Structural mechanism of bridge RNA-guided recombination.

Insertion sequence (IS) elements are the simplest autonomous transposable elements found in prokaryotic genomes1. We recently discovered that IS110 family elements encode a recombinase and a non-coding bridge RNA (bRNA) that confers modular specificity for target DNA and donor DNA through two programmable loops2. Here we report the cryo-electron microscopy structures of the IS110 recombinase in complex with its bRNA, target DNA and donor DNA in three different stages of the recombination reaction cycle. The IS110 synaptic complex comprises two recombinase dimers, one of which houses the target-binding loop of the bRNA and binds to target DNA, whereas the other coordinates the bRNA donor-binding loop and donor DNA. We uncovered the formation of a composite RuvC-Tnp active site that spans the two dimers, positioning the catalytic serine residues adjacent to the recombination sites in both target and donor DNA. A comparison of the three structures revealed that (1) the top strands of target and donor DNA are cleaved at the composite active sites to form covalent 5'-phosphoserine intermediates, (2) the cleaved DNA strands are exchanged and religated to create a Holliday junction intermediate, and (3) this intermediate is subsequently resolved by cleavage of the bottom strands. Overall, this study reveals the mechanism by which a bispecific RNA confers target and donor DNA specificity to IS110 recombinases for programmable DNA recombination.

Transcriptome screening followed by integrated physicochemical and structural analyses for investigating RNA-mediated berberine activity.

Non-coding RNAs are regarded as promising targets for the discovery of innovative drugs due to their abundance in the genome and their involvement in many biological processes. Phytochemicals (PCs) are the primary source of ligand-based drugs due to their broad spectrum of biological activities. Since many PCs are heterocyclic and have chemical groups potentially involved in the interaction with nucleic acids, detailed interaction analysis between PCs and RNA is crucial to explore the effect of PCs on RNA functions. In this study, an integrated approach for investigating interactions between PCs and RNAs were demonstrated to verify the RNA-mediated PCs functions by using berberine (BRB) as a model PC. RNA screening of a transcriptome library followed by sequence refinement found minimal RNA motif consisting of a cytosine bulge with U-A and G-U neighbouring base pairs for interaction with BRB. NMR-based structure determination and physicochemical analyses using chemical analogues of BRB demonstrated the importance of electrostatic and stacking interactions for sequence selective interaction and RNA stabilization. The selective interaction with a relatively small RNA motif based on a chemical structure of a planer heterocyclic highlights the biological activities of various PCs mediated by the interactions with particular functional RNAs. In addition, the systematic and quantitative investigations demonstrated in this study could be useful for the development of therapeutic chemicals targeting functional RNAs, based on the PCs, in the future.

Noncoding Y RNAs regulate the levels, subcellular distribution and protein interactions of their Ro60 autoantigen partner.

Noncoding Y RNAs are abundant in animal cells and present in many bacteria. These RNAs are bound and stabilized by Ro60, a ring-shaped protein that is a target of autoantibodies in patients with systemic lupus erythematosus. Studies in bacteria revealed that Y RNA tethers Ro60 to a ring-shaped exoribonuclease, forming a double-ringed RNP machine specialized for structured RNA degradation. In addition to functioning as a tether, the bacterial RNA gates access of substrates to the Ro60 cavity. To identify roles for Y RNAs in mammals, we used CRISPR to generate mouse embryonic stem cells lacking one or both of the two murine Y RNAs. Despite reports that animal cell Y RNAs are essential for DNA replication, cells lacking these RNAs divide normally. However, Ro60 levels are reduced, revealing that Y RNA binding is required for Ro60 to accumulate to wild-type levels. Y RNAs regulate the subcellular location of Ro60, since Ro60 is reduced in the cytoplasm and increased in nucleoli when Y RNAs are absent. Last, we show that Y RNAs tether Ro60 to diverse effector proteins to generate specialized RNPs. Together, our data demonstrate that the roles of Y RNAs are intimately connected to that of their Ro60 partner.

Discovery of ANTAR-RNAs and their Mechanism of Action in Mycobacteria.

