Evidence for a major role of antisense RNAs in cyanobacterial gene regulation

Jens Georg¹, Björn Vo¹, Ingeborg Scholz¹, Jan Mitschke¹, Annegret Wilde² & Wolfgang R Hess¹

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Synopsis

In addition to regulatory proteins, bacteria, as well as eukaryotes, possess a significant number of regulatory RNAs. In bacteria, the majority of regulatory RNAs appears to be encoded at genomic locations far away from their target genes and exhibit only partial base complementarity to their mRNA targets. However, a small number of regulatory RNAs is transcribed from the reverse complementary strand of an annotated gene and hence these exhibit full or partial overlaps with their potential targets (cis-encoded regulatory RNAs, asRNAs). It was known early on that such asRNAs control phage development and plasmid replication in bacteria (Wagner and Simons, 1994), yet recent work has much more advanced on trans-encoded regulatory RNAs, leaving information on the numbers, functions and systemic relevance of chromosomally encoded asRNAs behind.

There are three main technical problems in dealing with antisense transcription in bacteria: (i) the general lack of robust algorithms to predict them; (ii) the high risk of measuring experimental artifacts generated during cDNA synthesis in microarray analyses (Perocchi et al, 2007); (iii) a low level of transcription reported to occur virtually throughout the entire genome (Selinger et al, 2000), making it difficult to differentiate asRNAs with a regulatory function from transcriptional noise.

Here, we have tried to overcome all three obstacles by (i) rigorously interrogating all predictions made in a computational approach using tiled microarrays. To overcome the problem of unintended second strand synthesis (ii) we labeled RNA samples directly before their hybridization on the microarray and finally (iii) we focused predominantly on very highly expressed asRNAs.

A tiling microarray was developed, covering all genes and intergenic regions for which a terminator, and thus a candidate asRNA or ncRNA, was computationally predicted. The arrays were hybridized in quadruplicates with pooled RNA from nine different conditions, to detect also those transcripts, which are only induced under specific conditions. As a positive control, the asRNA IsrR (Duehring et al, 2006) was detected as one contiguous segment of the array (Figure 1). In the 20 kb genomic region, that also gives rise to the IsrR/isiA transcript pair, two further asRNAs were detected. The affected genes (as_sll1586 and as_ndhH) code for an unknown protein and NADH dehydrogenase subunit 7, respectively (Figure 1). 432 of 646 transcripts above the expression threshold of +1.0 corresponded to mono-, di-, and polycistronic mRNAs, whereas 60 originated from intergenic regions and were considered ncRNAs and 73 at least partially overlap sense transcripts and therefore were designated asRNAs.

Figure 1

Example for verification of microarray-detected asRNAs in a 20 kb region of the Synechocystis genome, from coordinate 1 500 000–1 520 000. (A) Individual probes are indicated by dots, sets of probes with similar absolute expression levels were joined into contiguous segments, separated from each other and from regions not covered by the array by vertical lines (for the full data set see Supplementary information 'Segmentation2500_final.pdf'). Annotated protein-coding genes are represented by blue boxes. At least three clearly detectable asRNAs (segments in red) originate in this region: IsrR (Duehring et al, 2006), an 90 nt asRNA to sll1586 and an asRNA to ndhH (slr0261). (B) Northern blot hybridizations based on high-resolution polyacrylamide gels and agarose gels. For each asRNA the hybridization (H), the corresponding lane in the RNA electrophoresis (R) and a molecular mass marker (M) is shown. As an additional experimental control, 5' ends of the two new asRNAs were mapped by 5' RACE to positions 1504239 (as_sll1586) and c1511138 (as_ndhH), providing a third line of evidence for the existence of these asRNAs (see also Table I).

