In cell mutational interference mapping experiment (in cell MIME) identifies the 5' polyadenylation signal as a dual regulator of HIV-1 genomic RNA production and packaging.

Non-coding RNA regulatory elements are important for viral replication, making them promising targets for therapeutic intervention. However, regulatory RNA is challenging to detect and characterise using classical structure-function assays. Here, we present in cell Mutational Interference Mapping Experiment (in cell MIME) as a way to define RNA regulatory landscapes at single nucleotide resolution under native conditions. In cell MIME is based on (i) random mutation of an RNA target, (ii) expression of mutated RNA in cells, (iii) physical separation of RNA into functional and non-functional populations, and (iv) high-throughput sequencing to identify mutations affecting function. We used in cell MIME to define RNA elements within the 5' region of the HIV-1 genomic RNA (gRNA) that are important for viral replication in cells. We identified three distinct RNA motifs controlling intracellular gRNA production, and two distinct motifs required for gRNA packaging into virions. Our analysis reveals the 73AAUAAA78 polyadenylation motif within the 5' PolyA domain as a dual regulator of gRNA production and gRNA packaging, and demonstrates that a functional polyadenylation signal is required for viral packaging even though it negatively affects gRNA production.

High-resolution cryo-electron microscopy structure of the Trypanosoma brucei ribosome.

Ribosomes, the protein factories of living cells, translate genetic information carried by messenger RNAs into proteins, and are thus involved in virtually all aspects of cellular development and maintenance. The few available structures of the eukaryotic ribosome reveal that it is more complex than its prokaryotic counterpart, owing mainly to the presence of eukaryote-specific ribosomal proteins and additional ribosomal RNA insertions, called expansion segments. The structures also differ among species, partly in the size and arrangement of these expansion segments. Such differences are extreme in kinetoplastids, unicellular eukaryotic parasites often infectious to humans. Here we present a high-resolution cryo-electron microscopy structure of the ribosome of Trypanosoma brucei, the parasite that is transmitted by the tsetse fly and that causes African sleeping sickness. The atomic model reveals the unique features of this ribosome, characterized mainly by the presence of unusually large expansion segments and ribosomal-protein extensions leading to the formation of four additional inter-subunit bridges. We also find additional rRNA insertions, including one large rRNA domain that is not found in other eukaryotes. Furthermore, the structure reveals the five cleavage sites of the kinetoplastid large ribosomal subunit (LSU) rRNA chain, which is known to be cleaved uniquely into six pieces, and suggests that the cleavage is important for the maintenance of the T. brucei ribosome in the observed structure. We discuss several possible implications of the large rRNA expansion segments for the translation-regulation process. The structure could serve as a basis for future experiments aimed at understanding the functional importance of these kinetoplastid-specific ribosomal features in protein-translation regulation, an essential step towards finding effective and safe kinetoplastid-specific drugs.

Mutational interference mapping experiment (MIME) for studying RNA structure and function.

RNA regulates many biological processes; however, identifying functional RNA sequences and structures is complex and time-consuming. We introduce a method, mutational interference mapping experiment (MIME), to identify, at single-nucleotide resolution, the primary sequence and secondary structures of an RNA molecule that are crucial for its function. MIME is based on random mutagenesis of the RNA target followed by functional selection and next-generation sequencing. Our analytical approach allows the recovery of quantitative binding parameters and permits the identification of base-pairing partners directly from the sequencing data. We used this method to map the binding site of the human immunodeficiency virus-1 (HIV-1) Pr55(Gag) protein on the viral genomic RNA in vitro, and showed that, by analyzing permitted base-pairing patterns, we could model RNA structure motifs that are crucial for protein binding.

Quantitative and predictive model of kinetic regulation by E. coli TPP riboswitches.

Riboswitches are non-coding elements upstream or downstream of mRNAs that, upon binding of a specific ligand, regulate transcription and/or translation initiation in bacteria, or alternative splicing in plants and fungi. We have studied thiamine pyrophosphate (TPP) riboswitches regulating translation of thiM operon and transcription and translation of thiC operon in E. coli, and that of THIC in the plant A. thaliana. For all, we ascertained an induced-fit mechanism involving initial binding of the TPP followed by a conformational change leading to a higher-affinity complex. The experimental values obtained for all kinetic and thermodynamic parameters of TPP binding imply that the regulation by A. thaliana riboswitch is governed by mass-action law, whereas it is of kinetic nature for the two bacterial riboswitches. Kinetic regulation requires that the RNA polymerase pauses after synthesis of each riboswitch aptamer to leave time for TPP binding, but only when its concentration is sufficient. A quantitative model of regulation highlighted how the pausing time has to be linked to the kinetic rates of initial TPP binding to obtain an ON/OFF switch in the correct concentration range of TPP. We verified the existence of these pauses and the model prediction on their duration. Our analysis also led to quantitative estimates of the respective efficiency of kinetic and thermodynamic regulations, which shows that kinetically regulated riboswitches react more sharply to concentration variation of their ligand than thermodynamically regulated riboswitches. This rationalizes the interest of kinetic regulation and confirms empirical observations that were obtained by numerical simulations.

