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Biological Circuits with Small RNA Switches

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Biological Circuits with Small RNA Switches Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW Stealth regulation: biological circuits with small RNA switches Susan Gottesman Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892, USA So you thinkyou finally understand the regulation of temporal RNAs (stRNAs) or microRNAs, and that these your favorite gene? The transcriptional regulators have RNAs are processed by some of the same protein cofac- been identified; the signaling cascades that regulate syn- tors as is RNAi, have put regulatory RNAs in the spot- thesis and activity of the regulators have been found. light in eukaryotes as well. Recent searches have con- Possibly you have found that the regulator is itself un- firmed that flies, worms, plants, and humans all harbor stable, and that instability is necessary for proper regu- significant numbers of small RNAs likely to play regu- lation. Time to lookfor a new project, or retire and rest latory roles. on your laurels? Not so fast—there’s more. It is rapidly Along with the rapid expansion in RNAs doing inter- becoming apparent that another whole level of regula- esting things, has come a proliferation of nomenclature. tion lurks, unsuspected, in both prokaryotic and eukary- Noncoding RNAs (ncRNA) has been used recently, as otic cells, hidden from our notice in part by the tran- the most general term (Storz 2002). Among the noncod- scription-based approaches that we usually use to study ing RNAs, the subclass of relatively small RNAs that gene regulation, and in part because these regulators are frequently act as regulators have been called stRNAs very small targets for mutagenesis and are not easily (small temporal RNAs, eukaryotes) and sRNAs (small found from genome sequences alone. These stealth regu- RNAs, prokaryotes), among others. Here, I will refer to lators, operating below our radar, if not that of the cell, the regulatory RNAs, which should be considered a sub- are small regulatory RNAs, acting to control the trans- set of the ncRNAs. lation and degradation of many messengers. These RNAs I review here the range of regulatory RNAs that have can be potent and multifunctional, allowing new signal- been identified and how they can be found, what we ing pathways to cross-regulate targets independently of know about how they work, drawing lessons from the the transcriptional signals for those targets, introducing plasmid antisense molecules, and how they transduce polarity within operons, and explaining some puzzles in regulatory signals. These stealthy RNAs may be the final well-studied regulatory circuits. level of unexpected regulatory circuitry in all of those The importance of small regulatory RNAs was first systems we thought we were beginning to understand; appreciated in the elegant studies of plasmid-encoded an- now that we know they are there, we have some hope of tisense RNAs. The few apparently unusual cases of non- understanding what it is they do and how they do it. coding regulatory RNAs encoded in the bacterial chro- mosome has expanded over the last decade, and the role What regulatory RNAS do we know about such RNA regulators play in both stimulating and inhib- and how do we find more? iting gene expression has been firmly established. As ge- nome sequences have become available for many bacte- Small regulatory RNAs are small, and possibly as a re- ria, it has become possible to search for additional mem- sult, have not generally been found in mutant hunts. bers of this regulatory family, and, eventually, to begin Therefore, realizing that they are there at all has been the to understand how they act at the molecular level. first challenge. Maybe not surprisingly, the small, well- Simultaneously, researchers in eukaryotic systems studied genomes of plasmid and bacteriophage were the were discovering the wonders of RNAi, a cellular strat- first place these molecules were noted and investigated. egy for protecting itself from RNA invaders, in which The plasmid-encoded RNA regulators are generally en- small double-stranded RNA molecules cause destruction coded by the antisense strand for their target gene or of homologous messages. The discovery that develop- transcript and, thus, are complementary to it over many mental mutants in Caenorhabditis elegans define genes nucleotides (nt). The ground-breaking and still fairly for two small RNA translational regulators, called small unique example in this field is the regulation of ColE1 replication by RNAI, elegantly studied by Tomizawa and coworkers (for review, see Eguchi et al. 1991; Zeiler and Simons 1996; discussion below). This 108-nt RNA inter- E-MAIL [email protected]; FAX (301) 496-3875. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ acts with the RNA primer for DNA replication, leading gad.1030302. to a decrease in the frequency with which the RNA GENES & DEVELOPMENT 16:2829–2842 © 2002 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/02 $5.00; www.genesdev.org 2829 Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Gottesman primer extends into a DNA primer and, therefore, low- Some of these RNAs resemble the snoRNAs that act as ering plasmid copy number. The use of antisense RNA to guides for tRNA and rRNA modification in eukaryotes, control plasmid copy number is widespread in both but a substantial number appear to be unique. The func- gram-positive and gram-negative organisms; in many of tion of these newly described ones remains unknown; these plasmids, it is synthesis of the replication protein presumably some are regulatory RNAs. that is subject to negative regulation rather than the rep- In eukaryotes, searches for small RNAs have been mo- lication primer itself. A second set of plasmid-encoded tivated by the recognition of two small RNAs, lin-4 and RNA-based regulatory systems are the antitoxin compo- let-7, as critical regulators of development in C. elegans. nents of post-segregational killing (PSK) modules or ad- Methods were designed to purify RNAs with similarity diction systems (Fig. 2, below; for review, see Gerdes et to these molecules (Lagos-Quintana et al. 2001, 2002; al. 1997; Engelberg-Kulka and Glaser 1999). These sys- Lau et al. 2001; Lee and Ambros 2001; Reinhart et al. tems help ensure plasmid stability by acting as lethal 2002). This approach has successfully found dozens of timers, killing cells that have lost the encoding plasmid. similar molecules, called microRNAs, in C. elegans, Toxin synthesis or activity is sequestered by an unstable Drosophila, Hela cells, mouse tissues, and Arabidopsis antitoxin; when new mRNA synthesis ceases with loss (summarized in Table 1); these molecules are frequently of the plasmid from the cell and the unstable antitoxin found to be embedded in a larger (65–70 nt) inverted re- decays, the toxin is free to kill the cell. For one set of peat structure, and to depend on processing by the such toxin/antitoxin systems, including the well-studied RNAse III homolog Dicer for maturation. Like lin-4 and hok/sok system from ColE1 (for review, see Gerdes et al. let-7, it is expected that these will act as translational 1997), the unstable antitoxin is a small RNA that inhib- repressors. Essentially all of these RNAs appear to be its, by a complicated series of events, the synthesis of the encoded outside of ORFs; some of the RNAs identified toxin. In other cases, the antitoxin is an unstable protein, are conserved among fairly disparate species. In these degraded by the ATP-dependent cytoplasmic proteases. organisms, conservation of intergenic regions can also be In addition to these plasmid systems, antisense RNAs used as an indicator of the presence of structured small have been found to regulate phage immunity and RNAs (Lee and Ambros 2001); approaches that depend growth, as well as transposition, again using RNAs en- on finding promoters and terminators are unlikely to be coded by the antisense strand of the target gene (for re- as useful in the near future. The other class of small view, see Wagner and Simons 1994; Zeiler and Simons RNAs that might have been identified in these searches 1996). are the interfering RNAs, or RNAi; little evidence for What about the bacterial host itself? As of 1999, about such double-stranded RNAs were found, supporting the a dozen small RNAs encoded by the Escherichia coli idea that although microRNAs do regulatory jobs in the chromosome had been identified. Almost all of these cell, RNAi is primarily used to protect the cell from in- were found either in searches for stable small RNAs or vaders. A broader search for somewhat larger RNAs in serendipitously—from phenotypes of multicopy plas- the mouse brain yielded multiple new guide RNAs as mids or by identification of unexpected transcripts near well as many of unknown function (Huttenhofer et al. genes of interest (for review, see Wassarman et al. 1999; 2001). Although it is not yet possible to make an accu- Gottesman et al. 2001; Wassarman 2002). This set of 12 rate estimate of the numbers of small RNAs in a eukary- RNAs in E. coli has expanded to >50 over the last two otic genome, those found thus far in nonexhaustive years, as a result of a concerted effort in a number of searches suggest many hundreds are likely. laboratories to define methods for finding small RNAs in It is clear that what has been learned thus far will genomes (summarized in Table 1). The searches in E. coli simplify future searches for these previously elusive utilized conservation at both the sequence and structural regulators. Using the characteristics of the most inter- level (covariation suggesting the existence of stems; Ri- esting of the currently known small RNAs, increasingly vas et al.
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