Riboswitches: still a lot of undiscovered country

In this era of electronic publishing there are not a lot of compelling reasons to keep one's personal library filled with shelves of dusty print journals. Over the past few years, my office and laboratory have been depopulated of most of these relics with the exception of a few issues of special significance for various reasons: an issue of Nature with the first hammerhead structure, issues of Science describing structures of the P4-P6 domain from a group I intron and of the 50S ribosomal subunit and, of course, some issues containing papers of my own. But, amongst the rump of this once substantial hoard are issues 2 and 5 of volume 1 of RNA. Why did these survive? Unlike most of the others, they have dog-eared pages, highlighted references, and post-its with comments about a technique I needed to look into or a result relevant to my research project at the time. As a graduate student at the time of the inception of this journal, RNA rapidly became the journal that I studied and thus an enormously valuable learning tool that greatly shaped my growth as a scientist. I suspect that I just could not bear to let go of these issues, not because of any one significant piece of research—as in the others—but just the fact that they were old friends that didn't need to go into the recycling bin just yet. Looking through the somewhat yellowed pages of these issues from the not-so-distant past, I find them remarkable in what I didn't see. On their cover is a brightly colored model of a group I intron from Francois Michel, but within their pages is an almost complete absence of RNA structure with the exception of a nice article on U-turns in GNRA tetraloops from Art Pardi. Indeed, when RNA was founded, very little was known about RNA structure beyond tRNA, tetraloops, and simple A-form duplexes. While there is plenty of research on RNA “oldies-but-goodies” such as tRNA, the spliceosome, and the ribosome in these early issues, entirely absent is the plethora of regulatory RNAs including small interfering RNAs, lncRNAs, CRISPR RNAs, or my current personal favorite, riboswitches. All of these RNAs, and many more, were discovered after the inception of RNA, and this journal has been able to chronicle work on characterizing them from discovery through unraveling the details of their mechanisms. This has certainly been true for our knowledge of riboswitches, which have progressed from a few intriguing genetic elements to an important model system for exploring RNA structure-function relationships over the past twenty years. In the late 1990's, various conserved elements were found in the leader sequences of bacterial mRNAs and identified as regulators of gene expression of unknown mechanism, including the S-box by Tina Henkin, the btuB leader by Robert Kadner, and the thi-box by Juan Miranda-Rios. Additionally, bioinformatic analyses of the rapidly increasing number of bacterial genomes uncovered others such as the RFN element in the leader of mRNAs associated with riboflavin biosynthesis by Mikhail Gelfand. In the absence of evidence for proteins interacting with these sequences to manage the regulatory response, such as in the Bacillus subtilis trp operon previously described by Charles Yanofsky, it was proposed in a prescient review by Gary Stromo that, like RNA aptamers raised through in vitro selection methods, these mRNAs could directly interact with small molecules. Ronald Breaker's group proved this hypothesis in 2002 for cobalamin binding to the btuB leader, followed by S-adenosylmethionine (SAM) binding the S-box by Tina Henkin, and thiamine-pyrophosphate (TPP) binding the thi-box and flavin mononucleotide (FMN) binding to the RFN element by Evgeny Nudler. These regulatory elements became known as riboswitches, formally defined as RNAs that bind small organic molecules or ions via an “aptamer domain” that instructs a second domain called the “expression platform” that in turn directs the expression outcome. By 2004, the first atomic-level pictures of how the aptamer domain of purine riboswitches binds their cognate effector emerged concurrently from my own laboratory and that of Dinshaw Patel. Since these discoveries, riboswitches have emerged as an important model system in the RNA field. In a little over a decade, the number of these elements discovered and validated—the bulk of which has come from the work of Ronald Breaker and colleagues—has surged past twenty-five, with more in the pipeline. This has provided a wealth of feedstock for investigation of RNA structure, folding, and function. Structural analysis of riboswitch aptamer domains in complex with cognate ligand has kept pace with discovery such that there is a crystal structure of a representative of almost every validated class. These structures currently comprise the majority of known small folded RNA domains (sequences of >200 nt that exhibit helical packing), substantially informing our understanding of RNA architecture. Their small size, high-fidelity folding, and easily observable activity make riboswitch aptamers ideal model systems for investigation of various biological and chemical properties of RNA using an increasing array of biochemical, biophysical, and computational tools. Riboswitches are also emerging as important RNAs in human health and engineering of biological systems. Because of their ability to bind small molecules with high affinity and specificity and their almost complete uniqueness to bacteria, riboswitches have been proposed by Blount and Breaker to be an important new class of targets for antimicrobial therapeutics and are being employed as model systems for structure-based design of molecules targeting RNA. Indeed, they discovered that roseoflavin is a naturally occurring compound that acts by targeting the FMN riboswitch, suggesting that this is a practical route for the creation of new antimicrobials. The utility of riboswitches in synthetic biology is also beginning to emerge. Recent work has exploited these RNAs to select for bacteria that overproduce metabolites, reveal novel aspects of biosynthetic pathways, and as sensors of intracellular metabolites. Despite this rapidly growing body of research on riboswitches, there remain significant underexplored areas as well as unanswered questions. The following is certainly not a comprehensive list of what remains to be learned about riboswitches, but is a set of the questions that represents important avenues of research on these RNAs.