Elaborate uORF/IRES features control expression and localization of human glycyl-tRNA synthetase.

The canonical activity of glycyl-tRNA synthetase (GARS) is to charge glycine onto its cognate tRNAs. However, outside translation, GARS also participates in many other functions. A single gene encodes both the cytosolic and mitochondrial forms of GARS but 2 mRNA isoforms were identified. Using immunolocalization assays, in vitro translation assays and bicistronic constructs we provide experimental evidence that one of these mRNAs tightly controls expression and localization of human GARS. An intricate regulatory domain was found in its 5'-UTR which displays a functional Internal Ribosome Entry Site and an upstream Open Reading Frame. Together, these elements hinder the synthesis of the mitochondrial GARS and target the translation of the cytosolic enzyme to ER-bound ribosomes. This finding reveals a complex picture of GARS translation and localization in mammals. In this context, we discuss how human GARS expression could influence its moonlighting activities and its involvement in diseases.

A universal RNA structural motif docking the elbow of tRNA in the ribosome, RNAse P and T-box leaders.

The structure and function of conserved motifs constituting the apex of Stem I in T-box mRNA leaders are investigated. We point out that this apex shares striking similarities with the L1 stalk (helices 76-78) of the ribosome. A sequence and structure analysis of both elements shows that, similarly to the head of the L1 stalk, the function of the apex of Stem I lies in the docking of tRNA through a stacking interaction with the conserved G19:C56 base pair platform. The inferred structure in the apex of Stem I consists of a module of two T-loops bound together head to tail, a module that is also present in the head of the L1 stalk, but went unnoticed. Supporting the analysis, we show that a highly conserved structure in RNAse P formerly described as the J11/12-J12/11 module, which is precisely known to bind the elbow of tRNA, constitutes a third instance of this T-loop module. A structural analysis explains why six nucleotides constituting the core of this module are highly invariant among all three types of RNA. Our finding that major RNA partners of tRNA bind the elbow with a same RNA structure suggests an explanation for the origin of the tRNA L-shape.

Visualization of multiple alignments, phylogenies and gene family evolution.

Software for visualizing sequence alignments and trees are essential tools for life scientists. In this review, we describe the major features and capabilities of a selection of stand-alone and web-based applications useful when investigating the function and evolution of a gene family. These range from simple viewers, to systems that provide sophisticated editing and analysis functions. We conclude with a discussion of the challenges that these tools now face due to the flood of next generation sequence data and the increasingly complex network of bioinformatics information sources.

Visualization of macromolecular structures.

Structural biology is rapidly accumulating a wealth of detailed information about protein function, binding sites, RNA, large assemblies and molecular motions. These data are increasingly of interest to a broader community of life scientists, not just structural experts. Visualization is a primary means for accessing and using these data, yet visualization is also a stumbling block that prevents many life scientists from benefiting from three-dimensional structural data. In this review, we focus on key biological questions where visualizing three-dimensional structures can provide insight and describe available methods and tools.

Localization of eukaryote-specific ribosomal proteins in a 5.5-Å cryo-EM map of the 80S eukaryotic ribosome.

Protein synthesis in all living organisms occurs on ribonucleoprotein particles, called ribosomes. Despite the universality of this process, eukaryotic ribosomes are significantly larger in size than their bacterial counterparts due in part to the presence of 80 r proteins rather than 54 in bacteria. Using cryoelectron microscopy reconstructions of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution, together with a 6.1-Å map of a translating Saccharomyces cerevisiae 80S ribosome, we have localized and modeled 74/80 (92.5%) of the ribosomal proteins, encompassing 12 archaeal/eukaryote-specific small subunit proteins as well as the complete complement of the ribosomal proteins of the eukaryotic large subunit. Near-complete atomic models of the 80S ribosome provide insights into the structure, function, and evolution of the eukaryotic translational apparatus.

Cryo-EM structure and rRNA model of a translating eukaryotic 80S ribosome at 5.5-A resolution.

Protein biosynthesis, the translation of the genetic code into polypeptides, occurs on ribonucleoprotein particles called ribosomes. Although X-ray structures of bacterial ribosomes are available, high-resolution structures of eukaryotic 80S ribosomes are lacking. Using cryoelectron microscopy and single-particle reconstruction, we have determined the structure of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Šresolution. This map, together with a 6.1-Šmap of a Saccharomyces cerevisiae 80S ribosome, has enabled us to model ∼98% of the rRNA. Accurate assignment of the rRNA expansion segments (ES) and variable regions has revealed unique ES-ES and r-protein-ES interactions, providing insight into the structure and evolution of the eukaryotic ribosome.