Non-coding RNAs play pivotal roles in bacterial signaling. However, RNAs from certain phyla (specially high-GC actinobacteria) still remain elusive. Here, by re-engineering the existing genome-wide search approach, we discover a family of structurally conserved RNAs that are present ubiquitously across actinobacteria, including mycobacteria. In vitro analysis shows that RNAs belonging to this family bind response-regulator proteins that contain the widely prevalent ANTAR domain. The Mycobacterium tuberculosis ANTAR protein gets phosphorylated by a histidine kinase and interacts with RNA only in its phosphorylated state. These newly identified RNAs reside only in certain transcripts and typically overlap with the ribosome-binding site, regulating translation of these transcripts. In this way, the RNAs directly link signaling pathways to translational control, thus expanding the mechanistic tool kit available for ANTAR-based control of gene expression. In mycobacteria, we find that RNAs targeted by ANTAR proteins majorly encode enzymes of lipid metabolism and associated redox pathways. This now allows us to identify the key genes that mediate ANTAR-dependent control of lipid metabolism. Our study establishes the identity and wide prevalence of ANTAR-target RNAs in mycobacteria, bringing RNA-mediated regulation in these bacteria to the center stage.

RNAconTest: comparing tools for noncoding RNA multiple sequence alignment based on structural consistency.

The importance of noncoding RNA sequences has become increasingly clear over the past decade. New RNA families are often detected and analyzed using comparative methods based on multiple sequence alignments. Accordingly, a number of programs have been developed for aligning and deriving secondary structures from sets of RNA sequences. Yet, the best tools for these tasks remain unclear because existing benchmarks contain too few sequences belonging to only a small number of RNA families. RNAconTest (RNA consistency test) is a new benchmarking approach relying on the observation that secondary structure is often conserved across highly divergent RNA sequences from the same family. RNAconTest scores multiple sequence alignments based on the level of consistency among known secondary structures belonging to reference sequences in their output alignment. Similarly, consensus secondary structure predictions are scored according to their agreement with one or more known structures in a family. Comparing the performance of 10 popular alignment programs using RNAconTest revealed that DAFS, DECIPHER, LocARNA, and MAFFT created the most structurally consistent alignments. The best consensus secondary structure predictions were generated by DAFS and LocARNA (via RNAalifold). Many of the methods specific to noncoding RNAs exhibited poor scalability as the number or length of input sequences increased, and several programs displayed substantial declines in score as more sequences were aligned. Overall, RNAconTest provides a means of testing and improving tools for comparative RNA analysis, as well as highlighting the best available approaches. RNAconTest is available from the DECIPHER website (http://DECIPHER.codes/Downloads.html).

Understanding the mechanistic basis of non-coding RNA through molecular dynamics simulations.

Noncoding RNA (ncRNA) has a key role in regulating gene expression, mediating fundamental processes and diseases via a variety of yet unknown mechanisms. Here, we review recent applications of conventional and enhanced Molecular Dynamics (MD) simulations methods to address the mechanistic function of large biomolecular systems that are tightly involved in the ncRNA function and that are of key importance in life sciences. This compendium focuses of three biomolecular systems, namely the CRISPR-Cas9 genome editing machinery, group II intron ribozyme and the ribonucleoprotein complex of the spliceosome, which edit and process ncRNA. We show how the application of a novel accelerated MD simulations method has been key in disclosing the conformational transitions underlying RNA binding in the CRISPR-Cas9 complex, suggesting a mechanism for RNA recruitment and clarifying the conformational changes required for attaining genome editing. As well, we discuss the use of mixed quantum-classical MD simulations in deciphering the catalytic mechanism of RNA splicing as operated by group II intron ribozyme, one of the largest ncRNA structures crystallized so far. Finally, we debate the future challenges and opportunities in the field, discussing the recent application of MD simulations for unraveling the functional biophysics of the spliceosome, a multi-mega Dalton complex of proteins and small nuclear RNAs that performs RNA splicing in humans. This showcase of applications highlights the current talent of MD simulations to dissect atomic-level details of complex biomolecular systems instrumental for the design of finely engineered genome editing machines. As well, this review aims at inspiring future investigations of several other ncRNA regulatory systems, such as micro and small interfering RNAs, which achieve their function and specificity using RNA-based recognition and targeting strategies.

RNA localization in bacteria.

