Genome-wide RNA pol II initiation and pausing in neural progenitors of the rat

Abstract

Background Global RNA sequencing technologies have revealed widespread RNA polymerase II (Pol II) transcription outside of gene promoters. Small 5′-capped RNA sequencing (Start-seq) originally developed for the detection of promoter-proximal Pol II pausing has helped improve annotation of Transcription Start Sites (TSSs) of genes as well as identification of non-genic regulatory elements. However, apart from the most well studied genomes of human and mouse, mammalian transcription has not been profiled with sufficiently high precision. Results We prepared and sequenced Start-seq libraries from rat ( Rattus norgevicus ) primary neural progenitor cells. Over 48 million uniquely mappable reads from two independent biological replicates allowed us to define the TSSs of 7365 known genes in the rn6 genome, reannotating 2503 TSSs by more than 5 base pairs, characterize promoter-associated antisense transcription, and profile Pol II pausing. By combining TSS data with polyA-selected RNA sequencing, we also identified thousands of potential new genes producing stable RNA as well as non-genic transcripts representing possible regulatory elements. Conclusions Our study has produced the first Start-seq dataset for the rat. Apart from profiling transcription initiation, our data reaffirm the prevalence of Pol II pausing across the rat genome and indicate conservation of pausing mechanisms across metazoan genomes. We suggest that pausing location, at least in mammals, is constrained by a distance from initiation of transcription, whether it occurs at or outside of a gene promoter. Abundant antisense transcription initiation around protein coding genes indicates that Pol II recruited to the vicinity of a promoter is distributed to available start sites of transcription at either DNA strand. Transcriptome profiling of neural progenitors presented here will facilitate further studies of other rat cell types as well as other organisms. Electronic supplementary material The online version of this article (10.1186/s12864-019-5829-4) contains supplementary material, which is available to authorized users.

Global approaches for profiling transcription initiation

“…One note for oligo-capping methods is that TAP is no longer commercially available, so alternative enzymes are required. The E.coli RNA 5′ pyrophosphohydrolase RppH has been successfully used for decapping in Start-seq ( Scheidegger et al, 2019 ), and a recombinant fusion of the Schizosaccharomyces pombe Dcp1-Dcp2 to its activator Edc1 has also shown promise in this regard ( Paquette et al, 2018 ). Oligo-capping methods also tend to have high input requirements.…”

Targeting the Transcriptome Through Globally Acting Components

“…Perhaps because Pol II pausing was originally described on inducible genes such as MYC and heat shock HSP70 ( O'Brien and Lis, 1991 ; Spencer and Groudine, 1990 ; Krumm et al, 1995 ), it was long believed to be a specialized mechanism that prepares highly inducible genes for activation. However, based on analyses of short RNA transcripts, Pol II pausing signatures are not confined to inducible genes, but are present on all genes, and likely accompany all transcription, whether initiating at or outside of promoters, including divergent transcription, intergenic transcription, enhancers, etc ( Scheidegger et al, 2019 ). This makes pausing unlikely to be a mechanism that universally prepares genes for activation, although it likely contributes to it (see below) ( Muse et al, 2007 ).…”

“…Many details of Pol II pausing such as its relationship with the burst mode of transcription ( Corrigan and Chubb, 2014 ; Fukaya et al, 2016 ; Tunnacliffe and Chubb, 2020 ) and its dynamics remain to be established. Whether Pol II pausing can be bypassed for individual transcription events or not, at least a certain percentage of Pol II complexes appear to undergo pausing at every start site of transcription ( Scheidegger et al, 2019 ). The so-called “nonpaused” genes still show the same small RNA pausing signatures in terms of their size distributions, but are either less active or have a lower pausing index ( Muse et al, 2007 ; Zeitlinger et al, 2007 ) (or higher traveling ratio ( Rahl et al, 2010 )) compared to other genes ( Nechaev et al, 2010 ; Scheidegger et al, 2019 ).…”

“…Whether Pol II pausing can be bypassed for individual transcription events or not, at least a certain percentage of Pol II complexes appear to undergo pausing at every start site of transcription ( Scheidegger et al, 2019 ). The so-called “nonpaused” genes still show the same small RNA pausing signatures in terms of their size distributions, but are either less active or have a lower pausing index ( Muse et al, 2007 ; Zeitlinger et al, 2007 ) (or higher traveling ratio ( Rahl et al, 2010 )) compared to other genes ( Nechaev et al, 2010 ; Scheidegger et al, 2019 ). Examination of genome-wide datasets shows that different genes within a dataset show higher or lower pausing index, but these signatures, just like some histone marks, are overall stable across cell lines ( Figure 1 ).…”

ERK2-topoisomerase II regulatory axis is important for gene activation in immediate early genes

“…In particular, stress-inducible genes respond to environmental signals and cellular needs, and their synchronized and rapid gene expression is crucial for cell survival, maintenance, and growth 2,5,[8][9][10] . Representative examples are serum/growth factorinducible immediate early genes (IEGs) 2,5 , heat shock genes [11][12][13] , neurotransmitter-inducible genes 4,14 , enhancer RNA genes 15,16 , and hypoxia-inducible genes 17,18 .…”