Search

Analysis of the profile of the tRNA modifications in severalArchaeaallowed us to observe a novel modified uridine in the V-loop of several tRNAs from two species:Pyrococcus furiosusandSulfolobus acidocaldarius. Recently, Ohira and colleagues characterized 2′-phosphouridine (Up) at position 47 in tRNAs of thermophilicSulfurisphaera tokodaii, as well as in several other archaea and thermophilic bacteria. From the presence of the genearkIcorresponding to the RNA kinase responsible for Up47 formation, they also concluded that Up47 should be present in tRNAs of other thermophilicArchaea. Reanalysis of our earlier data confirms that the unidentified residue in tRNAs of bothP. furiosusandS. acidocaldariusis indeed 2′-phosphouridine followed by m5C48. Moreover, we find this modification in several tRNAs of otherArchaeaand of the hyperthermophilic bacteriumAquifex aeolicus.

The 5′UTR part of coronavirus genomes plays key roles in the viral replication cycle and translation of viral mRNAs. The first 75–80 nt, also called the leader sequence, are identical for genomic mRNA and subgenomic mRNAs. Recently, it was shown that cooperative actions of a 5′UTR segment and the nonstructural protein NSP1 are essential for both the inhibition of host mRNAs and for specific translation of viral mRNAs. Here, sequence analyses of both the 5′UTR RNA segment and the NSP1 protein have been done for several coronaviruses, with special attention to the betacoronaviruses. The conclusions are: (i) precise specific molecular signatures can be found in both the RNA and the NSP1 protein; (ii) both types of signatures correlate between each other. Indeed, definite sequence motifs in the RNA correlate with sequence motifs in the protein, indicating a coevolution between the 5′UTR and NSP1 in betacoronaviruses. Experimental mutational data on 5′UTR and NSP1 from SARS-CoV-2 using cell-free translation extracts support these conclusions and show that some conserved key residues in the amino-terminal half of the NSP1 protein are essential for evasion to the inhibitory effect of NSP1 on translation.

The prediction of RNA three-dimensional structures remains an unsolved problem. Here, we report double-blind assessments of RNA structure predictions in CASP15, the first CASP exercise in which RNA modeling was assessed. Forty two predictor groups submitted models for at least one of twelve RNA-containing targets. These models were evaluated by the RNA-Puzzles organizers and, separately, by a CASP-recruited team using metrics (GDT, lDDT) and approaches (Z-score rankings) initially developed for assessment of proteins and generalized here for RNA assessment. The two assessments independently ranked the same predictor groups as first (AIchemy_RNA2), second (Chen), and third (RNAPolis and GeneSilico, tied); predictions from deep learning approaches were significantly worse than these top ranked groups, who did not use deep learning. Further analyses based on direct comparison of predicted models to cryogenic electron microscopy (cryo-EM) maps and X-ray diffraction data support these rankings. With the exception of two RNA-protein complexes, models submitted by CASP15 groups correctly predicted the global topology of the RNA targets. Comparisons of CASP15 submissions to designed RNA nanostructures as well as molecular replacement trials highlight the potential utility of current RNA modeling approaches for RNA nanotechnology and structural biology, respectively. Nevertheless, challenges remain in modeling fine details such as non-canonical pairs, in ranking among submitted models, and in prediction of multiple structures resolved by cryo-EM or crystallography.

In most bacterial type A RNase P RNAs (P RNAs), two major loop-helix tertiary contacts (L8–P4 and L18–P8) help to orient the two independently folding S- and C-domains for concerted recognition of precursor tRNA substrates. Here, we analyze the effects of mutations in these tertiary contacts in P RNAs from three different species: (i) the psychrophilic bacterium Pseudoalteromonas translucida (Ptr), (ii) the mesophilic radiation-resistant bacterium Deinococcus radiodurans (Dra), and (iii) the thermophilic bacterium Thermus thermophilus (Tth). We show by UV melting experiments that simultaneous disruption of these two interdomain contacts has a stabilizing effect on all three P RNAs. This can be inferred from reduced RNA unfolding at lower temperatures and a more concerted unfolding at higher temperatures. Thus, when the two domains tightly interact via the tertiary contacts, one domain facilitates structural transitions in the other. P RNA mutants with disrupted interdomain contacts showed severe kinetic defects that were most pronounced upon simultaneous disruption of the L8–P4 and L18–P8 contacts. At 37°C, the mildest effects were observed for the thermostable Tth RNA. A third interdomain contact, L9–P1, makes only a minor contribution to P RNA tertiary folding. Furthermore, D. radiodurans RNase P RNA forms an additional pseudoknot structure between the P9 and P12 of its S-domain. This interaction was found to be particularly crucial for RNase P holoenzyme activity at near-physiological Mg2+ concentrations (2 mM). We further analyzed an exceptionally stable folding trap of the G,C-rich Tth P RNA.