Base pairing constraints drive structural epistasis in ribosomal RNA sequences.

It has long been accepted that the structural constraints stemming from the 3D structure of ribosomal RNA (rRNA) lead to coevolution through compensating mutations between interacting sites. State-of-the-art methods for detecting coevolving sites, however, while reaching high levels of specificity and sensitivity for Watson-Crick (WC) pairs of the helices defining the secondary structure, only scarcely reveal tertiary interactions occurring at the level of the 3D structure. In order to understand the relative failure of coevolutionary methods to detect such interactions, we analyze 2,682 interacting sites derived from high-resolution structures, which include a comprehensive data set of rRNA sequences from Archaea and Bacteria. We report a striking difference in the amount of coevolution between WC and non-WC pairs. In order to understand this pattern, we derive fitness landscapes from the geometry of base pairing interactions and construct neutral networks of substitutions for each type of interaction. These networks show that coevolution is a property of WC pairs because, unlike non-WC pairs, their landscapes exhibit fitness valleys, a single mutation in a WC pair resulting in a fitness drop. Second, we used the inferred neutral networks to estimate the level of constraint acting on each type of base pair and show that it correlates negatively with the observed rate of substitutions for all non-WC pairs. WC pairs appear as outliers, fixing more substitutions than expected according to their level of constraint. We here propose that the rate of substitution in WC pairs is due to coevolution resulting from constraints acting at intermediate levels of organization, namely the one of the helical stem with its forming WC pairs. In agreement with this hypothesis, we report a significant excess of intrahelical, inter-WC pairs coevolution compared with interhelices pairs. Altogether, these results show that detailed biochemical knowledge is required and has to be incorporated into evolutionary reasoning in order to understand the fine patterns of variation at the molecular level. They also demonstrate that coevolutionary analysis provides almost exclusively 2D information and only little 3D signal.

The RNA structure alignment ontology.

Multiple sequence alignments are powerful tools for understanding the structures, functions, and evolutionary histories of linear biological macromolecules (DNA, RNA, and proteins), and for finding homologs in sequence databases. We address several ontological issues related to RNA sequence alignments that are informed by structure. Multiple sequence alignments are usually shown as two-dimensional (2D) matrices, with rows representing individual sequences, and columns identifying nucleotides from different sequences that correspond structurally, functionally, and/or evolutionarily. However, the requirement that sequences and structures correspond nucleotide-by-nucleotide is unrealistic and hinders representation of important biological relationships. High-throughput sequencing efforts are also rapidly making 2D alignments unmanageable because of vertical and horizontal expansion as more sequences are added. Solving the shortcomings of traditional RNA sequence alignments requires explicit annotation of the meaning of each relationship within the alignment. We introduce the notion of "correspondence," which is an equivalence relation between RNA elements in sets of sequences as the basis of an RNA alignment ontology. The purpose of this ontology is twofold: first, to enable the development of new representations of RNA data and of software tools that resolve the expansion problems with current RNA sequence alignments, and second, to facilitate the integration of sequence data with secondary and three-dimensional structural information, as well as other experimental information, to create simultaneously more accurate and more exploitable RNA alignments.

Assemble: an interactive graphical tool to analyze and build RNA architectures at the 2D and 3D levels.

SUMMARY: Assemble is an intuitive graphical interface to analyze, manipulate and build complex 3D RNA architectures. It provides several advanced and unique features within the framework of a semi-automated modeling process that can be performed by homology and ab initio with or without electron density maps. Those include the interactive editing of a secondary structure and a searchable, embedded library of annotated tertiary structures. Assemble helps users with performing recurrent and otherwise tedious tasks in structural RNA research. AVAILABILITY AND IMPLEMENTATION: Assemble is released under an open-source license (MIT license) and is freely available at http://bioinformatics.org/assemble. It is implemented in the Java language and runs on MacOSX, Linux and Windows operating systems.

RNA structure: bioinformatic analysis.

The range of functions ascribed to RNA molecules has grown considerably during recent years. Consequently, the analysis and comparison of RNA sequences have become recurrent tasks in molecular biology. Because the biological function of an RNA is expressed more by its folded architecture than by its sequence, original computational tools adapted to the multifaceted RNA functions have to be developed. Such tools, recently published, enable a user to solve classical problems related to RNA research: constructing 'structural' multiple alignments, inferring complete structures and structural motifs from RNA alignments, or searching structural homology in genomic databases.

Tools for the automatic identification and classification of RNA base pairs.

