is one of the most extensively studied organisms in molecular biology. For this bacterium, six searches for new sRNAs have been carried out (4
) and so far have extended the number of experimentally verified sRNAs to 55 (10
). In addition, numerous candidate sRNAs were predicted but have not been tested so far. The rationale for carrying out yet another search lies in that bioinformatics-based approaches must build on patterns that require prior knowledge of searched-for features. RNAs that fail to meet these criteria may escape detection. RNomics approaches, used successfully in a number of organisms (12
), identify sRNAs by cDNA shot-gun cloning of those RNA species that are present under a chosen set of conditions. Thus, RNomics is, in some respects, similar to the early approaches that identified distinct sRNAs by orthophosphate labeling and biochemical isolation [4.5S and 6S RNA (42
), Spot 42, tmRNA and M1 RNA of RNase P (43
)]. However, reverse transcription followed by cloning has since revolutionized isolation and sequence determination of numerous small RNAs in parallel, notably narrowly sized (~22 nt) subclasses of eukaryotic sRNAs, e.g. miRNAs and siRNAs (44
In addition to 20 known sRNAs, our cDNA libraries of cloned sRNAs (or fragments thereof) contain a number of new sRNAs, some of which are absent from the current list of approximately 1000 non-redundant candidates [Table ; (10
)]. Some well-characterized sRNAs were not found here, most probably due to specific expression patterns, e.g. OxyS [oxidative stress (48
)] and DsrA [cold shock (49
)]. Other RNAs might have escaped detection because of technical limitations, i.e. sRNAs may differ dramatically in their efficiency of C-tailing in cDNA library construction (17
), e.g. due to structures near the 3′ end. Thus, such technical biases may be responsible for the absence of some sRNAs, but improvement of the individual cloning steps and a greater sampling number might significantly reduce these problems in the future. The total number of experimentally verified sRNAs in E.coli
, including the present work, is now 62. In the cDNA libraries obtained, 20 of the previously found sRNAs were represented, from single to 60 independent sequences in contigs (Table S1). So far, the biological roles of the recently identified E.coli
sRNAs are unknown [exceptions are RyhB/FerA (50
) and CsrC (51
)], and so are their targets, presuming most of them to be riboregulators/antisense RNAs (52
Given that functions have yet to be assigned, what can we learn from the present study? RACE experiments and positional information indicate that the definition of an sRNA is a matter of perspective. For example, distinct sRNA species are not always indicative of independently synthesized RNAs (independent gene, bordered by promoter and terminator). Some sRNAs are derived from leader (e.g. SroA and SroG) or trailer regions of mRNAs (e.g. SroD, SroE and SroC). An interesting observation concerns SroE, predicted to be encoded in the hisS–gcpE
IGR by Rivas et al
). SroE is not a primary transcript (Fig. ), as its 5′ end is generated by cleavage within the stop codon of the preceding gcpE
ORF. Intriguingly, a recent study showed that the RelE toxin, in poor growth conditions, arrests translation by cleaving ribosome-bound mRNAs within stop codons (54
One can speculate that sRNAs may not only originate from independent genes, but may alternatively be generated and accumulated as independent functional units by processing from longer transcripts. Mattick and Gagen (55
) suggested non-coding regulatory RNAs (in eukaryotes) to be part of a parallel transcriptional output, e.g. they are processed out of mRNA introns and in turn play roles in regulatory cross-talk. Their location would ensure that they are produced along with the intron-containing mRNA. Similarly, bacterial sRNAs derived from mRNA leaders or trailers may have independent functions, exerted under conditions when the gene in question is active. SroA and SroG consist essentially exclusively of known riboswitch elements, THI and RFN, respectively (26
). Riboswitch elements are aptamer-like binding sites for small molecules (here thiamine and flavin derivatives) that trigger secondary structure changes of the mRNA leader in response to ligand binding, resulting in translational or attenuation-type regulation of the genes they precede. Since both elements accumulate strongly as independent sRNA species with predicted binding affinity, it is conceivable that they could carry out additional, independent roles such as titration of the ligand.
Further complexity arises from the observation that one and the same RNA may function as both mRNA and regulatory RNA. Staphylococcus aureus
RNAIII acts as both an mRNA encoding δ-hemolysin, and an antisense RNA regulator of the α-hemolysin mRNA (57
). Whether such ‘moonlighting’ sRNAs are present in E.coli
is unknown but remains an interesting possibility. Thus, in the absence of functional information, the current definition of an sRNA must rely on its mere presence in cells, and various themes are possible (Fig. ). This may even include sRNAs derived from coding regions; some of the mRNA-derived contigs in the cDNA library represent such candidates and are currently being tested.
Definitions of sRNAs. (A) Origin of a distinct sRNA species in an own sRNA gene or (B) through parallel transcriptional output, in an mRNA gene.
Riboregulators can be expected to be present in signifi cant excess over their targets when regulation occurs. Characteristically, plasmid-encoded antisense RNAs are constitutively expressed, unstable, and yet abundant (37
). Most chromosomally encoded sRNAs are induced, but intracellular stabilities are known for only a few. All RNAs tested were analyzed when they were most abundant, i.e they can be assumed to be in excess over putative targets, so that target interaction-dependent degradion pathways (e.g. by RNase III or RNase E) at this point are expected to have little effect on the measured half-life. However, if/when target genes are induced, and the target RNA concentration approaches that of the riboregulator, second-order decay rates may increasingly dominate over target-independent (first order) decay rates. Thus, although very long half-lives in rifampicin run-out experiments may be questionable due to major changes in physiology at late time points, it is clear that sRNAs cover the entire range from very unstable to stable (Fig. ).
In conclusion, this study used an RNomics approach to describe yet another set of new sRNAs in E.coli
, adding to the previously found sRNAs (4
). Since 62 sRNAs are now supported experimentally, it is clear that sRNAs (although the hallmarks of bona fide
sRNAs may still be poorly defined) constitute a significant fraction of the transcriptional output. In a wider perspective, the ubiquitous presence of new sRNAs in eukaryotes and archea (3
) has only recently been recognized. The exciting and challenging task in the years to come will be the elucidation of the roles these sRNAs play in all kingdoms of life.