tRNA molecules have well-defined sequence conservations that reflect the conserved tertiary pairs maintaining their architecture and functions during the translation processes. An analysis of aligned tRNA sequences present in the GtRNAdb database (the Lowe Laboratory, University of California, Santa Cruz) led to surprising conservations on some cytosolic tRNAs specific for alanine compared to other tRNA species, including tRNAs specific for glycine. First, besides the well-known G3oU70 base pair in the amino acid stem, there is the frequent occurrence of a second wobble pair at G30oU40, a pair generally observed as a Watson–Crick pair throughout phylogeny. Second, the tertiary pair R15/Y48 occurs as a purine–purine R15/A48 pair. Finally, the conserved T54/A58 pair maintaining the fold of the T-loop is observed as a purine–purine A54/A58 pair. The R15/A48 and A54/A58 pairs always occur together. The G30oU40 pair occurs alone or together with these other two pairs. The pairing variations are observed to a variable extent depending on phylogeny. Among eukaryotes, insects display all variations simultaneously, whereas mammals present either the G30oU40 pair or both R15/A48 and A54/A58. tRNAs with the anticodon 34A(I)GC36 are the most prone to display all those pair variations in mammals and insects. tRNAs with anticodon Y34GC36 have preferentially G30oU40 only. These unusual pairs are not observed in bacterial, nor archaeal, tRNAs, probably because of the avoidance of A34-containing anticodons in four-codon boxes. Among eukaryotes, these unusual pairing features were not observed in fungi and nematodes. These unusual structural features may affect, besides aminoacylation, transcription rates (e.g., 54/58) or ribosomal translocation (30/40).

Wobble GU pairs (or G•U) occur frequently within double-stranded RNA helices interspersed between standard G=C and A-U Watson-Crick pairs. Another type of G•U pair interacting via their Watson-Crick edges has been observed in the A site of ribosome structures between a modified U34 in the tRNA anticodon triplet and G+3 in the mRNA. In such pairs the electronic structure of the U is changed with a negative charge on N3(U), resulting in two H-bonds between N1(G)…O4(U) and N2(G)…N3(U). Here, we report that such pairs occur in other highly conserved positions in ribosomal RNAs of bacteria in the absence of U modification. An anionic cis Watson-Crick G•G pair is also observed and well conserved in the small subunit. These pairs are observed in tightly folded regions.

The outbreak of the COVID-19 pandemic has led to intensive studies of both the structure and replication mechanism of SARS-CoV-2. In spite of some secondary structure experiments being carried out, the 3D structure of the key function regions of the viral RNA has not yet been well understood. At the beginning of COVID-19 breakout, RNA-Puzzles community attempted to envisage the three-dimensional structure of 5′- and 3′-Un-Translated Regions (UTRs) of the SARS-CoV-2 genome. Here, we report the results of this prediction challenge, presenting the methodologies developed by six participating groups and discussing 100 RNA 3D models (60 models of 5′-UTR and 40 of 3′-UTR) predicted through applying both human experts and automated server approaches. We describe the original protocol for the reference-free comparative analysis of RNA 3D structures designed especially for this challenge. We elaborate on the deduced consensus structure and the reliability of the predicted structural motifs. All the computationally simulated models, as well as the development and the testing of computational tools dedicated to 3D structure analysis, are available for further study.

The silkworm Bombyx mori is a domestic insect for silk production and a lepidopteran model. The currently available genomes limit a full understanding of its genetic and phenotypic diversity. Here we assembled long-read genomes of 545 domestic and wild silkworms and constructed a high-resolution pan-genome dataset. We found that the silkworm population harbors extremely variable genomes containing 7,308 new gene families, 4,260 (22%) core gene families, and 3,432,266 non-redundant SVs. We deciphered a series of causal genes and variants associated with domestication, breeding, and ecological adaptation traits, and experimentally validated two of those genes using CRISPR-Cas9 or RNA interference. This unprecedented large-scale genomic resource allows for high-throughput screening of interesting traits for functional genomic research and breeding improvement of silkworms and may serve as a guideline for traits decoding in other species.

