François Major

The MC-Fold and MC-Sym pipeline infers RNA structure from sequence data.

The classical RNA secondary structure model considers A·U and G·C Watson–Crick as well as G·U wobble base pairs. Here we substitute it for a new one, in which sets of nucleotide cyclic motifs define RNA structures. This model allows us to unify all base pairing energetic contributions in an effective scoring function to tackle the problem of RNA folding. We show how pipelining two computer algorithms based on nucleotide cyclic motifs, MC-Fold and MC-Sym, reproduces a series of experimentally determined RNA three-dimensional structures from the sequence. This demonstrates how crucial the consideration of all base-pairing interactions is in filling the gap between sequence and structure. We use the pipeline to define rules of precursor microRNA folding in double helices, despite the presence of a number of presumed mismatches and bulges, and to propose a new model of the human immunodeficiency virus-1 -1 frame-shifting element.

An E2F/miR-20a Autoregulatory Feedback Loop

The E2F family of transcription factors is essential in the regulation of the cell cycle and apoptosis. While the activity of E2F1–3 is tightly controlled by the retinoblastoma family of proteins, the expression of these factors is also regulated at the level of transcription, post-translational modifications and protein stability. Recently, a new level of regulation of E2Fs has been identified, where micro-RNAs (miRNAs) from the mir-17–92 cluster influence the translation of the E2F1 mRNA. We now report that miR-20a, a member of the mir-17–92 cluster, modulates the translation of the E2F2 and E2F3 mRNAs via binding sites in their 3′-untranslated region. We also found that the endogenous E2F1, E2F2, and E2F3 directly bind the promoter of the mir-17–92 cluster activating its transcription, suggesting an autoregulatory feedback loop between E2F factors and miRNAs from the mir-17–92 cluster. Our data also point toward an anti-apoptotic role for miR-20a, since overexpression of this miRNA decreased apoptosis in a prostate cancer cell line, while inhibition of miR-20a by an antisense oligonucleotide resulted in increased cell death after doxorubicin treatment. This anti-apoptotic role of miR-20a may explain some of the oncogenic capacities of the mir-17–92 cluster. Altogether, these results suggest that the autoregulation between E2F1–3 and miR-20a is important for preventing an abnormal accumulation of E2F1–3 and may play a role in the regulation of cellular proliferation and apoptosis.

Autism-related deficits via dysregulated eIF4E-dependent translational control

Hyperconnectivity of neuronal circuits due to increased synaptic protein synthesis is thought to cause autism spectrum disorders (ASDs). The mammalian target of rapamycin (mTOR) is strongly implicated in ASDs by means of upstream signalling; however, downstream regulatory mechanisms are ill-defined. Here we show that knockout of the eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2)—an eIF4E repressor downstream of mTOR—or eIF4E overexpression leads to increased translation of neuroligins, which are postsynaptic proteins that are causally linked to ASDs. Mice that have the gene encoding 4E-BP2 (Eif4ebp2) knocked out exhibit an increased ratio of excitatory to inhibitory synaptic inputs and autistic-like behaviours (that is, social interaction deficits, altered communication and repetitive/stereotyped behaviours). Pharmacological inhibition of eIF4E activity or normalization of neuroligin 1, but not neuroligin 2, protein levels restores the normal excitation/inhibition ratio and rectifies the social behaviour deficits. Thus, translational control by eIF4E regulates the synthesis of neuroligins, maintaining the excitation-to-inhibition balance, and its dysregulation engenders ASD-like phenotypes.

Staufen1 regulates diverse classes of mammalian transcripts

It is currently unknown how extensively the double‐stranded RNA‐binding protein Staufen (Stau)1 is utilized by mammalian cells to regulate gene expression. To date, Stau1 binding to the 3′‐untranslated region (3′‐UTR) of ADP ribosylation factor (ARF)1 mRNA has been shown to target ARF1 mRNA for Stau1‐mediated mRNA decay (SMD). ARF1 SMD depends on translation and recruitment of the nonsense‐mediated mRNA decay factor Upf1 to the ARF1 3′‐UTR by Stau1. Here, we demonstrate that Stau1 binds to a complex structure within the ARF1 3′‐UTR. We also use microarrays to show that 1.1 and 1.0% of the 11 569 HeLa‐cell transcripts that were analyzed are upregulated and downregulated, respectively, at least two‐fold upon Stau1 depletion in three independently performed experiments. We localize the Stau1 binding site to the 3′‐UTR of four mRNAs that we define as natural SMD targets. Additionally, we provide evidence that the efficiency of SMD increases during the differentiation of C2C12 myoblasts to myotubes. We propose that Stau1 influences the expression of a wide variety of physiologic transcripts and metabolic pathways.

Function of Hexameric RNA in Packaging of Bacteriophage φ29 DNA In Vitro

A cyclic hexamer of the 120-base prohead RNA (pRNA) is needed for efficient in vitro packaging of the B. subtilis bacteriophage phi 29 genome. This capacity of pRNA to form higher multimers by intermolecular base pairing of identical subunits represents a new RNA structural motif. Dimers of pRNA are likely intermediates in formation of the cyclic hexamer. A three-dimensional model of the pRNA hexamer is presented.

