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.

Keywords: Promoters; RNA pol II; Small RNA; Transcription.

Fig. 1

Validation of TSS-RNA sequencing in rat neural precursors. a. A scheme of an updated protocol for the preparation of Start-seq libraries for Illumina sequencing. b. UCSC browser shot of highly expressed Actb gene showing tracks from this study including 5′-tracks for TSS-RNA for each strand (red and blue) and mRNA-sequencing (black), alongside Pol II ChIP-seq (using an antibody against the Pol II N-terminus) track from mature rat neurons [10] representing a rat Pol II dataset that is most closely related to the current cell type. c. A zoomed-in view of Actb promoter-proximal region showing 5′- (red) and 3′-end (gold) of TSS-RNAs on this gene. The annotated Actb TSS is shown in blue bar and is located 2 bp downstream of the 5′ TSS-RNA peak. d. Correlation plot for promoter-proximal counts between two independent biological Start-Seq replicates. TSS-RNAs on the gene (sense) strand were counted in a +/− 500 bp interval from each RefSeq-annotated TSS

Fig. 2

TSS RNA-based refinement of gene TSSs. a. Heatmaps of sense-strand TSS-RNA on 7112 rat genes ordered by decreasing TSS-RNA count within the promoter region, centered around annotated (left panel) and TSS-RNA peak-centered locations (right panel). b. Left pane. Metaplots of TSS-RNA 5′-ends for the same genes as in A, centered on TSSs defined using RefSeq (black) and TSS-RNA (red) TSS annotations. Right pane. Pol II ChIP-sequencing traces based on previously published data [10] centered against the same TSSs. c. Weblogo visualization of sequence enrichment of the genes centered around RefSeq annotation (top) and TSS-RNA reannotation (bottom panel). d. Correspondence between TSS annotations between two independent biological replicates, with each dot representing a gene and color coding indicating the number of TSS-RNA hits per gene as indicated. Genes are plotted against RefSeq-annotated TSSs. Strikethrough lines enclose genes considered for TSS reannotations based on each replicate, with the center quadrant containing genes that were reannotated

Fig. 3

Antisense transcription in the rat genome. a. A heatmap representing antisense transcription sorted by the distance of divergent peak from the gene TSS, indicated by arrow. Of the 7112 genes, 601 genes that did not contain divergent signal within 600 bp from the TSS were removed from the heatmap. Due to low signal from antisense, and especially convergent transcription, the image was enhanced with Pixelmator to highlight convergent transcription. b. Metagene plot of antisense transcription relative to the reannotated gene TSSs. c. Spearman r correlation matrix of sense and antisense transcription, per replicate. Counts for convergent and divergent transcription were defined on the appropriate strand within +/− 50 nt from the location with the highest signal. d. Weblogo representation of DNA sequence context centered around Convergent (top) and Divergent (bottom) peak locations

Fig. 4

Pol II pausing across the rat genome. a. Scatter plot showing the distribution of TSS-RNA versus RNA-seq FPKM signal for all genes, with pausing index indicated by color. c. Metaplot of TSS-RNA 3′-ends relative to the reannotated TSS. b. Metaplot of TSS-RNA lengths around the TSSs. All RNAs on the sense strand from a gene within +/− 150 bp interval were considered regardless of their initiation site. All genes versus top 25% highest expressed genes (based on RNA-seq signal FPKM) are shown, respectively, in grey and dark red. d. A UCSC browser shot showing Pol II pausing and transcription on Fabp7 gene, which has the highest expression in rat neural progenitors based on RNA-seq analysis. Inset shows a zoomed-in view of the gene’s promoter region

Fig. 5

Pol II pausing and distance from transcription initiation. a. Metaplot of TSS-RNAs initiating outside of the exact TSS region +/− 10 bp (greyed out), which was excluded from this analysis. The 5′-ends of RNAs are shown in dashed lines and their 3′-ends in solid lines. RNA initiating at more upstream regions are shown in red and at downstream regions in blue. b. 2-d scatter plot of initiation on two genes with top 20 FPKM values as in [31]. c. TSS-RNA length versus their initiation location around the TSS. Circles show mean (with SEM) for TSS-RNA lengths metaplot for RNAs initiating at the indicated locations (positions) relative to the TSS (vertical line). Grey line (right Y-axis) shows the number of RNAs mapping (metagene) to each location in Replicate 1. d. Weblogo plot of DNA sequence context around locations of Pol II pausing based on distance from the reannotated TSS (top) and relative to 3′-end of each RNA

Fig. 6

Examples of transcriptome elements identified in the rat genome. a. A potential regulatory element (enhancer) upstream of Sox2 gene defined based on bidirectional TSS-RNA signal and low RNA-seq signal. b. Example of a new annotation in the rat genome showing homology to Auts2 gene. Transcripts assembled from RNA-seq data are shown inside a bracket. Mouse and H. sapiens genes are shown underneath