In silico prediction is a well-established approach to derive a general shape of an RNA molecule based on its sequence or secondary structure. This paper reports an analysis of the stereochemical quality of the RNA three-dimensional models predicted using dedicated computer programs. The stereochemistry of 1,052 RNA 3D structures, including 1,030 models predicted by fully automated and human-guided approaches within 22 RNA-Puzzles challenges and reference structures, is analysed. The evaluation is based on standards of RNA stereochemistry that the Protein Data Bank requires from deposited experimental structures. Deviations from standard bond lengths and angles, planarity or chirality are quantified. A reduction in the number of such deviations should help in the improvement of RNA 3D structure modelling approaches.

Fluorogenic RNA aptamers are short nucleic acids able to specifically interact with small molecules and strongly enhance their fluorescence upon complex formation. Among the different systems recently introduced, Spinach, an aptamer forming a fluorescent complex with the 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), is one of the most promising. Using random mutagenesis and ultrahigh-throughput screening, we recently developed iSpinach, an improved version of the aptamer, endowed with an increased folding efficiency and thermal stability. iSpinach is a shorter version of Spinach, comprising five mutations for which the exact role has not yet been deciphered. In this work, we cocrystallized a reengineered version of iSpinach in complex with the DFHBI and solved the X-ray structure of the complex at 2 Å resolution. Only a few mutations were required to optimize iSpinach production and crystallization, underlying the good folding capacity of the molecule. The measured fluorescence half-lives in the crystal were 60% higher than in solution. Comparisons with structures previously reported for Spinach sheds some light on the possible function of the different beneficial mutations carried by iSpinach.

On November 18-19, 2016, the Human Frontier Science Program Organization (HFSPO) hosted a meeting of senior managers of key data resources and leaders of several major funding organizations to discuss the challenges associated with sustaining biological and biomedical (i.e., life sciences) data resources and associated infrastructure. A strong consensus emerged from the group that core data resources for the life sciences should be supported through coordinated international efforts that better ensure long-term sustainability and that appropriately align funding with scientific impact. Ideally, funding for such data resources should allow for access at no charge, as is presently the usual (and preferred) mechanism. Designing and implementing a plan for long-term accessibility is the vision. Below, the rationale for this vision is described, and some important considerations for developing a new international funding model to support core data resources for the life sciences are presented.

We report the results of a first, collective, blind experiment in RNA three-dimensional (3D) structure prediction, encompassing three prediction puzzles. The goals are to assess the leading edge of RNA structure prediction techniques; compare existing methods and tools; and evaluate their relative strengths, weaknesses, and limitations in terms of sequence length and structural complexity. The results should give potential users insight into the suitability of available methods for different applications and facilitate efforts in the RNA structure prediction community in ongoing efforts to improve prediction tools. We also report the creation of an automated evaluation pipeline to facilitate the analysis of future RNA structure prediction exercises.

The locus alx, which encodes a putative transporter, was discovered previously in a screen for genes induced under extreme alkaline conditions. Here we show that the RNA region preceding the alx ORF acts as a pH-responsive element, which, in response to high pH, leads to an increase in alx expression. Under normal growth conditions this RNA region forms a translationally inactive structure, but when exposed to high pH, a translationally active structure is formed to produce Alx. Formation of the active structure occurs while transcription is in progress under alkaline conditions and involves pausing of RNA polymerase at two distinct sites. Alkali increases the longevity of pausing at these sites and thereby interferes with formation of the inactive structure and promotes folding of the active one. The alx locus represents the first example of a pH-responsive riboregulator of gene expression, introducing a novel regulatory mechanism that involves RNA folding dynamics driven by pH.

