Divergently Transcribed ncRNAs in Escherichia coli: Refinement of the Transcription Starts Assumes Functional Diversification

Abstract

Non-coding regulatory RNAs (ncRNAs) comprise specialized group of essential genetically encoded biological molecules involved in the wide variety of cellular metabolic processes. The progressive increase in the number of newly identified ncRNAs and the defining of their genome location indicate their predominant nesting in intergenic regions and expression under the control of their own regulatory elements. At the same time, the regulation of ncRNA's transcription cannot be considered in isolation from the processes occurring in the immediate genetic environment. A number of experimental data indicate the notable impact of positional regulation of gene expression mediated by dynamic temporal DNA rearrangements accompanying transcription events in the vicinity of neighboring genes. This issue can be perceived as particularly significant for divergently transcribed ncRNAs being actually subjected to double regulatory pressure. Based on available results of RNAseq experiments for Escherichia coli, we screened out divergent ncRNAs and the adjacent genes for the exact positions of transcription start sites (TSSs) and relative efficiency of RNA production. This analysis revealed extension or shortening of some previously annotated ncRNAs resulting in modified secondary structure, confirmed stable expression of four ncRNAs annotated earlier as putative, and approved the possibility of expression of divergently transcribed ncRNAs containing repetitive extragenic palindromic (REP) elements. The biogenesis of secreted ncRNAs from divergently transcribed ffs, chiX, ralA, and ryhB is discussed taking into account positions of TSSs. Refinement of TSSs for the neighboring genes renders some ncRNAs as true antisense overlapping with 5'UTR of divergently transcribed mRNAs.

Keywords: REP elements; divergent transcription; genome annotation; non-coding RNAs; secreted RNAs.

FIGURE 1

Schematic presentation of various genomic contexts for ncRNAs annotated as separate transcription units. 5′ end of ncRNA assumed to coincide with transcription start site (TSS). “Stressed DNA” and positions of “transcription bubbles” illustrate topology-mediated effects.

FIGURE 2

Mapping of TSSs for divergently transcribed ncRNAs according to RNAseq data (Thomason et al., 2015; Ettwiller et al., 2016) and computational prediction (Shavkunov et al., 2009). X axis: coordinates in E. coli genome; Y axis: average number of normalized counts with perfect matching for given growth condition. For PlatProm predictions absolute score values are indicated on Y axis. RNA denoted according to the annotation in RegulonDB is shown in cyan; adjacent divergent gene is indicated in gray. Bars above and below X axis correspond to the upper and low DNA strands, respectively. Exactly matching TSSs for ncRNAs are presented in red and for divergently transcribed gene are indicated in blue. Transcriptional landscapes upstream and downstream relatively to the genomic regions depicted in Figure 2 are shown in Supplementary Figure S2.

FIGURE 3

Predictions of secondary structures for 5′-extended or shortened ncRNAs. RNA structure was modeled using server http://rna.urmc.rochester.edu/RNAstructureWeb/Servers (Reuter and Mathews, 2010). Transcripts originating from two TSSs, one corresponding to annotated start (RegulonDB) and the other inferred from RNAseq data (Thomason et al., 2015), were subjected as a query using default parameters. Annotated position of 3′-end was assumed for both variants. Energy of folding is indicated below the structures.

FIGURE 4

Mapping of TSSs for oxyS-oxyR genomic region according to RNAseq data (Thomason et al., 2015; Ettwiller et al., 2016). All designations are the same as in Figure 2. Transcriptional landscape upstream and downstream relatively to the genomic region depicted in Figure 4 is shown in Supplementary Figure S4.

FIGURE 5

Mapping of TSSs for divergently transcribed REP elements according to RNAseq data (Thomason et al., 2015; Ettwiller et al., 2016) and computational prediction (Shavkunov et al., 2009). X axis: coordinates in E. coli genome; Y axis: average number of normalized counts with perfect matching for given growth condition. For PlatProm predictions absolute score values are indicated on Y axis. REP element is denoted in green; adjacent genes are indicated in gray. Bars above and below X axis correspond to the upper and low DNA strands, respectively. Exactly matching TSSs related to divergently transcribed REP sequence are presented in red and for protein-coding genes are shown in blue.

FIGURE 6

Mapping of TSSs for divergent potential ncRNAs—G0-10698, G0-10703, G0-10702, and G0-10705 according to RNAseq data (Thomason et al., 2015; Ettwiller et al., 2016) and computational prediction (Shavkunov et al., 2009). X axis: coordinates in E. coli genome; Y axis: average number of normalized counts with perfect matching for given growth condition. For PlatProm predictions absolute score values are indicated on Y axis. RNA denoted according to the annotation in RegulonDB is shown in cyan; adjacent divergent gene is indicated in gray. Bars above and below X axis correspond to the upper and low DNA strands, respectively. Exactly matching TSSs for ncRNAs are presented in red and for divergently transcribed gene are shown in blue. Transcriptional landscapes upstream and downstream relatively to the genomic regions corresponding to G0-10698-asr and G0-10703- yhcG are shown in Supplementary Figure S5.