RNA-Puzzles: A CASP-like evaluation of RNA three-dimensional structure prediction

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.

Identification of a conserved RNA motif essential for she2p recognition and mRNA localization to the yeast bud

ABSTRACT In Saccharomyces cerevisiae, over twenty mRNAs localize to the bud tip of daughter cells, playing roles in processes as different as mating type switching and plasma membrane targeting. The localization of these transcripts depends on interactions between a cis-acting localization element(s) or zipcodes and the RNA-binding protein She2p. While previous studies identified four different localization elements in the bud-localized ASH1 mRNA, the main determinants for She2p recognition are still unknown. To investigate the RNA-binding specificity of She2p, we isolated She2p-binding RNAs by in vivo selection from libraries of partially randomized ASH1 localization elements. The RNAs isolated contained a similar loop-stem-loop structure with a highly conserved CGA triplet in one loop and a single conserved cytosine in the other loop. Mutating these conserved nucleotides or the stem separating them resulted in the loss of She2p binding and in the delocalization of a reporter mRNA. Using this information, we identified the same RNA motif in two other known bud-localized transcripts, suggesting that this motif is conserved among bud-localized mRNAs. These results show that mRNAs with zipcodes lacking primary sequence similarity can rely on a few conserved nucleotides properly oriented in their three-dimensional structure in order to be recognized by the same localization machinery.

Functional involvement of G8 in the hairpin ribozyme cleavage mechanism

The catalytic determinants for the cleavage and ligation reactions mediated by the hairpin ribozyme are integral to the polyribonucleotide chain. We describe experiments that place G8, a critical guanosine, at the active site, and point to an essential role in catalysis. Cross‐linking and modeling show that formation of a catalytic complex is accompanied by a conformational change in which N1 and O6 of G8 become closely apposed to the scissile phosphodiester. UV cross‐linking, hydroxyl‐radical footprinting and native gel electrophoresis indicate that G8 variants inhibit the reaction at a step following domain association, and that the tertiary structure of the inactive complex is not measurably altered. Rate–pH profiles and fluorescence spectroscopy show that protonation at the N1 position of G8 is required for catalysis, and that modification of O6 can inhibit the reaction. Kinetic solvent isotope analysis suggests that two protons are transferred during the rate‐limiting step, consistent with rate‐limiting cleavage chemistry involving concerted deprotonation of the attacking 2′‐OH and protonation of the 5′‐O leaving group. We propose mechanistic models that are consistent with these data, including some that invoke a novel keto–enol tautomerization.

RNAML: A Standard Syntax for Exchanging RNA Information

Analyzing a single data set using multiple RNA informatics programs often requires a file format conversion between each pair of programs, significantly hampering productivity. To facilitate the interoperation of these programs, we propose a syntax to exchange basic RNA molecular information. This RNAML syntax allows for the storage and the exchange of information about RNA sequence and secondary and tertiary structures. The syntax permits the description of higher level information about the data including, but not restricted to, base pairs, base triples, and pseudoknots. A class-oriented approach allows us to represent data common to a given set of RNA molecules, such as a sequence alignment and a consensus secondary structure. Documentation about experiments and computations, as well as references to journals and external databases, are included in the syntax. The chief challenge in creating such a syntax was to determine the appropriate scope of usage and to ensure extensibility as new needs will arise. The syntax complies with the eXtensible Markup Language (XML) recommendations, a widely accepted standard for syntax specifications. In addition to the various generic packages that exist to read and interpret XML formats, an XML processor was developed and put in the open-source MC-Core library for nucleic acid and protein structure computer manipulation.

RNA-Puzzles Round II: assessment of RNA structure prediction programs applied to three large RNA structures

This paper is a report of a second round of RNA-Puzzles, a collective and blind experiment in three-dimensional (3D) RNA structure prediction. Three puzzles, Puzzles 5, 6, and 10, represented sequences of three large RNA structures with limited or no homology with previously solved RNA molecules. A lariat-capping ribozyme, as well as riboswitches complexed to adenosylcobalamin and tRNA, were predicted by seven groups using RNAComposer, ModeRNA/SimRNA, Vfold, Rosetta, DMD, MC-Fold, 3dRNA, and AMBER refinement. Some groups derived models using data from state-of-the-art chemical-mapping methods (SHAPE, DMS, CMCT, and mutate-and-map). The comparisons between the predictions and the three subsequently released crystallographic structures, solved at diffraction resolutions of 2.5-3.2 Å, were carried out automatically using various sets of quality indicators. The comparisons clearly demonstrate the state of present-day de novo prediction abilities as well as the limitations of these state-of-the-art methods. All of the best prediction models have similar topologies to the native structures, which suggests that computational methods for RNA structure prediction can already provide useful structural information for biological problems. However, the prediction accuracy for non-Watson-Crick interactions, key to proper folding of RNAs, is low and some predicted models had high Clash Scores. These two difficulties point to some of the continuing bottlenecks in RNA structure prediction. All submitted models are available for download at http://ahsoka.u-strasbg.fr/rnapuzzles/.