Three programs have been developed to aid in the classification and visualization of RNA structure. BPViewer provides a web interface for displaying three-dimensional (3D) coordinates of individual base pairs or base pair collections. A web server, RNAview, automatically identifies and classifies the types of base pairs that are formed in nucleic acid structures by various combinations of the three edges, Watson-Crick, Hoogsteen and the Sugar edge. RNAView produces two-dimensional (2D) diagrams of secondary and tertiary structure in either Postscript, VRML or RNAML formats. The application RNAMLview can be used to rearrange various parts of the RNAView 2D diagram to generate a standard representation (like the cloverleaf structure of tRNAs) or any layout desired by the user. A 2D diagram can be rapidly reformatted using RNAMLview since all the parts of RNA (like helices and single strands) are dynamically linked while moving the selected parts. With the base pair annotation and the 2D graphic display, RNA motifs are rapidly identified and classified. A survey has been carried out for 41 unique structures selected from the NDB database. The statistics for the occurrence of each edge and of each of the 12 bp families are given for the combinations of the four bases: A, G, U and C. The program also allows for visualization of the base pair interactions by using a symbolic convention previously proposed for base pairs. The web servers for BPViewer and RNAview are available at http://ndbserver.rutgers.edu/services/. The application RNAMLview can also be downloaded from this site. The 2D diagrams produced by RNAview are available for RNA structures in the Nucleic Acid Database (NDB) at http://ndbserver.rutgers.edu/atlas/.

Genome engineering in the yeast pathogen Candida glabrata using the CRISPR-Cas9 system.

Among Candida species, the opportunistic fungal pathogen Candida glabrata has become the second most common causative agent of candidiasis in the world and a major public health concern. Yet, few molecular tools and resources are available to explore the biology of C. glabrata and to better understand its virulence during infection. In this study, we describe a robust experimental strategy to generate loss-of-function mutants in C. glabrata. The procedure is based on the development of three main tools: (i) a recombinant strain of C. glabrata constitutively expressing the CRISPR-Cas9 system, (ii) an online program facilitating the selection of the most efficient guide RNAs for a given C. glabrata gene, and (iii) the identification of mutant strains by the Surveyor technique and sequencing. As a proof-of-concept, we have tested the virulence of some mutants in vivo in a Drosophila melanogaster infection model. Our results suggest that yps11 and a previously uncharacterized serine/threonine kinase are involved, directly or indirectly, in the ability of the pathogenic yeast to infect this model host organism.

Transient compartmentalization of RNA replicators prevents extinction due to parasites.

The appearance of molecular replicators (molecules that can be copied) was probably a critical step in the origin of life. However, parasitic replicators would take over and would have prevented life from taking off unless the replicators were compartmentalized in reproducing protocells. Paradoxically, control of protocell reproduction would seem to require evolved replicators. We show here that a simpler population structure, based on cycles of transient compartmentalization (TC) and mixing of RNA replicators, is sufficient to prevent takeover by parasitic mutants. TC tends to select for ensembles of replicators that replicate at a similar rate, including a diversity of parasites that could serve as a source of opportunistic functionality. Thus, TC in natural, abiological compartments could have allowed life to take hold.

Specific recognition of the HIV-1 genomic RNA by the Gag precursor.

During assembly of HIV-1 particles in infected cells, the viral Pr55(Gag) protein (or Gag precursor) must select the viral genomic RNA (gRNA) from a variety of cellular and viral spliced RNAs. However, there is no consensus on how Pr55(Gag) achieves this selection. Here, by using RNA binding and footprinting assays, we demonstrate that the primary Pr55(Gag) binding site on the gRNA consists of the internal loop and the lower part of stem-loop 1 (SL1), the upper part of which initiates gRNA dimerization. A double regulation ensures specific binding of Pr55(Gag) to the gRNA despite the fact that SL1 is also present in spliced viral RNAs. The region upstream of SL1, which is present in all HIV-1 RNAs, prevents binding to SL1, but this negative effect is counteracted by sequences downstream of SL4, which are unique to the gRNA.

Predicting and modeling RNA architecture.

A general approach for modeling the architecture of large and structured RNA molecules is described. The method exploits the modularity and the hierarchical folding of RNA architecture that is viewed as the assembly of preformed double-stranded helices defined by Watson-Crick base pairs and RNA modules maintained by non-Watson-Crick base pairs. Despite the extensive molecular neutrality observed in RNA structures, specificity in RNA folding is achieved through global constraints like lengths of helices, coaxiality of helical stacks, and structures adopted at the junctions of helices. The Assemble integrated suite of computer tools allows for sequence and structure analysis as well as interactive modeling by homology or ab initio assembly with possibilities for fitting within electronic density maps. The local key role of non-Watson-Crick pairs guides RNA architecture formation and offers metrics for assessing the accuracy of three-dimensional models in a more useful way than usual root mean square deviation (RMSD) values.