Conserved Pseudoknots in lncRNA MEG3 Are Essential for Stimulation of the p53 Pathway.

Long non-coding RNAs (lncRNAs) are key regulatory molecules, but unlike with other RNAs, the direct link between their tertiary structure motifs and their function has proven elusive. Here we report structural and functional studies of human maternally expressed gene 3 (MEG3), a tumor suppressor lncRNA that modulates the p53 response. We found that, in an evolutionary conserved region of MEG3, two distal motifs interact by base complementarity to form alternative, mutually exclusive pseudoknot structures ("kissing loops"). Mutations that disrupt these interactions impair MEG3-dependent p53 stimulation in vivo and disrupt MEG3 folding in vitro. These findings provide mechanistic insights into regulation of the p53 pathway by MEG3 and reveal how conserved motifs of tertiary structure can regulate lncRNA biological function.

A Single Mechanism of Biogenesis, Initiated and Directed by PIWI Proteins, Explains piRNA Production in Most Animals.

In animals, PIWI-interacting RNAs (piRNAs) guide PIWI proteins to silence transposons and regulate gene expression. The mechanisms for making piRNAs have been proposed to differ among cell types, tissues, and animals. Our data instead suggest a single model that explains piRNA production in most animals. piRNAs initiate piRNA production by guiding PIWI proteins to slice precursor transcripts. Next, PIWI proteins direct the stepwise fragmentation of the sliced precursor transcripts, yielding tail-to-head strings of phased precursor piRNAs (pre-piRNAs). Our analyses detect evidence for this piRNA biogenesis strategy across an evolutionarily broad range of animals, including humans. Thus, PIWI proteins initiate and sustain piRNA biogenesis by the same mechanism in species whose last common ancestor predates the branching of most animal lineages. The unified model places PIWI-clade Argonautes at the center of piRNA biology and suggests that the ancestral animal-the Urmetazoan-used PIWI proteins both to generate piRNA guides and to execute piRNA function.

Elucidating Evolutionary Mechanisms and Variants of the Hammerhead Ribozyme Using In Vitro Selection.

The Hammerhead Ribozyme (HHR) is a ubiquitous RNA enzyme that catalyzes site-specific intramolecular cleavage. While mutations to its catalytic core have traditionally been viewed as detrimental to its activity, several discoveries of naturally occurring variants of the full-length ribozyme challenge this notion, suggesting a deeper understanding of HHR evolution and functionality. By systematically introducing mutations at key nucleotide positions within the catalytic core, we generated single-, double-, and triple-mutation libraries to explore the sequence requirements and evolution of a full-length HHR. In vitro selection revealed many novel hammerhead variants, some of which possess mutations at nucleotides previously considered to be essential. We also demonstrate that the evolutionary trajectory of each nucleotide in the catalytic core directly correlates with their functional importance, potentially giving researchers a novel method to assess the sequence requirements of functional nucleic acids.

The hammerhead self-cleaving motif as a precursor to complex endonucleolytic ribozymes.

Connections between distinct catalytic RNA motifs through networks of mutations that retain catalytic function (neutral networks) were likely central to the evolution of biocatalysis. Despite suggestions that functional RNAs collectively form an interconnected web of neutral networks, little evidence has emerged to demonstrate the existence of such intersecting networks in naturally occurring RNAs. Here we show that neutral networks of two naturally occurring, seemingly unrelated endonucleolytic ribozymes, the hammerhead (HH) and hairpin (HP), intersect. Sequences at the intersection of these networks exhibit catalytic functions corresponding to both ribozymes by potentially populating both catalytic folds and enable a smooth crossover between the two. Small and structurally simple endonucleolytic motifs like the HH ribozyme could, through mutational walks along their neutral networks, encounter novel catalytic phenotypes, and structurally flexible, bifunctional sequences at the intersection of these networks could have acted as nodes for evolutionary diversification in an RNA world. Considering the simplicity and small size of the HH ribozyme, we propose that this self-cleaving motif could have been a precursor to other more complex endonucleolytic ribozymes. More generally, our results suggest that RNAs that possess distinct sequences, structures, and catalytic functions, can potentially share evolutionary history through mutational connections in sequence space.

The molecular structure of long non-coding RNAs: emerging patterns and functional implications.