One of the most important discoveries in the field of microbiology in the last two decades is that bacterial cells have intricate subcellular organization. This understanding has emerged mainly from the depiction of spatial and temporal organization of proteins in specific domains within bacterial cells, e.g., midcell, cell poles, membrane and periplasm. Because translation of bacterial RNA molecules was considered to be strictly coupled to their synthesis, they were not thought to specifically localize to regions outside the nucleoid. However, the increasing interest in RNAs, including non-coding RNAs, encouraged researchers to explore the spatial and temporal localization of RNAs in bacteria. The recent technological improvements in the field of fluorescence microscopy allowed subcellular imaging of RNAs even in the tiny bacterial cells. It has been reported by several groups, including ours that transcripts may specifically localize in such cells. Here we review what is known about localization of RNA and of the pathways that determine RNA fate in bacteria, and discuss the possible cues and mechanisms underlying these distribution patterns.

Telomeric noncoding RNA TERRA is induced by telomere shortening to nucleate telomerase molecules at short telomeres.

Elongation of a short telomere depends on the action of multiple telomerase molecules, which are visible as telomerase RNA foci or clusters associated with telomeres in yeast and mammalian cells. How several telomerase molecules act on a single short telomere is unknown. Herein, we report that the telomeric noncoding RNA TERRA is involved in the nucleation of telomerase molecules into clusters prior to their recruitment at a short telomere. We find that telomere shortening induces TERRA expression, leading to the accumulation of TERRA molecules into a nuclear focus. Simultaneous time-lapse imaging of telomerase RNA and TERRA reveals spontaneous events of telomerase nucleation on TERRA foci in early S phase, generating TERRA-telomerase clusters. This cluster is subsequently recruited to the short telomere from which TERRA transcripts originate during S phase. We propose that telomere shortening induces noncoding RNA expression to coordinate the recruitment and activity of telomerase molecules at short telomeres.

An RNA degradation machine sculpted by Ro autoantigen and noncoding RNA.

Many bacteria contain an ortholog of the Ro autoantigen, a ring-shaped protein that binds noncoding RNAs (ncRNAs) called Y RNAs. In the only studied bacterium, Deinococcus radiodurans, the Ro ortholog Rsr functions in heat-stress-induced ribosomal RNA (rRNA) maturation and starvation-induced rRNA decay. However, the mechanism by which this conserved protein and its associated ncRNAs act has been obscure. We report that Rsr and the exoribonuclease polynucleotide phosphorylase (PNPase) form an RNA degradation machine that is scaffolded by Y RNA. Single-particle electron microscopy, followed by docking of atomic models into the reconstruction, suggests that Rsr channels single-stranded RNA into the PNPase cavity. Biochemical assays reveal that Rsr and Y RNA adapt PNPase for effective degradation of structured RNAs. A Ro ortholog and ncRNA also associate with PNPase in Salmonella Typhimurium. Our studies identify another ribonucleoprotein machine and demonstrate that ncRNA, by tethering a protein cofactor, can alter the substrate specificity of an enzyme.

The potential of 3D-FISH and super-resolution structured illumination microscopy for studies of 3D nuclear architecture: 3D structured illumination microscopy of defined chromosomal structures visualized by 3D (immuno)-FISH opens new perspectives for studies of nuclear architecture.

Three-dimensional structured illumination microscopy (3D-SIM) has opened up new possibilities to study nuclear architecture at the ultrastructural level down to the ~100 nm range. We present first results and assess the potential using 3D-SIM in combination with 3D fluorescence in situ hybridization (3D-FISH) for the topographical analysis of defined nuclear targets. Our study also deals with the concern that artifacts produced by FISH may counteract the gain in resolution. We address the topography of DAPI-stained DNA in nuclei before and after 3D-FISH, nuclear pores and the lamina, chromosome territories, chromatin domains, and individual gene loci. We also look at the replication patterns of chromocenters and the topographical relationship of Xist-RNA within the inactive X-territory. These examples demonstrate that an appropriately adapted 3D-FISH/3D-SIM approach preserves key characteristics of the nuclear ultrastructure and that the gain in information obtained by 3D-SIM yields new insights into the functional nuclear organization.

A top-down analysis of Xa- and Xi-territories reveals differences of higher order structure at ≥ 20 Mb genomic length scales.

