Editorial: RNA–ligand interactions

Abstract

Since its discovery, RNA has surprised and fascinated the scientific community with its multifaceted roles in cell biology. Early observations linking RNA to protein biosynthesis emerged during the late nineteen thirties and were followed by the seminal discovery of ribosomes. A revolutionary proposal during the mid forties first associated ribosomes and protein biosynthesis. Twenty or so years passed until messenger and transfer RNAs were discovered, and the genetic code was elucidated. These discoveries cemented the central dogma of biology, where the transfer of genetic information from a string of nucleotides in DNA to a chain of amino acids in proteins is mediated via RNA. Although RNA was recognized to play key roles in protein expression, it was held as a passive carrier of genetic information for decades. The discovery of RNA enzymes (ribozymes) in the nineteen eighties had changed this view and paved the way for a major paradigm shift. The impressive potential of RNA catalysis not only categorized RNA as a respectable functional biomolecule, but also provided the necessary foundation for advancing the thesis of an RNA world. In this hypothetical prebiotic environment, RNA played a dual role, serving both as a carrier of genetic information and as the catalytic molecule. These are exciting days for RNA researchers. A sense of another paradigm shift is emerging. High-resolution structures of ribosomes, where RNA is unequivocally shown to be the key component responsible for peptide bond formation, shed light on this wonderful biological machine, its inner workings and modes of inhibition. RNA interference (RNAi), a cellular response to double stranded RNA leading to sequence-specific gene silencing, highlights a new and unexpected role for small RNA molecules in gene regulation. This intriguing discovery has already revolutionized basic biological research and is at the foundation of current industrial endeavors. Additionally, brand new experimental evidence, showing the specific interaction between low molecular weight metabolites and mRNAs related to their biosynthetic pathways, begins to fill gaps in our basic understanding of biosynthesis and its regulation. RNA, once again, is taking center stage. The ability of RNA to specifically interact with large and small molecules is key to its diverse biological functions. Revealing the structural and dynamic features of RNA–ligand recognition events, as well as their temporal and spatial confinement, will directly impact our ability to ultimately control cell function at the RNA level. It will also open up new opportunities to combat pathogens by specifically targeting their RNA or RNA–protein complexes. With this in mind we have put together this special issue of Biopolymers/Nucleic Acid Sciences, focusing on RNA–ligand interactions. The following articles cover a range of exciting topics that provide a sound source for both experts in the field as well as for those who are curious about this rapidly developing area. Knowledge of the recognition modes utilized by natural, semi-synthetic and artificial low molecular weight RNA ligands is crucial for the future design of novel RNA binders. Hermann provides a comprehensive overview of known RNA binders and, when available, a structural insight into the specific molecular interactions involved in ligand–RNA recognition. Not surprisingly, many of the naturally occurring RNA binders known to date target the ribosome. This large and essential cellular machine possesses several Achilles' heels. These vulnerable points are elegantly exploited by far smaller natural products that meddle with the delicate process of protein biosynthesis. These low molecular weight RNA ligands display impressive structural and functional group diversity, a sophistication that is yet to be matched by artificial synthetic RNA binders. With some exceptions, most drug-like small molecules designed to target RNA sites exhibit lower affinity and, more importantly, lower target specificity. Nevertheless, by taking advantage of the increasing amount of structural information, chemists are bound to ultimately discover potent and selective molecules that target RNA and ribonucleoprotein complexes. Hermann's updated and well-organized summary provides an excellent reference for both experts and novices who are intrigued by RNA–small molecule interactions. Yonath and her group describe the architecture of the ribosome, its inner working as well as its inhibition by a variety of naturally occurring and synthetic antibiotics. Structural insight into the heart of this incredible molecular machine has become possible at relatively high resolution only in recent years and has revolutionized our view of protein biosynthesis. Yonath takes the reader through the evolution of ribosomal crystallography and shares the milestones that helped to materialize this enormous undertaking. This investment paid back by providing amazing insights into the molecular details of protein biosynthesis, its key steps and vulnerable points. Due to its central function, the ribosome is a prime target for many antibiotics. Such small organic molecules, described by Yonath as “sticks in the wheels of the translation machinery,” easily interfere with the otherwise very effective process of protein synthesis. Solving the crystal structures of ribosome–antibiotics complexes revealed the antibiotics' binding sites and provided insight into their modes of action. Interestingly, the antibiotics have been found to utilize a limited number of strategic sites to exert their action, despite their structural diversity. Nevertheless, the ribosome remains a goldmine for RNA–ligand interactions, and it is hoped that these data will assist medicinal chemists in creating the next generation of low molecular weight antibiotics. The final part of this overview outlines a proposed unified mechanism for ribosome-mediated protein biosynthesis that sheds light on peptide-bond formation as well as the synchronized positioning and movement of various modules within this large assembly. In spite of the enormous progress made in recent years, it is clear that many fundamental questions remain open. Exciting times are ahead of us! Aminoglycoside antibiotics are mentioned in this volume numerous times as the most studied RNA binders. The interest in this unique family of natural products stems from three major reasons: (a) their potent bactericidal activity, (b) their ability to selectively target the bacterial decoding A-site, and (c) their special place among low molecular weight ligands, being the only family of RNA selective binders. Vicens and Westhof discuss the high resolution structures of aminoglycosides bound to the 30S ribosomal particle, aminoglycosides complexed to short oligonucleotides containing the A-site, and aminoglycosides bound to resistance enzymes. They take this structural analysis to a new dimension by delineating several key aspects: (a) the molecular basis for aminoglycosides–rRNA recognition highlighting these antibiotics as a family of A-site specific ligands, (b) a molecular level