Long non-coding RNAs (lncRNAs) are recently-discovered transcripts that regulate vital cellular processes and are crucially connected to diseases. Despite their unprecedented molecular complexity, it is emerging that lncRNAs possess distinct structural motifs. Remarkably, the 3D shape and topology of full-length, native lncRNAs have been visualized for the first time in the last year. These studies reveal that lncRNA structures dictate lncRNA functions. Here, we review experimentally determined lncRNA structures and emphasize that lncRNA structural characterization requires synergistic integration of computational, biochemical and biophysical approaches. Based on these emerging paradigms, we discuss how to overcome the challenges posed by the complex molecular architecture of lncRNAs, with the goal of obtaining a detailed understanding of lncRNA functions and molecular mechanisms in the future.

RNA 3D structure prediction guided by independent folding of homologous sequences.

BACKGROUND: The understanding of the importance of RNA has dramatically changed over recent years. As in the case of proteins, the function of an RNA molecule is encoded in its tertiary structure, which in turn is determined by the molecule's sequence. The prediction of tertiary structures of complex RNAs is still a challenging task. RESULTS: Using the observation that RNA sequences from the same RNA family fold into conserved structure, we test herein whether parallel modeling of RNA homologs can improve ab initio RNA structure prediction. EvoClustRNA is a multi-step modeling process, in which homologous sequences for the target sequence are selected using the Rfam database. Subsequently, independent folding simulations using Rosetta FARFAR and SimRNA are carried out. The model of the target sequence is selected based on the most common structural arrangement of the common helical fragments. As a test, on two blind RNA-Puzzles challenges, EvoClustRNA predictions ranked as the first of all submissions for the L-glutamine riboswitch and as the second for the ZMP riboswitch. Moreover, through a benchmark of known structures, we discovered several cases in which particular homologs were unusually amenable to structure recovery in folding simulations compared to the single original target sequence. CONCLUSION: This work, for the first time to our knowledge, demonstrates the importance of the selection of the target sequence from an alignment of an RNA family for the success of RNA 3D structure prediction. These observations prompt investigations into a new direction of research for checking 3D structure "foldability" or "predictability" of related RNA sequences to obtain accurate predictions. To support new research in this area, we provide all relevant scripts in a documented and ready-to-use form. By exploring new ideas and identifying limitations of the current RNA 3D structure prediction methods, this work is bringing us closer to the near-native computational RNA 3D models.

United we stand: big roles for small RNA gene clusters.

Prokaryotes and eukaryotes evolved relatively similar RNA-based molecular mechanisms to fight potentially deleterious nucleic acids coming from phages, transposons, or viruses. Short RNAs guide effector complexes toward their targets to be silenced or eliminated. These short immunity RNAs are transcribed from clustered loci. Unexpectedly and strikingly, bacterial and eukaryotic immunity RNA clusters share substantial functional and mechanistic resemblances in fighting nucleic acid intruders.

RNA-Seq employing a novel rRNA depletion strategy reveals a rich repertoire of snoRNAs in Euglena gracilis including box C/D and Ψ-guide RNAs targeting the modification of rRNA extremities.

Previous mRNA transcriptome studies of Euglena gracilis have shown that this organism possesses a large and diverse complement of protein coding genes; however, the study of non-coding RNA classes has been limited. The natural extensive fragmentation of the E. gracilis large subunit ribosomal RNA presents additional barriers to the identification of non-coding RNAs as size-selected small RNA libraries will be dominated by rRNA sequences. In this study we have developed a strategy to significantly reduce rRNA amplification prior to RNA-Seq analysis thereby producing a ncRNA library allowing for the identification of many new E. gracilis small RNAs. Library analysis reveals 113 unique new small nucleolar (sno) RNAs and a large collection of snoRNA isoforms, as well as the first significant collection of nuclear tRNAs in this organism. A 3' end AGAUGN consensus motif and conserved structural features can now be defined for E. gracilis pseudouridine guide RNAs. snoRNAs of both classes were identified that target modification of the 3' extremities of rRNAs utilizing predicted base-pairing interactions with internally transcribed spacers (ITS), providing insight into the timing of steps in rRNA maturation. Cumulatively, this represents the most comprehensive analysis of small ncRNAs in Euglena gracilis to date.

History of the ribosome and the origin of translation.

We present a molecular-level model for the origin and evolution of the translation system, using a 3D comparative method. In this model, the ribosome evolved by accretion, recursively adding expansion segments, iteratively growing, subsuming, and freezing the rRNA. Functions of expansion segments in the ancestral ribosome are assigned by correspondence with their functions in the extant ribosome. The model explains the evolution of the large ribosomal subunit, the small ribosomal subunit, tRNA, and mRNA. Prokaryotic ribosomes evolved in six phases, sequentially acquiring capabilities for RNA folding, catalysis, subunit association, correlated evolution, decoding, energy-driven translocation, and surface proteinization. Two additional phases exclusive to eukaryotes led to tentacle-like rRNA expansions. In this model, ribosomal proteinization was a driving force for the broad adoption of proteins in other biological processes. The exit tunnel was clearly a central theme of all phases of ribosomal evolution and was continuously extended and rigidified. In the primitive noncoding ribosome, proto-mRNA and the small ribosomal subunit acted as cofactors, positioning the activated ends of tRNAs within the peptidyl transferase center. This association linked the evolution of the large and small ribosomal subunits, proto-mRNA, and tRNA.