Human accelerated region 1 (HAR1) is a short DNA region identified recently to have evolved the most rapidly among highly constrained regions since the divergence from our common ancestor with chimpanzee. It is transcribed as part of a noncoding RNA specifically expressed in the developing human neocortex. Employing a panoply of enzymatic and chemical probes, our analysis of HAR1 RNA proposed a secondary structure model differing from that published. Most surprisingly, we discovered that the substitutions between the chimpanzee and human sequences led the human HAR1 RNA to adopt a cloverleaf-like structure instead of an extended and unstable hairpin in the chimpanzee sequence. Thus, the rapid evolutionary changes resulted in a profound rearrangement of HAR1 RNA structure. Altogether, our results provide a structural context for elucidating HAR1 RNA function.

The aim of the RNA Ontology Consortium (ROC) is to create an integrated conceptual framework—an RNA Ontology (RO)—with a common, dynamic, controlled, and structured vocabulary to describe and characterize RNA sequences, secondary structures, three-dimensional structures, and dynamics pertaining to RNA function. The RO should produce tools for clear communication about RNA structure and function for multiple uses, including the integration of RNA electronic resources into the Semantic Web. These tools should allow the accurate description in computer-interpretable form of the coupling between RNA architecture, function, and evolution. The purposes for creating the RO are, therefore, (1) to integrate sequence and structural databases; (2) to allow different computational tools to interoperate; (3) to create powerful software tools that bring advanced computational methods to the bench scientist; and (4) to facilitate precise searches for all relevant information pertaining to RNA. For example, one initial objective of the ROC is to define, identify, and classify RNA structural motifs described in the literature or appearing in databases and to agree on a computer-interpretable definition for each of these motifs. To achieve these aims, the ROC will foster communication and promote collaboration among RNA scientists by coordinating frequent face-to-face workshops to discuss, debate, and resolve difficult conceptual issues. These meeting opportunities will create new directions at various levels of RNA research. The ROC will work closely with the PDB/NDB structural databases and the Gene, Sequence, and Open Biomedical Ontology Consortia to integrate the RO with existing biological ontologies to extend existing content while maintaining interoperability.

The three-way junctions contained in X-ray structures of folded RNAs have been compiled and analyzed. Three-way junctions with two helices approximately coaxially stacked can be divided into three main families depending on the relative lengths of the segments linking the three Watson-Crick helices. Each family has topological characteristics with some conservation in the non-Watson-Crick pairs within the linking segments as well as in the types of contacts between the segments and the helices. The most populated family presents tertiary interactions between two helices as well as extensive shallow/minor groove contacts between a linking segment and the third helix. On the basis of the lengths of the linking segments, some guidelines could be deduced for choosing a topology for a three-way junction on the basis of a secondary structure. Examples and prediction bas‘ed on those rules are discussed.

Electron paramagnetic resonance (EPR) spectroscopy is used to study the binding of MnII ions to a tertiary stabilized hammer-head ribozyme (tsHHRz) and to compare it with the binding to the minimal hammerhead ribozyme (mHHRz). Continuous wave EPR measurements show that the tsHHRz possesses a single high-affinity MnII binding site with a KD of ≤10 nM at an NaCl concentration of 0.1 M. This dissociation constant is at least two orders of magnitude smaller than the KD determined previously for the single high-affinity MnII site in the mHHRz. In addition, whereas the high-affinity MnII is displaced from the mHHRz upon binding of the aminoglycoside antibiotic neomycin B, it is not from the tsHHRz. Despite these pronounced differences in binding, a comparison between the electron spin echo envelope modulation and hyperfine sublevel correlation spectra of the minimal and tertiary stabilized HHRz demonstrates that the structure of both binding sites is very similar. This suggests that the MnII is located in both ribozymes between the bases A9 and G10.1 of the sheared G · A tandem base pair, as shown previously and in detail for the mHHRz. Thus, the much stronger MnII binding in the tsHHRz is attributed to the interaction between the two external loops, which locks in the RNA fold, trapping the MnII in the tightly bound conformation, whereas the absence of long-range loop–loop interactions in the mHHRz leads to more dynamical and open conformations, decreasing MnII binding.

The class I ligase ribozyme catalyzes a Mg++-dependent RNA-ligation reaction that is chemically analogous to a single step of RNA polymerization. Indeed, this ribozyme constitutes the catalytic domain of an accurate and general RNA polymerase ribozyme. The ligation reaction is also very rapid in both single- and multiple-turnover contexts and thus is informative for the study of RNA catalysis as well as RNA self-replication. Here we report the initial characterization of the three-dimensional architecture of the ligase. When the ligase folds, several segments become protected from hydroxyl-radical cleavage, indicating that the RNA adopts a compact tertiary structure. Ribozyme folding was largely, though not completely, Mg++ dependent, with a K1/2[Mg] < 1 mM, and was observed over a broad temperature range (20°C –50°C). The hydroxyl-radical mapping, together with comparative sequence analyses and analogy to a region within 23S ribosomal RNA, were used to generate a three-dimensional model of the ribozyme. The predictive value of the model was tested and supported by a photo-cross-linking experiment.

