Good old-fashioned (anti)sense

Two decades ago, in the early years of recombinant DNA technology, scientists posited the elegant proposal that expression of a eukaryotic target gene could be blocked by engineering production of a complementary “antisense” RNA that would base pair with the target transcript and somehow interrupt its function (1). And it worked, in organisms as divergent as fruit flies (2), cellular slime molds (3), and plants (4, 5), but not always as expected. In plants, for example, occasionally an ectopically expressed “sense” strand was as effective as antisense at suppressing expression of the target gene (6). An underlying naivete lay in the reasonable expectation from the central dogma of genetics that RNA normally behaves itself as an intermediate between gene and protein and leaves the regulatory business to transcription and translation factors. A few reports of small regulatory RNAs in Escherichia coli (7, 8) were not recognized at the time as harbingers of unforeseen RNA-based regulatory schemes that lurk in the depths of diverse cells. In the ensuing years, it has become obvious that both eukaryotic and prokaryotic organisms possess intricate regulatory systems based on noncoding RNAs (ncRNAs) (6, 9, 10). Those early antisense experiments in transgenic eukaryotes inadvertently tapped into unrecognized endogenous pathways of surprising complexity and remarkable epigenetic consequence (6, 10). The observed effects on gene suppression were not, as expected, mediated by a physical block resulting from extensive base pairing with the target transcript: the active species are now recognized as highly processed 22-nt fragments that act in the context of an elaborate protein complex (10). In this issue of PNAS, Duhring et al. (11) report a natural regulatory event in a cyanobacterium that comes close to the early expectations for straightforward antisense regulation: a 177-nt ncRNA is transcribed in antisense to the central portion of an iron-regulated transcript and blocks its accumulation, presumably through extensive base pairing. The iron-regulated cyanobacterial gene in question, isiA, itself caused a stir previously in the scientific community. Elucidation of the function of IsiA came as a delightful surprise to researchers in the field of photosynthesis in 2001, emerging after all black boxes of the process ostensibly had been opened and most reactions were understood at atomic and picosecond resolution. Under iron-limited conditions, the isiA gene is de-repressed in cyanobacteria, and its product forms what has been characterized as a “giant” antenna around trimers of the photosystem I (PSI) complex, compensating through an increased light-harvesting field for the limited number of PSI reaction centers that can be produced in low-iron environments (12, 13). The IsiA ring contains 18 IsiA monomers, binds 180 chlorophyll molecules, and completely surrounds PSI in iron-starved cyanobacteria. That such a big structure could go unnoticed for so long by such an attentive scientific field was surprise enough, but now the mechanism of regulation of that complex by iron and other triggers is providing additional novelty. Working with a cyanobacterial model organism for photosynthesis, Synechocystis sp. strain PCC 6803, Duhring et al. (11) show that IsiR, an ncRNA complementary to the central portion of the isiA transcript, is regulated inversely with isiA (11). Overexpression or depletion of IsiR has the expected effects of suppressing or stimulating, respectively, the presence of isiA transcript and elaboration of the IsiA antenna around PSI (Fig. 1). Regulation of isiA is known to use the ferric uptake repressor, Fur; the new results show that manipulation of IsiR levels has effects epistatic to Fur, meaning that IsiR acts downstream of Fur-mediated transcriptional regulation. Fig. 1. Antisense suppression of isiA expression under iron-replete conditions. The isiA locus is shown at the top of the figure, indicated by a large green arrow. The downstream isiB gene, cotranscribed with isiA as a discistronic operon, is not shown. Fur (brown ... The involvement of an ncRNA in bacterial gene regulation, and specifically in response to iron limitation, is not unique to cyanobacteria. In E. coli, an ncRNA called RhyB provides a quick switch-off of a suite of dispensable iron-binding proteins when that metal is limiting, leaving more iron available for essential proteins (9). Both the type of physiological response and the mechanism of ncRNA action are different between IsiR and RhyB. In cyanobacteria, iron starvation is a slow process, and measures are mustered effectively to counter short-term