Evolution of the ribosome at atomic resolution.

The origins and evolution of the ribosome, 3-4 billion years ago, remain imprinted in the biochemistry of extant life and in the structure of the ribosome. Processes of ribosomal RNA (rRNA) expansion can be "observed" by comparing 3D rRNA structures of bacteria (small), yeast (medium), and metazoans (large). rRNA size correlates well with species complexity. Differences in ribosomes across species reveal that rRNA expansion segments have been added to rRNAs without perturbing the preexisting core. Here we show that rRNA growth occurs by a limited number of processes that include inserting a branch helix onto a preexisting trunk helix and elongation of a helix. rRNA expansions can leave distinctive atomic resolution fingerprints, which we call "insertion fingerprints." Observation of insertion fingerprints in the ribosomal common core allows identification of probable ancestral expansion segments. Conceptually reversing these expansions allows extrapolation backward in time to generate models of primordial ribosomes. The approach presented here provides insight to the structure of pre-last universal common ancestor rRNAs and the subsequent expansions that shaped the peptidyl transferase center and the conserved core. We infer distinct phases of ribosomal evolution through which ribosomal particles evolve, acquiring coding and translocation, and extending and elaborating the exit tunnel.

Small RNAs from a Big Genome: The piRNA Pathway and Transposable Elements in the Salamander Species Desmognathus fuscus.

Most of the largest vertebrate genomes are found in salamanders, a clade of amphibians that includes 686 species. Salamander genomes range in size from 14 to 120 Gb, reflecting the accumulation of large numbers of transposable element (TE) sequences from all three TE classes. Although DNA loss rates are slow in salamanders relative to other vertebrates, high levels of TE insertion are also likely required to explain such high TE loads. Across the Tree of Life, novel TE insertions are suppressed by several pathways involving small RNA molecules. In most known animals, TE activity in the germline is primarily regulated by the Piwi-interacting RNA (piRNA) pathway. In this study, we test the hypothesis that salamanders' unusually high TE loads reflect the loss of the ancestral piRNA-mediated TE-silencing machinery. We characterized the small RNA pool in the female and male adult gonads, testing for the presence of small RNA molecules that bear the characteristics of TE-targeting piRNAs. We also analyzed the amino acid sequences of piRNA pathway proteins from salamanders and other vertebrates, testing whether the overall patterns of sequence divergence are consistent with conserved pathway function across the vertebrate clade. Our results do not support the hypothesis of piRNA pathway loss; instead, they suggest that the piRNA pathway is expressed in salamanders. Given these results, we propose hypotheses to explain how the extraordinary TE loads in salamander genomes could have accumulated, despite the expression of TE-silencing machinery.

Structural Insights into Conformation Differences between DNA/TNA and RNA/TNA Chimeric Duplexes.

Threose nucleic acid (TNA) is an artificial genetic polymer capable of heredity and evolution, and is studied in the context of RNA chemical etiology. It has a four-carbon threose backbone in place of the five-carbon ribose of natural nucleic acids, yet forms stable antiparallel complementary Watson-Crick homoduplexes and heteroduplexes with DNA and RNA. TNA base-pairs more favorably with RNA than with DNA but the reason is unknown. Here, we employed NMR, ITC, UV, and CD to probe the structural and dynamic properties of heteroduplexes of RNA/TNA and DNA/TNA. The results indicate that TNA templates the structure of heteroduplexes, thereby forcing an A-like helical geometry. NMR measurement of kinetic and thermodynamic parameters for individual base pair opening events reveal unexpected asymmetric "breathing" fluctuations of the DNA/TNA helix. The results suggest that DNA is unable to fully adapt to the conformational constraints of the rigid TNA backbone and that nucleic acid breathing dynamics are determined from both backbone and base contributions.

The new world of RNA diagnostics and therapeutics.

