Molecular Systems Biology, 2009, Vol.5(1), pp.n/a-n/a
Information on the numbers and functions of naturally occurring antisense RNAs (asRNAs) in eubacteria has thus far remained incomplete. Here, we screened the model cyanobacterium sp. PCC 6803 for asRNAs using four different methods. In the final data set, the number of known noncoding RNAs rose from 6 earlier identified to 60 and of asRNAs from 1 to 73 (28 were verified using at least three methods). Among these, there are many asRNAs to housekeeping, regulatory or metabolic genes, as well as to genes encoding electron transport proteins. Transferring cultures to high light, carbon‐limited conditions or darkness influenced the expression levels of several asRNAs, suggesting their functional relevance. Examples include the asRNA to , which accumulates in a light‐dependent manner and may be required for processing the L11 r‐operon and the SyR7 noncoding RNA, which is antisense to the 5′ UTR, possibly modulating murein biosynthesis. Extrapolated to the whole genome, ∼10% of all genes in are influenced by asRNAs. Thus, chromosomally encoded asRNAs may have an important function in eubacterial regulatory networks. 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 (‐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 ‐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 , 2007); (iii) a low level of transcription reported to occur virtually throughout the entire genome (Selinger , 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 , 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/ 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. 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 , 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). The ncRNA SyR7 overlaps with the 5′ UTR of the gene over its full length. The level of SyR7 was more than 20 times higher than that of the mRNA under three different conditions. However, the SyR7/ ratio declined dramatically to ∼1 on a shift to HL (Figure 6A). The enzyme encoded by is required for murein biosynthesis. Therefore, we assume that the translation of is controlled by SyR7 and that under HL 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 targets can form RNA–RNA duplexes, which are degraded by dsRNA‐specific RNases (Hernandez , 2005; Duehring , 2006; Darfeuille , 2007; Kawano , 2007; Fozo , 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 RNAβ in (Stork , 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 , 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 operon in (Andre , 2008). Extrapolated to the whole genome, we estimated the total number of chromosomally encoded asRNAs in to be at least 300. Chromosomally encoded ‐asRNAs are much more frequent than originally thought and seem to outnumber intergenic ncRNAs. Antisense RNAs may affect 8–10% of all genes in , 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. The presence of specific antisense transcripts (asRNAs) was scrutinized in Synechocystis sp. PCC 6803 by two different types of microarrays, a biocomputational prediction, Northern hybridizations and 5′ RACE experiments. Our results raise the number of strongly expressed asRNAs known from this model cyanobacterium from one previously described to 73 and of non‐coding RNAs transcribed from free‐standing genes from six to 60. The expression levels of several asRNAs were influenced upon transfer of cultures to high light, carbon limitation or darkness, suggesting their functional relevance. Extrapolated to the whole genome, ∼10% of all genes in Synechocystis are influenced by asRNAs. Thus, chromosomally encoded asRNAs may play a much more important role in eubacterial regulatory networks than previously expected.
Antisense Rna ; Cyanobacteria ; Microarray ; Noncoding Rna ; Synechocystis