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Earlier mathematical modeling of sRNA-based gene regulation suggested a particular niche for regulatory RNA in allowing cells to transition quickly yet reliably between distinct states, consistent with the widespread appearance of bacterial sRNAs in stress regulatory networks (Mehta et al, 2008). To derive functional and quantitative data in an efficient way, we constructed a second microarray for measuring changes in expression levels of mRNAs together with their cognate asRNAs and derived the expression ratio as a proxy for the possible impact of the putative riboregulator. In detail, we show that transfer of cultures to stress conditions, which are highly relevant for a photosynthetic organism, causes distinct and characteristic changes in this ratio. For six selected asRNA/mRNA pairs and for the SyR7 ncRNA, we confirmed the changes in expression levels further by Northern blot hybridization (Figure 6).

Figure 6

Quantitative analysis of expression microarray data and their verification. For each panel, a Northern blot is shown reproducing the results obtained for the individual small RNA in the expression microarray analysis. RNA was analyzed from cultures kept under control conditions (C), darkness for 1 h (D), high light for 30 min (HL), or depletion for CO₂ for 6 h (-CO₂). As a control for equal loading either 5S ribosomal RNA or the RNase P RNA (rnpB) was hybridized. The diagram shows the average of normalized probe set signal intensities from three biological replicates and two technical replicates each. The ratios of asRNA/mRNA signal intensities are indicated by filled circles. (A) Analysis of the SyR7 ncRNA, which is overlapping the murF 5' UTR. Two TSS for murF, P1, and P2 are indicated. (B) Analysis of the asRNA to gene slr0882. (C) Analysis of the asRNA to gene sll1289. The second Northern hybridization from a low-resolution gel confirms a 600 nt long asRNA under the dark condition.

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The ncRNA SyR7 overlaps with the 5' UTR of the murF gene over its full length. The level of SyR7 was more than 20 times higher than that of the murF mRNA under three different conditions. However, the SyR7/murF ratio declined dramatically to 1 on a shift to HL (Figure 6A). The enzyme encoded by murF is required for murein biosynthesis. Therefore, we assume that the translation of murF is controlled by SyR7 and that under HL de novo synthesis of MurF is required for accelerated cell wall biosynthesis. Similar characteristic changes were also obtained for the other asRNA/mRNA pairs studied in more detail (Figure 6B and C).

These selected examples show that a multitude of asRNA functions and mechanisms appear possible. It is well established that asRNAs and their cis-targets can form RNA–RNA duplexes, which are degraded by dsRNA-specific RNases (Hernandez et al, 2005; Duehring et al, 2006; Darfeuille et al, 2007; Kawano et al, 2007; Fozo et al, 2008). Hence antisense transcription is a powerful natural tool in repressing gene expression. There is a growing number of examples which support the idea of bacterial asRNAs serving as novel types of transcriptional terminators such as the 427 nt asRNA RNA in Vibrio anguillarum (Stork et al, 2007). Another possible level of regulation is represented by asRNAs, which directly modulate transcriptional activity. There is strong evidence to suggest that divergently located promoters can interfere with each other (Prescott and Proudfoot, 2002), and the length of transcripts generated from the divergently located promoter (Sneppen et al, 2005) is one important factor for this interaction. Here, we observed 180 nt as the average ncRNA length, whereas the lengths of the asRNAs ranged from 65 nt to 700 nt, with many asRNAs longer than 300 nt, lending support to the idea that some of them may have a function in transcriptional interference. An example of the transcriptional interference mechanism is an 1000 nt long asRNA involved in the sulfur-dependent expression of the ubiG operon in Clostridium acetobutylicum (Andre et al, 2008).

Extrapolated to the whole genome, we estimated the total number of chromosomally encoded asRNAs in Synechocystis to be at least 300. Chromosomally encoded cis-asRNAs are much more frequent than originally thought and seem to outnumber intergenic ncRNAs. Antisense RNAs may affect 8–10% of all genes in Synechocystis, a number that lies within the range of asRNAs in eukaryotic genomes. It is very likely that chromosomally encoded asRNAs constitute an important component of another, not yet fully appreciated, level of gene regulation in bacteria.