The 5th Workshop IRE on Translational Oncology was held in Rome (Italy) on 27-28 March at the IRCCS Regina Elena National Cancer Institute. This meeting entitled "The New World of RNA diagnostics and therapeutics" highlightes the significant progress in the RNA field made over the last years. Research moved from pure discovery towards the development of diagnostic biomarkers or RNA-base targeted therapies seeking validation in several clinical trials. Non-coding RNAs in particular have been the focus of this workshop due to their unique properties that make them attractive tools for the diagnosis and therapy of cancer.This report collected the presentations of many scientists from different institutions that discussed recent oncology research providing an excellent overview and representative examples for each possible application of RNA as biomarker, for therapy or to increase the number of patients that can benefit from precision oncology treatment.In particular, the meeting specifically emphasized two key features of RNA applications: RNA diagnostic (Blandino, Palcau, Sestito, Díaz Méndez, Cappelletto, Pulito, Monteonofrio, Calin, Sozzi, Cheong) and RNA therapeutics (Dinami, Marcia, Anastasiadou, Ryan, Fattore, Regazzo, Loria, Aharonov).

Acquisition of Dual Ribozyme-Functions in Nonfunctional Short Hairpin RNAs through Kissing-Loop Interactions.

The acquisition of functions via the elongation of nucleotides is an important factor in the development of the RNA world. In our previous study, we found that the introduction of complementary seven-membered kissing loops into inactive R3C ligase ribozymes revived their ligation activity. In this study, we applied the kissing complex formation-induced rearrangement of RNAs to two nonfunctional RNAs by introducing complementary seven-membered loops into each of them. By combining these two forms of RNAs, the ligase activity (derived from the R3C ligase ribozyme) as well as cleavage activity (derived from the hammerhead ribozyme) was obtained. Thus, effective RNA evolution toward the formation of a life system may require the achievement of "multiple" functions via kissing-loop interactions, as indicated in this study. Our results point toward the versatility of kissing-loop interactions in the evolution of RNA, i.e., two small nonfunctional RNAs can gain dual functions via a kissing-loop interaction.

A scenario for the emergence of protoviroids in the RNA world and for their further evolution into viroids and viroid-like RNAs by modular recombinations and mutations.

Viroids are tiny, circular, and noncoding RNAs that are able to replicate and systemically infect plants. The smallest known pathogens, viroids have been proposed to represent survivors from the RNA world that likely preceded the cellular world currently dominating life on the earth. Although the small, circular, and compact nature of viroid genomes, some of which are also endowed with catalytic activity mediated by hammerhead ribozymes, support this proposal, the lack of feasible evolutionary routes and the identification of hammerhead ribozymes in a large number of DNA genomes of organisms along the tree of life have led some to question such a proposal. Here, we reassess the origin and subsequent evolution of viroids by complementing phylogenetic reconstructions with molecular data, including the primary and higher-order structure of the genomic RNAs, their replication, and recombination mechanisms and selected biological information. Features of some viroid-like RNAs found in plants, animals, and possibly fungi are also considered. The resulting evolutionary scenario supports the emergence of protoviroids in the RNA world, mainly as replicative modules, followed by a further increase in genome complexity based on module/domain shuffling and combination and mutation. Such a modular evolutionary scenario would have facilitated the inclusion in the protoviroid genomes of complex RNA structures (or coding sequences, as in the case of hepatitis delta virus and delta-like agents), likely needed for their adaptation from the RNA world to a life based on cells, thus generating the ancestors of current infectious viroids and viroid-like RNAs. Other noninfectious viroid-like RNAs, such as retroviroid-like RNA elements and retrozymes, could also be derived from protoviroids if their reverse transcription and integration into viral or eukaryotic DNA, respectively, are considered as a possible key step in their evolution. Comparison of evidence supporting a general and modular evolutionary model for viroids and viroid-like RNAs with that favoring alternative scenarios provides reasonable reasons to keep alive the hypothesis that these small RNA pathogens may be relics of a precellular world.

High-Throughput Evolutionary Comparative Analysis of Long Intergenic Noncoding RNAs in Multiple Organisms.

Comparative genomic and transcriptomic analyses can help prioritize and facilitate the functional analysis of long noncoding RNAs (lncRNAs). Evolinc-II is a bioinformatic pipeline that automates comparative analyses, searching for sequence and structural conservation for thousands of lncRNAs at once. In addition, Evolinc-II takes a phylogenetic approach to infer key evolutionary events that may have occurred during the emergence of each query lncRNA. Here, we describe how to use command line or GUI (CyVerse's Discovery Environment) versions of Evolinc-II to identify lncRNA homologs and prioritize them for functional analysis.

Aminoacylation of short hairpin RNAs through kissing-loop interactions indicates evolutionary trend of RNA molecules.