limitation (14). The IsiR fast switch in Synechocystis serves not to conserve iron but to prevent assembly of the massive IsiA antenna unless it is genuinely needed and to halt its assembly quickly when iron-replete conditions are restored. The major distinction of cyanobacterial IsiR from RhyB and other ncRNAs in E. coli is that the enteric examples are not perfect matches to their targets (9). Rather, they are encoded in trans and have only limited regions of complementarity to the transcripts they regulate. A protein partner, such as the RNA chaperone Hfq (15), presents the ncRNA to its target and facilitates base pairing (9). Although the current data do not exclude interaction of IsiR with a protein partner, there is no need to invoke one for formation of an IsiR/isiA RNA duplex. In E. coli, Hfq and its bound ncRNA typically interact with the target transcript near the 5′ end, and inhibitory ncRNAs often occlude the ribosome-binding site and/or initiation codon (9). Because cyanobacterial IsiR is complementary to the central region of the isiA message, a physical block of translation is unlikely to be involved. Rather, the pattern of appearance and disappearance of IsiR is consistent with a mechanism in which the duplexed RNAs are targeted for degradation (11). One mechanism of Hfq-ncRNA-mediated regulation in E. coli also seems to employ targeted degradation of a message, likely through RNaseE (9). The trans-encoded ncRNAs of E. coli may have multiple targets, as is true for RhyB (9). In contrast, IsiR seems to be a dedicated partner with one role: posttranscriptional control that prevents isiA expression from slipping past the Fur repressor in the presence of iron. IsiA is induced by two stresses in addition to iron limitation: high light intensity and oxidative stress. The three conditions are interrelated in that both iron limitation and high light intensity indirectly cause oxidative stress (14). IsiR acts to regulate isiA expression in all three situations (11). Keeping isiA off in the absence of these stresses may be very important to the cell, because isiA can be very strongly induced, becoming the most abundant message after exposure to H2O2 (16). Does E. coli use one-on-one antisense regulation to control specific genes, and do cyanobacteria employ a trans-encoded partial-match strategy for regulation of genes by ncRNA? The global searches for ncRNAs in E. coli have focused on intergenic regions, such that perfect-match antisense would be excluded from detection by experimental design (9). Only one comprehensive bioinformatics search has been conducted for a cyanobacterium, specifically for Synechocystis sp. strain PCC 6803 (17); many small ncRNAs that might work like the Hfq partners of E. coli are predicted or shown to be present. Hfq, which is similar to eukaryotic Sm and Sm-like proteins that are involved in RNA splicing, has potential homologs in some cyanobacteria (15, 17). Moreover, an RNA–protein complex can be immunoprecipitated from cyanobacterial extracts by anti-Sm antiserum (18). Nonetheless, regulatory activity of an ncRNA associated with an RNA chaperone has not yet been demonstrated in a cyanobacterium. The current level of investigation does not exclude either universality of both types of ncRNA or fundamental differences in the ncRNA mechanisms that may have evolved in divergent bacterial lineages. One could make teleological arguments for the utility of gene-by-gene dedicated circuits in cyanobacteria, where the major nutrients (light, water, and CO2) are very reliable in their patterns of abundance and only a few nutrients, such as reduced nitrogen, iron, phosphorous, and sulfur, are required in more than trace amounts (14, 19, 20). Bacteria lack the machinery associated with posttranscriptional and transcriptional gene silencing that accounts for the suppressive effects of artificially expressed antisense RNA in eukaryotes (6, 9, 10). Nonetheless, reports have shown gene suppression by regulatable ectopic expression of perfect-match antisense RNA in Staphylococcus aureus (21) and the cyanobacterium Synechococcus elongatus PCC 7942 (22). The effectiveness of the antisense strategy in these species may reflect the serendipitous mimicry of an endogenous regulatory mechanism that is reminiscent of that in eukaryotic systems, with the important difference of reversibility. In the natural Synechocystis sp. situation and in the laboratory antisense manipulations demonstrated for S. aureus and S. elongatus, transcript suppression is transient, depends completely on the presence of the antisense ncRNA, and serves as a switch to effect an on or off in expression of the target gene.