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Cell. 2015 Apr 23;161(3):526-540. doi: 10.1016/j.cell.2015.03.027.

Mammalian NET-Seq Reveals Genome-wide Nascent Transcription Coupled to RNA Processing.

Author information

1
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
2
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal.
3
Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 226-8501Yokohama, Japan.
4
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal. Electronic address: carmo.fonseca@medicina.ulisboa.pt.
5
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK. Electronic address: nicholas.proudfoot@path.ox.ac.uk.

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.

PMID:
25910207
PMCID:
PMC4410947
DOI:
10.1016/j.cell.2015.03.027
[Indexed for MEDLINE]
Free PMC Article

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