The unique G3:U70 base pair in the acceptor stem of tRNAAla has been shown to be a critical recognition site by alanyl-tRNA synthetase (AlaRS). The base pair resides on one of the arms of the L-shaped structure of tRNA (minihelix) and the genetic code has likely evolved from a primordial tRNA-aaRS (aminoacyl-tRNA synthetase) system. In terms of the evolution of tRNA, incorporation of a G:U base pair in the structure would be important. Here, we found that two independent short hairpin RNAs change their conformation through kissing-loop interactions, finally forming a minihelix-like structure, in which the G3:U70 base pair is incorporated. The RNA system can be properly aminoacylated by the minimal Escherichia coli AlaRS variant with alanylation activity (AlaRS442N). Thus, characteristic structural features produced via kissing-loop interactions may provide important clues into the evolution of RNA.

SELEX and SHAPE reveal that sequence motifs and an extended hairpin in the 5' portion of Turnip crinkle virus satellite RNA C mediate fitness in plants.

Noncoding RNAs use their sequence and/or structure to mediate function(s). The 5' portion (166 nt) of the 356-nt noncoding satellite RNA C (satC) of Turnip crinkle virus (TCV) was previously modeled to contain a central region with two stem-loops (H6 and H7) and a large connecting hairpin (H2). We now report that in vivo functional selection (SELEX) experiments assessing sequence/structure requirements in H2, H6, and H7 reveal that H6 loop sequence motifs were recovered at nonrandom rates and only some residues are proposed to base-pair with accessible complementary sequences within the 5' central region. In vitro SHAPE of SELEX winners indicates that the central region is heavily base-paired, such that along with the lower stem and H2 region, one extensive hairpin exists composing the entire 5' region. As these SELEX winners are highly fit, these characteristics facilitate satRNA amplification in association with TCV in plants.

A Method for RNA Structure Prediction Shows Evidence for Structure in lncRNAs.

To compare the secondary structure profiles of RNA molecules we developed the CROSSalign method. CROSSalign is based on the combination of the Computational Recognition Of Secondary Structure (CROSS) algorithm to predict the RNA secondary structure profile at single-nucleotide resolution and the Dynamic Time Warping (DTW) method to align profiles of different lengths. We applied CROSSalign to investigate the structural conservation of long non-coding RNAs such as XIST and HOTAIR as well as ssRNA viruses including HIV. CROSSalign performs pair-wise comparisons and is able to find homologs between thousands of matches identifying the exact regions of similarity between profiles of different lengths. In a pool of sequences with the same secondary structure CROSSalign accurately recognizes repeat A of XIST and domain D2 of HOTAIR and outperforms other methods based on covariance modeling. The algorithm is freely available at the webpage http://service.tartaglialab.com//new_submission/crossalign.

Global Analysis of Small RNA Dynamics during Seed Development of Picea glauca and Arabidopsis thaliana Populations Reveals Insights on their Evolutionary Trajectories.

While DNA methylation carries genetic signals and is instrumental in the evolution of organismal complexity, small RNAs (sRNAs), ~18-24 ribonucleotide (nt) sequences, are crucial mediators of methylation as well as gene silencing. However, scant study deals with sRNA evolution via featuring their expression dynamics coupled with species of different evolutionary time. Here we report an atlas of sRNAs and microRNAs (miRNAs, single-stranded sRNAs) produced over time at seed-set of two major spermatophytes represented by populations of Picea glauca and Arabidopsis thaliana with different seed-set duration. We applied diverse profiling methods to examine sRNA and miRNA features, including size distribution, sequence conservation and reproduction-specific regulation, as well as to predict their putative targets. The top 27 most abundant miRNAs were highly overlapped between the two species (e.g., miR166,-319 and-396), but in P. glauca, they were less abundant and significantly less correlated with seed-set phases. The most abundant sRNAs in libraries were deeply conserved miRNAs in the plant kingdom for Arabidopsis but long sRNAs (24-nt) for P. glauca. We also found significant difference in normalized expression between populations for population-specific sRNAs but not for lineage-specific ones. Moreover, lineage-specific sRNAs were enriched in the 21-nt size class. This pattern is consistent in both species and alludes to a specific type of sRNAs (e.g., miRNA, tasiRNA) being selected for. In addition, we deemed 24 and 9 sRNAs in P. glauca and Arabidopsis, respectively, as sRNA candidates targeting known adaptive genes. Temperature had significant influence on selected gene and miRNA expression at seed development in both species. This study increases our integrated understanding of sRNA evolution and its potential link to genomic architecture (e.g., sRNA derivation from genome and sRNA-mediated genomic events) and organismal complexity (e.g., association between different sRNA expression and their functionality).