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. 2015 Jan 1;197(1):18-28.
doi: 10.1128/JB.02096-14. Epub 2014 Sep 29.

Global transcriptional start site mapping using differential RNA sequencing reveals novel antisense RNAs in Escherichia coli

Maureen K Thomason  1 , Thorsten Bischler  2 , Sara K Eisenbart  3 , Konrad U Förstner  2 , Aixia Zhang  1 , Alexander Herbig  4 , Kay Nieselt  4 , Cynthia M Sharma  5 , Gisela Storz  6
Affiliations

Global transcriptional start site mapping using differential RNA sequencing reveals novel antisense RNAs in Escherichia coli

Maureen K Thomason et al. J Bacteriol. .

Abstract

While the model organism Escherichia coli has been the subject of intense study for decades, the full complement of its RNAs is only now being examined. Here we describe a survey of the E. coli transcriptome carried out using a differential RNA sequencing (dRNA-seq) approach, which can distinguish between primary and processed transcripts, and an automated prediction algorithm for transcriptional start sites (TSS). With the criterion of expression under at least one of three growth conditions examined, we predicted 14,868 TSS candidates, including 5,574 internal to annotated genes (iTSS) and 5,495 TSS corresponding to potential antisense RNAs (asRNAs). We examined expression of 14 candidate asRNAs by Northern analysis using RNA from wild-type E. coli and from strains defective for RNases III and E, two RNases reported to be involved in asRNA processing. Interestingly, nine asRNAs detected as distinct bands by Northern analysis were differentially affected by the rnc and rne mutations. We also compared our asRNA candidates with previously published asRNA annotations from RNA-seq data and discuss the challenges associated with these cross-comparisons. Our global transcriptional start site map represents a valuable resource for identification of transcription start sites, promoters, and novel transcripts in E. coli and is easily accessible, together with the cDNA coverage plots, in an online genome browser.

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Figures

FIG 1
FIG 1
Summary of the biological, library, and Illumina sequencing replicates that were subjected to dRNA-seq analysis in this study.
FIG 2
FIG 2
Automated TSS prediction across three different growth conditions using TSSpredator. (A) Distribution of predicted TSS across the biological conditions M63 0.4, LB 0.4, and LB 2.0. (B) Distribution of predicted TSS in the primary, secondary, internal, orphan, and antisense TSS classes (pTSS, sTSS, iTSS, oTSS, and asTSS, respectively).
FIG 3
FIG 3
Examples of genes with newly detected pTSS. Screenshots showing the relative cDNA coverage plots for representative −TEX or +TEX libraries for the M63 0.4, LB 0.4, and 2.0 growth conditions across the genomic regions encompassing the lspA (A) and plsX (B) genes. The x axis depicts the genomic coordinates, while the y axis indicates the relative cDNA scores (normalized number of mapped cDNA reads). Red arrows indicate the previously unannotated TSS detected by our analysis. Promoter sequences for the new TSS, including the −10 and −35 sequences (boxed) and bases corresponding to TSS (red) are depicted below each plot.
FIG 4
FIG 4
Comparison of asTSS. (A) Distribution of only asTSS, only pTSS or sTSS, and NCBI-annotated asRNAs in RPKM expression bins. The RPKM expression values were calculated based on cDNA read counts within 50-nt windows starting at the TSS. (B) Distribution of TSS classified exclusively as asTSS across the three biological conditions M63 0.4, LB 0.4, and LB 2.0. (C) Pairwise comparison of asTSS identified by our study and in previously published studies by Conway et al. (19), Dornenburg et al. (14), Raghavan et al. (16), Shinhara et al. (13), Mendoza-Vargas et al. (20), and Salgado et al. (21). The total numbers of annotated asTSS are shown on the main diagonal of the matrix. asTSS from the studies in the rows are compared to the studies in columns, and the number of TSS with exact matches is reported in the matrix entries. The background color depicts the percentage of overlapping asTSS relative to the total number of asTSS from the study in the particular row.
FIG 5
FIG 5
Northern blot detection and cDNA coverage plots of selected candidate asRNAs from the top three expression bins. In all cases, wild-type E. coli strain MG1655, the corresponding deletion strain for the particular asRNA as well as an rnc deletion strain, and an RNase E (rne-131) mutant strain were grown in LB or M63 supplemented with glucose until they reached the indicated OD600. Samples were processed for Northern analysis and probed with a riboprobe specific for the asRNAs. The bands corresponding to the asRNAs are indicated with black stars. Schematics of cDNA coverage plots and genomic locations encoding the respective candidate asRNAs are shown on the right, with the position and direction of the asTSS indicated by red arrows. y axes indicating relative cDNA coverage have the same scale for the forward and reverse strands.
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