The active and inactive X (Xa;Xi) territory with its seemingly highly compacted Barr body in nuclei of female mammalian cells provide a key example for studies of structure/function relationships in homologous chromosomes with different functional properties. Here we used about 300 human X-specific large insert clones to generate probe sets, which target physically or functionally defined sub-chromosomal segments. We combined 3D multicolor FISH with quantitative 3D image analysis in order to compare the higher order organization in Xi-and Xa-territories in human diploid fibroblasts (HDFs) at various length scales ranging from about 50 Mb down to 1 Mb. Xi-territories were characterized by a rounder shape as compared to the flatter and more extended shape of Xa-territories. The overall compaction of the entire Xi-territory, including the Barr body, was only 1.2-fold higher than the Xa-territory. Significant differences, however, were noted between distinct subchromosomal segments: At 20 Mb length scales higher compaction in Xi-territories was restricted to specific segments, but higher compaction in these segments was not correlated with gene density, transcriptional activity, LINE content or histone markers locally enriched in Xi-territories. Notably, higher compaction in Xi-territories observed for 20 Mb segments was not reflected accordingly by inclosed segments of 1-4 Mb. We conclude that compaction differences result mainly from a regrouping of ~1 Mb chromatin domains rather than from an increased condensation of individual domains. In contrast to a previous report, genes subject to inactivation as well as escaping from inactivation were not excluded from the interior of the Barr body.

Auto-assembly of E. coli DsrA small noncoding RNA: Molecular characteristics and functional consequences.

RNA molecules are important factors involved in different cellular processes and have a multitude of roles in the cell. These roles include serving as a temporary copy of genes used for protein synthesis or functions in translational machinery. Interestingly, RNA is so far the only biological molecule that serves both as a catalyst (like proteins) and as information storage (like DNA). However, in contrast to proteins well known to be able to self-associate in order to maintain the architecture of the cell, such RNA polymers are not prevalent in cells and are usually not favored by the flexibility of this molecule. In this work, we present evidence that such a polymer of a natural RNA, the DsrA RNA, exists in the bacterial cell. DsrA is a small noncoding RNA (87 nucleotides) of Escherichia coli that acts by base-pairing to mRNA in order to control the translation and the turnover of some mRNA, including rpoS mRNA, which encodes the sigma(s) RNA polymerase subunit involved in bacterial stress response. A putative model is proposed for the structure of this RNA polymer. Although the function of this polymerization is not known completely, we propose that the formation of such a structure could be involved in the regulation of DsrA ncRNA concentration in vivo or in a quality control mechanism used by the cell to eliminate misfolded RNAs.

A novel representation of RNA secondary structure based on element-contact graphs.

BACKGROUND: Depending on their specific structures, noncoding RNAs (ncRNAs) play important roles in many biological processes. Interest in developing new topological indices based on RNA graphs has been revived in recent years, as such indices can be used to compare, identify and classify RNAs. Although the topological indices presented before characterize the main topological features of RNA secondary structures, information on RNA structural details is ignored to some degree. Therefore, it is necessity to identify topological features with low degeneracy based on complete and fine-grained RNA graphical representations. RESULTS: In this study, we present a complete and fine scheme for RNA graph representation as a new basis for constructing RNA topological indices. We propose a combination of three vertex-weighted element-contact graphs (ECGs) to describe the RNA element details and their adjacent patterns in RNA secondary structure. Both the stem and loop topologies are encoded completely in the ECGs. The relationship among the three typical topological index families defined by their ECGs and RNA secondary structures was investigated from a dataset of 6,305 ncRNAs. The applicability of topological indices is illustrated by three application case studies. Based on the applied small dataset, we find that the topological indices can distinguish true pre-miRNAs from pseudo pre-miRNAs with about 96% accuracy, and can cluster known types of ncRNAs with about 98% accuracy, respectively. CONCLUSION: The results indicate that the topological indices can characterize the details of RNA structures and may have a potential role in identifying and classifying ncRNAs. Moreover, these indices may lead to a new approach for discovering novel ncRNAs. However, further research is needed to fully resolve the challenging problem of predicting and classifying noncoding RNAs.

Bypassing translation initiation.

A high-resolution cryo-EM reconstruction of a ribosome-bound dicistrovirus IRES (Schüler et al., 2006) and the crystal structure of its ribosome binding domain (Pfingsten et al., 2006) provide new insights into an exceptional eukaryotic translation mechanism.