Non-Watson-Crick base pairs mediate specific interactions responsible for RNA-RNA self-assembly and RNA-protein recognition. An unambiguous and descriptive nomenclature with well-defined and nonoverlapping parameters is needed to communicate concisely structural information about RNA base pairs. The definitions should reflect underlying molecular structures and interactions and, thus, facilitate automated annotation, classification, and comparison of new RNA structures. We propose a classification based on the observation that the planar edge-to-edge, hydrogen-bonding interactions between RNA bases involve one of three distinct edges: the Watson-Crick edge, the Hoogsteen edge, and the Sugar edge (which includes the 2'-OH and which has also been referred to as the Shallow-groove edge). Bases can interact in either of two orientations with respect to the glycosidic bonds, cis or trans relative to the hydrogen bonds. This gives rise to 12 basic geometric types with at least two H bonds connecting the bases. For each geometric type, the relative orientations of the strands can be easily deduced. High-resolution examples of 11 of the 12 geometries are presently available. Bifurcated pairs, in which a single exocyclic carbonyl or amino group of one base directly contacts the edge of a second base, and water-inserted pairs, in which single functional groups on each base interact directly, are intermediate between two of the standard geometries. The nomenclature facilitates the recognition of isosteric relationships among base pairs within each geometry, and thus facilitates the recognition of recurrent three-dimensional motifs from comparison of homologous sequences. Graphical conventions are proposed for displaying non-Watson-Crick interactions on a secondary structure diagram. The utility of the classification in homology modeling of RNA tertiary motifs is illustrated.

Subdomain IlId from the hepatitis C virus (HCV) internal ribosome entry site (IRES) has been shown to be essential for cap-independent translation. We have conducted a structural study of a 27-nt fragment, identical in sequence to IlId, to explore the structural features of this subdomain. The proposed secondary structure of IlId is comprised of two 3 bp helical regions separated by an internal loop and closed at one end by a 6-nt terminal loop. NMR and molecular modeling were used interactively to formulate a validated model of the three-dimensional structure of IlId. We found that this fragment contains several noncanonical structural motifs and non-Watson-Crick base pairs, some of which are common to other RNAs. In particular, a motif characteristic of the rRNA alpha-sarcin/ricin loop was located in the internal loop. The terminal loop, 5'-UUGGGU, was found to fold to form a trinucleotide loop closed by a trans-wobble U.G base pair. The sixth nucleotide was bulged out to allow stacking of this U.G pair on the adjacent helical region. In vivo mutational analysis in the context of the full IRES confirmed the importance of each structural motif within IIId for IRES function. These findings may provide clues as to host cellular proteins that play a role in IRES-directed translation and, in particular, the mechanism through which host ribosomes are sequestered for viral function.

The antisense RNA CopA binds to the leader region of the repA mRNA (target: CopT). Previous studies on CopA-CopT pairing in vitro showed that the dominant product of antisense RNA-mRNA binding is not a full RNA duplex. We have studied here the structure of CopA-CopT complex, combining chemical and enzymatic probing and computer graphic modeling. CopI, a truncated derivative of CopA unable to bind CopT stably, was also analyzed. We show here that after initial loop-loop interaction (kissing), helix propagation resulted in an extended kissing complex that involves the formation of two intermolecular helices. By introducing mutations (base-pair inversions) into the upper stem regions of CopA and CopT, the boundaries of the two newly formed intermolecular helices were delimited. The resulting extended kissing complex represents a new type of four-way junction structure that adopts an asymmetrical X-shaped conformation formed by two helical domains, each one generated by coaxial stacking of two helices. This structure motif induces a side-by-side alignment of two long intramolecular helices that, in turn, facilitates the formation of an additional intermolecular helix that greatly stabilizes the inhibitory CopA-CopT RNA complex. This stabilizer helix cannot form in CopI-CopT complexes due to absence of the sequences involved. The functional significance of the three-dimensional models of the extended kissing complex (CopI-CopT) and the stable complex (CopA-CopT) are discussed.

