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

Abstract

SummaryTranscription 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.

Isolation of the protein and RNA content of active sites of transcription from mammalian cells

“…Similarly, there exist many methods for isolating and characterizing newly-synthesized RNA, for example "GRO-seq" 15 , "mNET-seq" 16,17 , "chromatin RNA-seq" 18 , "poly(A)-depleted RNA-seq" 19 , "nascent-seq" 20 , and the metabolic tagging of newly-made RNA using 4-thiouridine 21,22 . Each of these has its own particular shortcomings, and all are relatively laborious and/or require a high sequencing depth; most also focus on particular parts of the transcriptome (for a comparison of RNA-seq methods, see Table 1).…”

PHF13: A new player involved in RNA polymerase II transcriptional regulation and co-transcriptional splicing

“…S5P is required for efficient co-transcriptional splicing and has been reported to be involved in spliceosome assembly at the 5 0 and 3 0 splice sites triggering a splicing checkpoint and RNAPII pausing at intron-exon junctions. [15][16][17][29][30][31] Mammalian NET-seq demonstrated RNAPII S5P enrichment at 5 0 and 3 0 splice sites 15 and phospho-specific RNAPII immunoprecipitations have revealed that RNAPII S5P interacts with key proteins involved spliceosomal assembly. 15,16,29 For a detailed reviews on co-transcriptional splicing, we refer the reader to Saldi et al (2016) and Jonkers et al (2015) 25,32 .…”

“…4,8,[14][15][16] Thus, RNAPII S5P localizes to different chromatin regions, including repressed bivalent genes, 14 active promoters/promoter proximal regions 3 and at splice site junctions. 3,15 Furthermore, most of these RNAPII S5P contexts are affiliated with a pausing of RNAPII, 14,17,18 indicating that S5P is additionally important for co-transcriptional processing of pre-mRNA. Similarly, PHF13 localizes to each of these regions and interacts with RNAPII phosphorylated at serine 5 and serine 7, H3K4me2/3, PRC2 and several splicing factors 1 linking PHF13 to the various described functions of RNAPII S5P.…”

An end in sight? Xrn2 and transcriptional termination by RNA polymerase II

“…RNA interference (RNAi) of the human 5ʹ-3ʹ exonuclease, Xrn2, or mutation of its budding yeast equivalent, Rat1, was originally shown to disrupt termination on transfected plasmids and endogenous genes respectively [6,7]. Although the role of Rat1 was recently re-affirmed [8], RNAi of Xrn2 did not reveal a general termination defect at the 3ʹ end of protein-coding genes [9]. However, termination was subsequently shown to be affected when a catalytically inactive version of Xrn2 is expressed in an RNAi background [10].…”

“…Mammalian native elongating transcript sequencing (mNET-seq) was used to establish the effects of Xrn2 loss on transcription [9,14]. This method provides a picture of Pol II location over the genome at single nucleotide resolution and, unlike run-on based methods, detects polymerases irrespective of their ability to transcribe.…”