understanding of the mechanism utilized by aminoglycosides for meddling with the fidelity of the translation process, and (c) a molecular level insight into a variety of resistance mechanisms that include antibiotics-modifying enzymes as well as point mutation or methylation of key A-site nucleotides. It is gratifying to see how a coherent picture emerges where the biochemistry of aminoglycosides can finally be rationalized at a molecular level. This incredible insight is extremely helpful in designing new antibiotics that can combat pathogens and their resistance mechanisms. The chapter by Vicens and Westhof is a “must read” for any aminoglycosides enthusiast. A thorough understanding of antibiotics–RNA binding requires, in addition to structural data, knowledge of thermodynamic parameters. In a chapter entitled “Thermodynamics of Aminoglycoside-rRNA Recognition,” Pilch and colleagues present their meticulous calorimetric, spectroscopic, osmotic stress and computational studies. Their results open a window into the complex interplay between the molecular forces that govern the rRNA affinity and selectivity of aminoglycosides. For example, in their pH- and buffer-dependent binding studies, the authors identify protonation reactions as significant events in aminoglycoside–RNA binding. In the absence of protonation effects, the RNA binding free energy reflects unfavorable contributions (entropic cost of bimolecular complexation and RNA conformational changes) that are overwhelmed by favorable contributions resulting from the polyelectrolyte effect and a multitude of noncovalent aminoglycoside–RNA interactions. Another strength of such comparative biophysical studies is demonstrated in the ability to decipher the contribution of specific functional groups or sugar residues to the ligand–RNA binding free energy. It is apparent that a combination of structural work with a thorough biophysical analysis can significantly enhance the predictive ability of ligand “designers” in their search for new potent and selective RNA binders. While many of the chapters presented in this issue emphasize low molecular weight ligands as RNA binders, RNA-binding peptides have taught us a great deal about RNA recognition. Relatively short amino acid sequences, excised out of the RNA binding domain of larger proteins, typically exhibit exceptionally high affinity and selectivity to their cognate RNA targets. These favorable binding characteristics, well understood in terms of the necessary biological function, are yet to be matched by synthetic small molecules. Das and Frankel examine the boundaries of RNA-binding peptide sequence space in a structural context, emphasizing the peptide secondary structure and the known structural features of the RNA-peptide complexes. The authors summarize extensive combinatorial library experiments illustrating that many solutions exist for this recognition problem. Generally, such exercises in molecular diversity not only lead to the discovery of RNA-binding peptides with higher affinity than their natural counterpart, but also teach us which residues are essential and which are “mutable” as well as how stabilizing a necessary secondary structure can influence the RNA binding characteristics. Frankel's overview presents a challenge and inspiration to synthetic chemists. It is hoped that additional screens combined with the tools of peptidomimetic chemistry can help transform these fascinating RNA binders into novel ligands with therapeutic potential. Double stranded RNA (dsRNA) has been drawing increasing attention lately, mostly due to its participation in key processes such as RNA editing and RNAi. Despite the significance of this unique duplex, the contemporary understanding of the rules that govern its recognition by proteins is rather undeveloped. Beal and coworkers present the current knowledge in this field, emphasizing the significance of double stranded RNA binding motifs (dsRBM) and the interplay between general affinity to dsRNA and sequence specificity. Two proteins that are currently studied in the author's lab, the protein kinase regulated by RNA (PKR) and an adenosine deaminase that operates on dsRNA (ADAR2), are discussed in detail, together with affinity cleavage tools developed for their study. In addition to discussing protein-dsRNA binding, Beal and colleagues present their efforts to develop small organic molecules that specifically target dsRNA. Their approach to this challenging task relies on threading intercalation. This binding mode takes advantage of dsRNA “breathing” (most prominent next to deformed sites such as loops and bulges) and facilitates the delivery of recognition elements to the different grooves. There is little doubt that this field will continue to grow, at an accelerated pace, as the appreciation for the roles dsRNA plays in RNA biochemistry broadens. RNA is rapidly emerging as an important target for the development of small molecule therapeutics. Luedtke and Tor describe the discovery and characterization of new anti-viral agents that bind to the HIV-1 Rev Response Element (RRE), a key viral RNA site. The RRE may prove to be an ideal target for interfering with viral replication, as it is highly conserved even between diverse groups of HIV isolates. Systematic evaluation of both the RRE affinity and selectivity of numerous small molecule inhibitors is essential for deciphering the parameters that govern effective RRE recognition. Fluorescence-based methods play a key role in such studies and the authors share their experience in designing and employing these tools. The functional inhibition of the RRE can be evaluated by conducting peptide displacement experiments from a solid-phase immobilized fluorescent Rev–RRE complex. The same assay can determine the RRE specificity of inhibitors by re-evaluating the activity of each inhibitor in the presence of competing nucleic acids. While the author's discussion surrounds a single RNA target, the lessons learned are of general significance. As new RNA targets of therapeutic relevance continue to be identified, the ability to rapidly screen potential small molecule effectors for their RNA affinity and selectivity, as well as for their ability to exert the necessary biological function, will become even more important. Ligands that possess high affinity and specificity for a biologically important RNA site should continue to provide important leads for drug development and teach us about the parameters that govern effective RNA–small molecule recognition. RNA molecules play key roles in vital biological processes and are attractive targets for therapeutic intervention. Successful design of specific RNA binders requires the intimate knowledge of RNA structure, folding and recognition. As apparent from this issue's contributions, our understanding of the modes in which RNA is recognized by various ligands has advanced significantly, although alot remains to be unveiled. It is hoped that this collection of articles will inspire more scientists to get involved in an emerging field that assures future fundamental discoveries as well as a substantial impact on medicinal chemistry.