The wobble GoU pairs have been implicated in several biological processes where RNA molecules play a key role. We review the geometrical and conformational properties of wobble GoU pairs on the basis of available crystal structures of RNAs at high resolution. The similarities with the wobble A+oC pairs and UoU pairs are illustrated, while the differences with the recently discovered bifurcated G x U pairs are contrasted.

The crystal structure of the RNA duplex [r(CGUGAUCG)dC]2 has been solved at a resolution of 0.97 A. The model has been refined to R-work and R-free of 14.88% and 19.54% for 23,838 independent reflections. The base-pairing scheme forces the 5'-rC to be excluded from the helix and to be disordered. In the crystals, the sequence promotes the formation of two GoU wobble pairs that cluster around a crystallographic threefold axis in two different ways. In the first contact type, the GoU pairs are exclusively surrounded by water molecules, whereas in the other contact type, the three amino groups of the guanine residues of the symmetry-related GoU pairs trap a sulfate ion. This work provides the first example of the interaction of a GoU pair with a sulfate ion in a helical context. Despite the negative charge on the polynucleotide backbone, the guanine amino N2 is able to attract negatively charged groups that could, in the folding of complex RNA molecules, belong to a negative phosphodiester group from a neighboring strand and, in a RNA-protein complex, to a negative carboxyl group of an aspartate or glutamate side chain.

Retroviruses encapsidate their genome as a dimer of homologous RNA molecules noncovalently linked close to their 5' ends. The dimerization initiation site (DIS) of human immunodeficiency virus type 1 (HIV-1) RNA is a hairpin structure that contains in the loop a 6-nt self-complementary sequence flanked by two 5' and one 3' purines. The self-complementary sequence, as well as the flanking purines, are crucial for dimerization of HIV-1 RNA, which is mediated by formation of a "kissing-loop" complex between the DIS of each monomer. Here, we used chemical modification interference, lead-induced cleavage, and three-dimensional modeling to compare dimerization of subtype A and B HIV-1 RNAs. The DIS loop sequences of these RNAs are AGGUGCACA and AAGCGCGCA, respectively. In both RNAs, ethylation of most but not all phosphate groups in the loop and methylation of the N7 position of the G residues in the self-complementary sequence inhibited dimerization. These results demonstrate that small perturbations of the loop structure are detrimental to dimerization. Conversely, methylation of the N1 position of the first and last As in the loop were neutral or enhanced dimerization, a result consistent with these residues forming a noncanonical sheared base pair. Phosphorothioate interference, lead-induced cleavage, and Brownian-dynamics simulation revealed an unexpected difference in the dimerization mechanism of these RNAs. Unlike subtype B, subtype A requires binding of a divalent cation in the loop to promote RNA dimerization. This difference should be taken into consideration in the design of antidimerization molecules aimed at inhibiting HIV-1 replication.

