream exon transcripts are associated with Pol II CTD phosphorylated on the serine 5 position (S5P), which is accumulated over downstream exons. Also, depletion of termination factors substantially reduces Pol II pausing at gene ends, leading to termination defects. Notably, termination factors play an additional promoter role by restricting non-productive RNA synthesis in a Pol II CTD S2P-specific manner. Our results suggest that CTD phosphorylation patterns established for yeast transcription are significantly different in mammals. Taken together, mNET-seq provides dynamic and detailed snapshots of the complex events underlying transcription in mammals.

Original languageEnglish
Pages (from-to)526-540
Number of pages15
JournalCell
Volume161
Issue number3
DOIs
Publication statusPublished - Apr 23 2015
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)

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