I still remember vividly a phone call in the spring of 1995. I was then modeling an RNA structure in the darkened room where the Evans & Sutherland PS300 was buzzing loudly. Tom Cech was on the line and, after the usual greetings, he asked with his usual jovial and direct way, “Eric, we want to start a new journal dedicated to RNA; are you willing to become a journal editor?” I was stunned and speechless. My knowledge of RNA biology was scanty. I knew quite well all the twenty or so RNA structures that existed back then, as I had used their structural information to build RNA models in three dimensions, such as the one I was building when Tom called, or that of the core of group I introns I had built with Francois Michel a few years earlier. I remember that particular model to be quite appreciated by the community for rationalizing experimental data and helping to devise new experiments. But I did not think I qualified to become a journal editor! Tom insisted, and answered my novice questions about the job of an editor. Excited but still not fully realizing what was happening, I accepted and never regretted it. Tom's insightful initiative followed a logical move, since several RNA leaders had established the RNA Society in 1993 and organized the first RNA meeting, as we now know them, in Madison, Wisconsin, at the end of May 1996. The RNA journal, hence also supported by the RNA Society, became a critical avenue for publishing the exciting research that had been shared first as posters or talks at the popular RNA meetings. Over the years, the RNA meetings, always extremely well attended, took place in various places around the world with the lively poster sessions being always a fantastic playground for meeting authors and enriching one's list of potential referees. The poster rooms were rustling about RNAs at the origin of life, sequence alignments, dynamics of nucleic acids, magnesium ions, RNA evolution and folding, RNA purification and stability, novel RNAs being discovered in cells. Many of those posters appeared later as articles in the RNA journal. During the oral sessions, one could learn anything that was new in RNA biology in general, splicing, nonsense mediated decay, RNA editing or maturation. In this short note, realizing the amazing expansion of RNA biology, I would like to focus on the developments of RNA structural biology. In 1995, the number of X-ray RNA structures (alone or in complex with protein) in the Protein Data Bank amounted to about 1% of the present content of total RNA and RNP structures. One year later, a breakthrough in RNA crystallography happened with the crystal structure of the P4-P6 domain of the Tetrahymena group I intron, a structure twice the size of a tRNA with novel types of RNA contacts, from the labs of Tom Cech and Jennifer Doudna. Thus, it was a critical moment in the overall perception of the central role of RNA in molecular biology; and Tom was a very active and central figure in the push for promoting research in RNA chemistry, biochemistry, structure, and biological functions. With the beautiful structure of P4-P6, many interested scientists knew that large RNAs could also be crystallized starting from in vitro synthesis and production. And the repertoire of surprising and beautiful RNA architectures expanded at a very quick pace. Over the years, almost every single cover of the RNA Meeting abstract book and of the RNA journal displayed a novel crystal structure of an RNA or RNA/protein complex. The number of RNA and RNP structures in the PDB grew from the 31 available in 1995 to about five times more in 2000 (155 structures), to 15 times in 2005 (462 structures) and, at the beginning of 2015, to close to 60 times the number of RNA and RNP structures (2495) 20 years earlier. Certainly, not all those structures are independent structures; several are variants in sequence or ligand binding. However, about half of the total number of structures is at a resolution worse than 3.3 A. The late 1990s and early 2000s were the big days of ribozymes; and group I introns came to epitomize catalytic RNAs. Surprisingly, however, after the P4-P6 structure, structures of group I introns from various species appeared at a slow pace, starting with Tetrahymena in 1998 and 2003, Azoarcus in 2004, and the phage Twort in 2005. More recently (2014), the structure of the lariat capping ribozyme was also solved. Despite several structures of nucleolytic ribozymes (hammerhead [1994], hepatitis delta virus [1998], hairpin [2001], and recently the twister [2014]), the understanding pathway was tortuous and it took 12 years (1994–2006) to obtain a fully biologically relevant structure of the hammerhead ribozyme. Several factors had to be deconvoluted for reaching some consensus in our chemical understanding; among others, the interplay between folding and catalysis, the distinction between structural and catalytic divalent ions, and the roles of bases through protonation and deprotonation in the chemical steps. Although it took only 10 years between the first structures of the bacterial 30S and 50S ribosomal particles and the structure of the eukaryotic ribosome, it took seven years between the first crystal structure of an RNaseP and of its holoenzyme. For the family of group II introns, since the first crystal structure in 2008, not much fundamentally novel has appeared at the structural level. A field that yielded an amazingly rich chemically and architecturally diverse harvest is that of the riboswitches discovered in 2002. The PDB contains more than 100 structures of the aptamer domain of riboswitches, with and without ligands and under various conditions, with about 20 of them solved at a resolution better than 2.0 A. Those riboswitch aptamer domains recognize a huge variety of ligands, from the tiny fluoride ion to the cyclic second messengers and the large adenosylcobalamin of vitamin B12, and offer striking examples of the potential of folded RNA molecules for molecular recognition. Thus, with only the four main bases and without any chemical modification, one could recognize almost any atomic or molecular entity. All those structures demonstrated that RNA could reach its full molecular recognition potential through convoluted and complex three-dimensional architectural folds exploiting all the stereochemical capabilities of the RNA backbone, recurrent RNA modules, and especially non-Watson–Crick base pairs. Clearly, everything that chemistry allows is at some stage exploited in biology. The recent demonstration that tautomerism of G and U bases contributes to the intrinsic lower limit of fidelity errors in translation upholds that view. The roles of the modified tRNA bases in the anticodon loop constitute now a major stream of research in cellular stress. Thus, those first 20 years of RNA structural research taught us, among other things: (i) that all hydrogen bonding capabilities of the bases are exploited for forming the base pairs necessary for the beautifully intricate architectural folds that RNA can adopt to bind other RNAs, proteins, or encapsulate ligands, (ii) that protonation and deprotonation of bases contribute to the catalysis of nucleolytic ribozymes, (iii) that tautomeric forms of bases lead to Watson–Crick-like base pairs that fool the translational fidelity mechanisms of ribosomes and polymerases. Structural biology of RNA, especially with X-ray crystallography, made possible these advances in our understanding. A huge amount of effort put forth by several researchers went into the experimental and theoretical development of tools for deriving accurate and cogent crystallographic structures with biological relevance. The success of crystallography is undoubtedly due to the fact that it is firmly and solidly grounded in physics and chemistry. However, despite the numerous mathematical or chemical safeguards and warnings available, errors in stereochemistry, in RNA folds or in solvent or ligand identifications do occur and are, unfortunately, rarely corrected afterwards in the main depository databases that are constantly used by scientists all over the world for their research work. RNA structural biology is nowadays an extremely complex field, arduous experimentally and theoretically, with previous structures being used in the integration of more complex assemblies in cryo-electron microscopy and in X-ray crystallography. In this respect, journal editors and referees have a major role to play in order to prevent incorrect structures to reach the literature and, almost worse, the depository databases. In order to continue to maintain the highest level of usefulness and accuracy in the structural databases, I would like to urge referees (for the RNA journal and for any journal) to request complete and detailed X-ray statistics tables, validation reports and quality indicators, coordinates, and electron-density maps for proper evaluation and, as usual, to help the authors. Despite fierce competition for publication, I am sure authors will comply with such requests. The serious and fair peer-review process offers enough rail guards to prevent any mishandling of the X-ray data obtained after so much dedication and efforts by the authors. I feel it apt to conclude with words written by Alex Wlodawer and coworkers in 2013 (FEBS J), “As a community, we have to make sure that the high level of advancement is preserved and expanded, and also that the future generations of structural biologists are prepared to gain deeper insight from the massive amounts of data, and to take crystallography to areas that today may not even be foreseen.”

Selenocysteine biosynthesis and its cotranslational incorporation into selenoproteins are achieved by a complex molecular machinery (reviewed in Huttenhofer & Bock, 1998). A major wheel in this mechanism is the tRNASec, which plays a pivotal role. It is first charged with serine by the conventional SerRS, the seryl-residue being further converted in situ to the selenocysteyl-residue by the selenocysteine synthase enzyme. The charged selenocysteyl-tRNASec delivers selenocysteine to the nascent polypeptide chain in response to a reprogrammed UGA codon. The classical elongation factors EF-Tu (in bacteria) or EF1-α (in eukaryotes) do not intervene at this stage. Instead, this process requires a selenocysteine-specific translation factor, called SELB in bacteria, but for which no eukaryotic homologue has been cloned yet. Interestingly, antideterminants against EF-Tu binding were found in the Escherichia coli tRNASec (Rudinger et al., 1996). Based on the several functions that this tRNA has to accomplish, it is reasonable to expect that the tRNASec secondary structure should exhibit distinctive structural features deviating from classical elongator tRNAs. In this regard, functional studies, structural probing, and sequence comparisons confirmed the earlier proposal that the bacterial tRNASec needs an 8-bp long amino acceptor stem and a 6-bp long D-stem to function, instead of the canonical 7 bp and 3/4 bp, respectively (Leinfelder et al., 1988; Baron et al., 1990, 1993; Tormay et al., 1994).

Over 1000 nucleotides may separate the ribozyme core of some group I introns from their 3' splice junctions. Using the sunY intron of bacteriophage T4 as a model system, we have investigated the mechanisms by which proximal splicing events are suppressed in vitro, as well as in vivo. Exon ligation as well as cleavage at the 5' splice site are shown to require long-range pairing between one of the peripheral components of the ribozyme core and some of the nucleotides preceding the authentic 3' splice junction. Consistent with our three-dimensional modeling of the entire sunY ribozyme, we propose that this novel interaction is necessary to drive 5' exon-core transcripts into an active conformation. A requirement for additional stabilizing interactions, either RNA-based or mediated by proteins, appears to be a general feature of group I self-splicing. A role for these interactions in mediating putative alternative splicing events is discussed.